Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
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Autonomic Neuroscience: Basic and Clinical
journal homepage: www.elsevier.com/locate/autneu
Review
Vas deferens neuro-effector junction: From kymographic tracings to
structural biology principles
L. Camilo Navarrete a, Nelson P. Barrera a, J. Pablo Huidobro-Toro b,⁎
a
b
Laboratorio de Estructura de Proteínas de Membrana y Señalización, Núcleo Milenio de Biología Estructural, NuBEs, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Chile
Laboratorio de Nucleótidos, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Chile
a r t i c l e
i n f o
a b s t r a c t
Article history:
Received 27 November 2013
Received in revised form 14 May 2014
Accepted 20 May 2014
The vas deferens is a simple bioassay widely used to study the physiology of sympathetic neurotransmission and
the pharmacodynamics of adrenergic drugs. The role of ATP as a sympathetic co-transmitter has gained increasing
attention and furthered our understanding of its role in sympathetic reflexes. In addition, new information has
emerged on the mechanisms underlying the storage and release of ATP. Both noradrenaline and ATP concur to
elicit the tissue smooth muscle contractions following sympathetic reflexes or electrical field stimulation of the
sympathetic nerve terminals. ATP and adenosine (its metabolic byproduct) are powerful presynaptic regulators
of co-transmitter actions. In addition, neuropeptide Y, the third member of the sympathetic triad, is an endogenous
modulator. The peptide plus ATP and/or adenosine play a significant role as sympathetic modulators of
transmitter's release. This review focuses on the physiological principles that govern sympathetic co-transmitter
activity, with special interest in defining the motor role of ATP. In addition, we intended to review the recent structural biology findings related to the topology of the P2X1R based on the crystallized P2X4 receptor from Danio
rerio, or the crystallized adenosine A2A receptor as a member of the G protein coupled family of receptors as prototype neuro modulators. This review also covers structural elements of ectonucleotidases, since some members
are found in the vas deferens neuro-effector junction. The allosteric principles that apply to purinoceptors are
also reviewed highlighting concepts derived from receptor theory at the light of the current available structural
elements. Finally, we discuss clinical applications of these concepts.
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Sympathetic cotransmission
Purinergic mechanisms
P2X receptor modeling
GPCR modeling
Ectonucleotidases
Contents
1.
2.
3.
4.
5.
6.
Introduction and historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anatomy and physiology of the tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sympathetic nerve terminals; pharmacological evidence support co-transmission and purinergic mechanisms . . . . . . . . . . .
Presynaptic elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Organization of the nerve endings and its varicosities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
The presynaptic terminal, the site of transmitter storage, release and multiple regulatory mechanisms . . . . . . . . . . .
4.3.
Transmitter synthesis and the control of the biosynthetic process . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Co-storage and release of co-transmitters from the same vesicle? Alternative evidence derived from endogenous modulators
4.5.
Vesicular transport mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.
Neuromodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.
Ectonucleotide-triphosphohydrolases (ENTPDase), ectoATPases, and related enzymes . . . . . . . . . . . . . . . . . .
The postjunctional smooth muscle membrane, site of effector mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . .
The structural biology of signaling elements in purinergic sympathetic transmission . . . . . . . . . . . . . . . . . . . . . .
6.1.
Structure of ligand gated ionic channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Allosterism as a regulatory mode of P2XRs at the structural level . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
G-protein coupled receptors (GPCRs) in sympathetic signaling; involvement of adenosine (ADO) and P2Y receptors . . . . .
6.4.
Ecto enzymes in purinergic signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author at: Laboratory of Nucleotide Pharmacology, Millennium Nucleus of Structural Biology, NuBEs, Department of Biology, Faculty of Chemistry and Biology,
University of Santiago, Chile, Alameda B. O'Higgins 3363, Santiago, Chile. Tel.: +56 2 2718 1144.
E-mail address: [email protected] (J.P. Huidobro-Toro).
http://dx.doi.org/10.1016/j.autneu.2014.05.010
1566-0702/© 2014 Elsevier B.V. All rights reserved.
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L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
6.5.
Ectonucleotide-triphosphohydrolase, ENTPDases (CD 39) . . . . . . . . . .
6.6.
5′ecto nucleotidase, 5′eNT, (CD 73) . . . . . . . . . . . . . . . . . . . .
6.7.
Alkaline phosphatase, APs . . . . . . . . . . . . . . . . . . . . . . . .
7.
Towards an integral view of the purinergic component of sympathetic co-transmission
8.
Novel role of the epithelium in sympathetic excitability . . . . . . . . . . . . . .
9.
Clinical perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction and historical perspective
The vas deferens preparation is a multipurpose bioassay that played a
pivotal role as an experimental tissue model to study the physiology and
pharmacology of autonomic drugs and sympathetic neurotransmission.
The rodent vas deferens preparation has been considered the sympathetic bioassay par excellence, largely surpassing the use of the isolated
heart, nictitating membrane, or systemic blood pressure determinations,
among other reasons, because of its reduced cost, easy surgical access,
simplicity and expedite preparation. The tissue is easily mounted on
the classical physiological organ baths widely used by pharmacologists
and physiologists (Huković, 1997). In addition, the physiological motor
responses, elicited by either electrical stimulation of its nerve terminals
or drug additions to the bath, may be recorded without major distortions
for over 6–10 nonstop hours. As such, this bioassay either from the rat,
mouse, or the guinea pig has been useful to study the pharmacology of
adrenergic and psychoactive drugs, as well as principles of sympathetic
transmission and latter the tissue has played a pivotal role to study the
principles of sympathetic co-transmission (Burnstock and Verkhratsky,
2010). Its popularity as a bioassay to assess the pharmacodynamics of
autonomic drugs is supported, among other biological reasons, by its
high catecholamine content. This tissue contains the largest concentration of norepinephrine (NE) after the adrenal gland, and a correspondingly dense sympathetic nerve terminal network (Smith and Winkler,
1972). Recently, the advent of crystallized purinergic and adrenergic receptors, plus the widespread use of bioinformatics, has made possible
the visualization of structural elements that govern sympathetic cotransmission to further understand the molecular and even atomic
basis of sympathetic transmission. This review is intended to focus on
the cell biology, physiology and pharmacology of sympathetic processes
and discusses the principles of structural biology of purinergic and adenosine receptors and some of the ectonucleotidases present in the tissue
known to hydrolyze ATP (adenosine 5′triphosphate) mainly to adenosine (ADO or A) an ATP degradation byproduct with notable activity as
a sympathetically derived neuromodulator.
S. Huković was the first to use the guinea-pig vas deferens preparation while training in Oxford during 1959; he introduced methodological aspects, which have been only slightly modified over the years.
Using this preparation he addressed issues related to the mechanisms
of action of cocaine and reserpine (Huković, 1961), two key tools used
to study the peripheral sympathetic nerve endings. This seminal paper
was chosen as a landmark in pharmacology by the British J. Pharmacology Society in its Golden Jubilee issue (Huković, 1997). Along the years,
this simple preparation provided experimental evidence for some of the
major concepts of autonomic neurotransmission. Another paper featured in the same commemorative issue of the British Pharmacological
Society, was the mouse vas deferens, used as a novel and reliable
morphine-sensitive bioassay (Henderson et al., 1972), which proved
instrumental in the search for endogenous opiate receptor ligands. In
fact, the purification, and the later brain distribution of enkephalin
activity were widely supported by the use of this bioassay (Hughes
et al., 1975). Naloxone, the opiate receptor antagonist was used to
identify brain fractions naloxone-sensitive that guided the chemical
identification of the two enkephalins.
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By the same time, the vas deferens bioassay played a central role in
the dissection of endogenous negative and positive presynaptic control
mechanisms, which apart from the opiates sub served a relevant physiological role as modulators controlling sympathetic neurotransmission
strength. The first α-adrenoceptor classification (Smith and Winkler,
1972) and its regional variation in the distribution of rat vas deferens
were first reported by Vardolov and Pennefather (1976). The concept
of negative and positive autoreceptors was extended years later to
ATP and adenosine (ADO) receptors (Westfall et al., 1978; 2002;
Huidobro-Toro and Parada, 1989), that also contribute to the strength
of sympathetic co-transmission through presynaptic autoreceptors.
Note that ADO which is considered by many investigators as the final
product of the ATP metabolic degradation route in this neuro-effector
junction is not biologically inactive since it has potent presynaptic inhibitory actions (Huidobro-Toro and Parada, 1989), as will be discussed
further in advance. In the coming years, the notion that multiple modulators, likely auto/paracrine compounds also modulate sympathetic
transmission was spread and included studies with physiological relevant signals such as dopamine (Morishita and Katsuragi, 1999), acetylcholine (Sjöstrand, 1973; Iram and Hoyle, 2005), peptides such as
angiotensin II or bradykinin (Ellis and Burnstock, 1989), or calcitonin
gene related peptide (CGRP, Donoso et al., 2012). The current thinking
is that these chemicals diffuse to the neuro-effector junction from
neighboring cells to modulate transmitter release. Gasotransmitters including nitric oxide (NO), were also described as neuro-regulators
(Vladimirova et al., 1994; Jen et al., 1997). For a review that includes
adaptive mechanisms see Quintas and Noël (2009).
Moreover, the tissue motor responses correlate reasonably well with
intracellular signaling cascades, allowing the integration of cellular
events with functional aspects of tissue responses. For example, the corelease of motor transmitter's concept is abundantly supported by
multiple studies showing both NE and ATP released during exocytosis
by nerve terminals and linked to the tissue motor effects (Sneddon and
Burnstock, 1984; Von Kugelgen and Starke, 1991a). Years later, the
major population of the P2X receptor subtypes plus α1-adrenoceptor
subtypes involved in the tissue motor responses was clearly identified
by pharmacological analysis and later cloned (Valera et al., 1994; Burt
et al., 1998) an issue that will be detailed further in the review. Furthermore, the use of selective drugs that interfere with transduction pathways has allowed the identification of the multiple intracellular
signaling cascades, which allows to integrate the principles of cell biology with functional tissue responses and receptor signaling pathways
(North, 2002; Burnstock, 2007).
This contribution intended to review the current physiology of
purinergic transmission, with special relevance to the role of ATP as a
sympathetic co-transmitter. Moreover, based on the crystallized structure of P2X4 and A2A receptors, ectonucleotidases and NE membrane
transporter, we aim to provide a molecular insight to the principles of
sympathetic co-transmission at the light of current structural biology
findings. The combination of physiology/pharmacology with structural
biology principles is critical to diligently unravel the molecular basis
and the organization of the cell structure associated to sympathetic
co-transmission. This updated view on the sympathetic nervous system
that operates in the vas deferens neuro-effector junction offers novel
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10
L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
and exciting opportunities to understand the function of the P2X receptors (P2XRs) and α-adrenergic receptors at the structural level, underpinning the molecular basis of many regulatory mechanisms.
2. Anatomy and physiology of the tissue
The vas deferens is the conduct for sperm transport; it is a hollow
paired organ that connects the testes epididymis with the urethra. Histologically it comprises three smooth muscle layers, two of which are
oriented longitudinally plus a prominent middle circular layer; the
lumen is surrounded by a stratified columnar epithelial cell layer
(reviewed by Dixon et al., 1998). The tissue receives bilateral sympathetic nerve fibers mostly arising from the hypogastric ganglion and
from neurons in the hypogastric nerve trunk (Quintas and Noël,
2009). Sympathetic nerve terminals form a dense network between
the muscle layers; these endings are characterized by a dense network
of positive immunostaining for tyrosine-hydroxylase and neuropeptide
Y (Fehér and Burnstock, 1987). The hypogastric nerve originates the
pelvic plexus; it is basically composed of preganglionic fibers that innervate principal and accessory pelvic ganglia (Kolbeck and Steers, 1993). A
minor parasympathetic innervation (Keast, 1992; Hoyle, 1996), plus
sensory terminals (Maggi and Meli, 1988; Kaleczyc, 1998) has been
also described but its functional significance is less evident for the tissue
motor responses; it likely innervates the lamina propia. The cholinergic
nerve fibers contain several neuropeptides including vasoactive intestinal polypeptide, somatostatin, CGRP and likely NO, in view of NO synthase detection (Koslov and Andersson, 2013). Capsaicin-sensitive
sensory nerve fibers, which originate from pelvic neurons in a discrete
location in the pelvic ganglia, have been described and shown to synthesize and secrete CGRP (Keast, 1992). A population of 6hydroxydopamine-resistant nerves has also been described and might
correspond to parasympathetic and/or sensory afferent fibers arising
from lumbar and even sacral dorsal root ganglia which travel to the tissue together with hypogastric or pelvic nerves (Jackson and Cunnane,
2002).
3. Sympathetic nerve terminals; pharmacological evidence support
co-transmission and purinergic mechanisms
From a functional point of view, the tissue motor responses are due
to NE and ATP acting as main sympathetic co-transmitters (Burnstock
and Verkhratsky, 2010). The finding that NE and ATP are packed in synaptic vesicles and released upon nerve terminal depolarization
(Sneddon and Burnstock, 1984; Donoso et al., 2012) plus the fact that
both transmitters act in a concerted way in the smooth muscles fostered
the concept of sympathetic co-transmission. Supporting this notion,
ATP activates mainly depolarizing P2X1Rs in this tissue, which are excitatory trimeric ion channels gated essentially by ATP (Burnstock, 2007).
On the other hand, NE acts through a dominant population of α1Aadrenoceptors with a minor component of α1D coupled to protein Gq,
plus α2A and αA2D-adrenoceptors, coupled to Gs or Gi. The α1adrenoceptors activate phospholipase C generating inositol 1,4,5
triphosphate (IP3) and diacylglycerol as the main intracellular second
messengers, triggering a metabolic signaling pathway complementary
to that triggered by co-transmitter ATP (Burt et al., 1998; Cleary et al.,
2002, 2003).
Moreover, and as expected for a complex signaling system involving
at least two co-transmitters, the resultant muscular response of the
smooth muscle elicited by nerve ending depolarization, evidenced two
components, the phasic and tonic counterparts (see kymograph recordings in Fig. 1). Using pharmacological antagonists, it was determined
that ATP mediates the fast excitatory junction potentials which initiates
the phasic component of the nerve induced motor contraction, and
corresponds to the purinergic component of the neuro-effector motor
tone. The latter is mediated essentially though P2X1Rs sensitive to
suramin antagonism, a non-subtype selective purinergic antagonist.
The phasic purinergic motor component is more prominent in the prostatic segment, although it also operates in the tissue segment closer to
the epididymal end (Huidobro-Toro and Parada, 1988; Von Kugelgen
and Starke, 1991a; Donoso et al., 1994). The secondary, tonic, adrenergic
component is more prominent towards the epididymal portion of the
ductus and is sensitive to prazosin, a selective although non-subtype
specific α1-adrenoceptor antagonist. Prototypical kymograph recordings of the two components of the nerve-evoked contractions in isolated segments of the epididymal and prostatic portions of the rat vas
deferens in the absence and in the presence of either suramin or
prazosin are shown in Fig. 1. As anticipated , pharmacological dissection
of the purinergic and adrenergic components revealed the involvement
of both co-transmitters in the motor responses although the relative
magnitude of the purinergic versus the adrenergic component varies
along the vas deferens length. This regional variation in the relative
relevance of the purinergic versus the adrenergic component from the
epididymal towards the prostatic end of the ductus has opened spirited
discussions on the mechanisms underpinned. The cellular basis that
accounts for these observations are under scrutiny; we hypothesized
that it may involve differential distribution of these receptors in the epididymal versus the prostatic of the vas deferens, or other local tissue
variables related to a differential density distribution of the sympathetic
varicosities along the ductus can account for these differences.
Nevertheless, the joint application of both transmitters elicited a
synergic motor response as might be anticipated for co-transmitters
(Huidobro-Toro and Parada, 1988). Post-junctional organization details
of the vas deferens neuro-effector junction are depicted in scheme
presented in Fig. 2.
With regard to ATP and its receptors, it is convenient to recall their
nomenclature and distinguish purinergic from ADO or simply A receptors. ADO acts on a set of 4 A receptors initially termed P1 (all belonging
to the G protein coupled receptors, GPCR family) to separate them from
the P2 responses elicited by ADP/ATP or pyrimidine's such as UTP/UDP
(Burnstock and Kennedy, 1985). The P2 receptors are further
subdivided into the P2X and P2Y families. The former composed of 7
clones which are homo or heterotrimeric ionic channels activated almost exclusively by ATP and related adenosine triphosphates. The latter
P2Y family comprises 8 clones, all belonging to the GPCRs, activated by
ATP, UTP, ADP, or UDP and nucleotide sugars such as UDG-glucose or
UDP-galactose (Burnstock, 2007). A cartoon of the topology of these receptors at an equivalent structural scale, based on homology modeling
is presented schematically in the upper part of Fig. 2.
In addition, neuropeptide Y (NPY) acts as a pre and postjunctional
modulator of the tissue motor responses (Allen et al., 1982;
Huidobro-Toro, 1985; Huidobro-Toro et al., 1985), adding further interest to sympathetic transmission and its regulation (see schematic
cartoon in Fig. 2B). NPY is stored in the large, dense cored vesicles together with NE and thought to be co-released with ATP and/or NE. At
present, there is no indication that NPY is stored exclusively on the
large vesicles; on the contrary, data supports co-storage with at least
NE. The modulator role of NPY is complex since it acts on pre and
postjunctional sites, effects mediated by a selective subset of NPY receptors (NPYRs, also belong to the GPCR family). While NPY has a role as
postjunctional neuromodulator of sympathetic transmission through
NPY1Rs (Bitran et al., 1991 and Torres et al., 1992), it also acts on the
presynaptic terminal as a negative modulator of sympathetic transmitter release through NPY2R activation (see illustration of a sympathetic
button in Fig. 2).
The diagram shown in Fig. 2 illustrates a sympathetic varicosity and
summarizes functional data illustrating the essential principles of sympathetic co-transmission: ATP and NE elicit the tissue motor responses
through the activation of postjunctional P2X1R and α1A-adrenoceptors,
respectively. Transmitter release is a tightly regulated process which involves multiple receptor mechanisms. As expected for a highly regulated
biological system, both ATP and NE, apart from acting as co-transmitters,
play a role as sympathetic modulators activating a subset of presynaptic
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11
Fig. 1. Kymographic tracings of muscular tension elicited by electrical field stimulation (EFS) in prostatic and epididymal segments of the rat vas deferens in control and preparations treated in-vitro with either suramin or prazosin. a) Muscular tracings from the prostatic segment of a vas deferens elicited by a single pulse EFS (70 V, 1 ms duration, closed circles) in a prostatic
preparation showing the phasic purinergic component in blue and the tonic, adrenergic component in pink. 100 μM suramin application strongly inhibited the phasic while 0.3 μM
prazosin abrogated the tonic adrenergic phase. b) Recordings from the same vas deferens but from a segment of the epididymal segment of the rat ductus. Drug treatments as in a; suramin
blocked the phasic but not the tonic component while prazosin blocked preferentially the tonic component. c) and d) correspond to the muscular contraction elicited following a 15 Hz
train of pulses delivered during 30 s in prostatic and epididymal preparations respectively in the absence and later in the presence of suramin and/or prazosin. While prazosin reduced
mainly the tonic adrenergic component, suramin reduced the phasic, purinergic component, or blocked the full response in the prostatic but not the epididymal segment of the tissue.
Horizontal bars depict the 15 Hz EFS procedure. Tension and time calibration of the recordings are depicted at the left side of each set of recordings.
regulatory receptors, which involve P2Y1R, P2Y12R, P2Y13Rs as well as
P2X2 or P2X3Rs, α2-adrenoceptors, plus the NPY2Rs. Pre and post junctional NPY1R and NPY2Rs are also indicated. In addition, adenosine, a
metabolic ATP degradation product, has transporters and presynaptic receptors of the A1, A2A and A2B subtypes plus ADO transporters to recycle
the purine.
4. Presynaptic elements
4.1. Organization of the nerve endings and its varicosities
Mandatory to understand neurotransmission is to have an updated
view of the organization of the nerve terminal, a key anatomical
compartment of the neuro-effector junction. As already introduced,
the sympathetic efferens relies on the sympathetic triad: the cotransmitters NE and ATP plus NPY acting as the modulator (Fehér and
Burnstock, 1987). These neurochemicals are stored in and released
from synaptic vesicles along the sympathetic varicosities of the sympathetic neuron ending. Recent progress in cellular biology teaches
that synaptic varicosities are extraordinarily specialized nerve compartments. These contain small and large synaptic vesicles, sites for
vesicle docking, transporters to recycle transmitters, presynaptic
autoreceptors and other regulatory mechanisms to tightly regulate
the release process voltage sensitive ion channels (Bennett et al.,
1998). Likewise, the postjunctional membrane has receptors for
each transmitter and modulator derived from the same or adjacent
neuron in addition to key enzymes that hydrolyze ATP as a sympathetic
co-transmitter.
4.2. The presynaptic terminal, the site of transmitter storage, release and
multiple regulatory mechanisms
One of the key elements that define anatomically a synapse and lend
support to the co-transmitter roles of ATP and NE is the structural and
biochemical identification of synaptic vesicles containing these molecules; vesicles in the varicosity also contain dopamine β-hydroxylase
activity, necessary for NE synthesis. A variety of microscopic techniques
have consistently visualized two types of synaptic vesicles in the sympathetic nerve endings of this tissue as well as many sympathetic organs:
the small and the large dense cored synaptic vesicles (Neuman et al.,
1984). It is not at all clear whether each transmitter is separately stored
in the small vesicles and therefore each transmitter is individually released, or both transmitters are co-stored in common vesicles that contain ATP and NE (Stjärne, 2001). Notwithstanding, not all varicosities
discharge at the same time with each nerve stimuli nor secrete the
same proportion of co-transmitters (Westfall et al., 2002). The large vesicles are currently thought to secrete mainly NPY co-stored with either
NE or ATP, or both simultaneously. See schematic cartoon shown in
Fig. 2 lower panel, which depicts for educational purposes each transmitter stored in a separate vesicle along with their corresponding
transporters.
The transmitter release process requires a sophisticated protein machinery part of which is located in the outer membrane of the synaptic
vesicle; the other part pertains to the presynaptic nerve membrane constituting a specialized site for transmitter release referred as the
docking vesicular site. The protein complex called vSNARE (vesicular
soluble N-ethylmaleimide sulfhydryl factor attachment receptors) is essential for the vesicular exocytic release process (Ramakrishnan et al.,
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12
L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
Fig. 2. Schematic illustration of the vas deferens neuro-effector junction pre and postjunctional membranes with distinct structural proteins typical of a sympathetic terminal. Framed
upper panel: scaled topographical representation of a P2XR, an ectoATPase (ENTDPase), 5′ectonucleotidase (Ecto-5-NT), a G protein coupled receptor (GPCR) and its accompanying trimer
G protein, and a presynaptic membrane transporter. The DAT, dopamine transporter served as a template for the NET transported shown. All structures were designed according to scaled
modeling. Lower panel: schematic representation of a sympathetic terminal. The presynaptic membrane is shown in pink and shows various synaptic vesicles with its characteristic membrane proteins. For pedagogical purposes, each transmitter is illustrated stored in a separate vesicle endowed with its selective transporter: VMAT for the NE and VNUT for ATP; the vesicles
are somewhat acidic due to proton (H+) accumulation. In addition, the presynaptic membrane contains a different NE transporter (NET), ADO transporter and likely a related VNUT, ATP/
ADP transporter. Presynaptic regulatory receptors include α2-adrenoceptors for NE, multiple ATP auto receptor subtypes (A. P2X2R and P2X3R and/or P2X2/3R; B. P2Y1R and P2Y12R and/
or P2Y13R), ADO receptors (C. A1R, A2AR and A2BR) and the NPY-2 receptor. The post junctional membrane is schematized in red and shows the set of transmitter receptors required for
smooth muscle depolarization. Consistent with the notion of co-transmission, α1A/D-adrenoceptors and the P2X1R are main determinants of smooth muscle contractility. In addition the
cartoon shows ecto nucleotide hydrolases (ENTD), 5′ nucleotidase (ecto 5′eNT) which as a final result originates ADO, a ligand for presynaptic regulatory receptors and alkaline phosphatase. As a consequence of the neuro-effector junction activity, the tissue overflow is enriched in transmitter and metabolites plus soluble ENTDPases and alkaline phosphatase among other
enzymes (not shown). Note that the receptors and enzyme structures were shaped according to homologous template crystals and are scaled among them. Numbers indicate the multiple
feed-back mechanisms related to the control of co-transmitter release by either ATP, NE or NPY. Roman numerals represent the various steps involved in the release process: I. P2Y12R and/
or P2Y13R activation inhibits NPY release. II. NPY-2R activation induces NPY inhibition release. While the activation of A2AR and A2BR (III), P2X2R and P2X3R and/or P2X2/3R (IV) increase
the NA release; the P2Y12 and/or P2Y13 (V), NPY2R (VI), α-2 adrenoceptor inhibit the process. NPY-2R activation inhibits ATP release. A. P2X2/P2X3Rs, B. P2YRs, including P2Y1, 12 or 13,
C. Ado receptors like A1R, A2A or A2BRs.
2012). Newer cell biology findings indicate that these proteins are not
exclusive of exocytosis, some of them also participate in protein trafficking and/or vesicle sorting in several cell types. Some of the vSNARE
complex proteins such as synaptophysin and synaptotagmin have been
identified by immuno reactive staining in vas deferens varicosities
(Brain et al., 1997; Sung et al., 2003). The ruling hypothesis for vesicular
release relies on plasma membrane fusion with synaptic vesicles, a process that requires the entrance of calcium via presynaptic voltagedependent N-type calcium channels. The divalent cation is essential
for the fusion of complementary vesicle proteins with proteins from
terminal ending at the aforementioned docking sites. SNARE helical
domains cytoplasmic interactions form preferred coiled-coil interactions which are favored because of thermodynamical stability
(Ramakrishnan et al., 2012; Risselada and Grubmüller, 2012). The
synaptic vesicle is mildly acidic (see schemes in Figs. 2 and 3), a feature that is physiologically relevant since protons are allosteric
modulators of P2XRs (Coddou et al., 2011).
4.3. Transmitter synthesis and the control of the biosynthetic process
Early in the 50s, it was established that the synthesis of NE starts
with the oxidation of tyrosine, an essential diet amino acid. Tyrosine hydroxylase is a well characterized NE-varicosity enzyme marker (Jen
et al., 1997). In addition, this enzyme is the rate limiting step endowed
with a negative allosteric mechanism since it is strongly inhibited by excess NE, a process known as substrate inhibition, a primary regulatory
step in NE biosynthesis (Quinsey et al., 1998). Hydroxylated tyrosine
is decarboxylated to form dopamine, which is converted to NE by action
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13
Fig. 3. The pre/postsynaptic purinergic triad of a sympathetic nerve ending and sequential enzymatic ATP hydrolysis to generate adenosine (ADO). The cartoon illustrates three purinergic
structural elements that define the functional activity of purinergic transmission: receptors, degradation enzymes plus a purinergic transporter. These elements may all be localized pre or
postjunctionally or either some are presynaptic while others are postjuctional. The receptors may be positioned pre and/or post as indicated in Fig. 2, or as shown in this cartoon. The yellowish element of the pre and postjunctional membranes indicates lipid raft domains, as we know that the P2X1R and some ENTDPases in this tissue are micro regionalized into lipid rafts,
membrane micro regions enriched in glycosphingolipids and cholesterol that anchor these proteins. The diagram also shows a gradient of ATP through ADO (colored circles) due to the
enzymatic degradation of the nucleotide; this gradient is related to differential activation of presynaptic receptors. While ATP acts on P2X1R and likely on a related VNUT transporter, ADP
targets P2YRs and ADO the ADO receptors and transporters. Arrows indicate that the activation of P2X2 or P2X3 or its heterodimers has a strong negative presynaptic regulation of NE
release, P2YRs block release of NE and NPY, while the activation of the several ADO receptors has a compounded effect on transmitter release inhibiting or favoring co-transmitter release
NE release. The postjunctional P2X1R is restricted to lipid rafts and causes depolarization of the smooth muscles leading to muscular contractions. A VNUT-like transporter is postulated in
the presynaptic membrane to recycle released ATP in analogy to the several adenosine transporters present in this neuro-effector junction.
of dopamine β-hydroxylase, an enzyme localized either in the cytoplasm as in synaptic vesicles as already referred. Since VMAT (vesicular
monoamine transporter) has almost the same affinity for dopamine and
NE (Parsons, 2000), part of NE is synthesized de novo in the vesicles.
Likewise, ATP generated by oxidative phosphorylation in the mitochondria of varicosities is stored in vesicles through VNUT (vesicular nucleotide transporter), a nucleotide selective transporter first described in the
brain (Sawada et al., 2008; Iwatsuki et al., 2009). The demonstration of a
dense network of ir-NPY fibers in the vas deferens, coupled to its detection in the dense-core vesicles, offered an explanation for the role of
NPY as a sympathetic neuromodulator (Donoso et al., 1988), as will be
further detailed. For a review focused on the co-storage of ATP, NE,
and NPY, see Huidobro-Toro and Donoso (2004)
4.4. Co-storage and release of co-transmitters from the same vesicle?
Alternative evidence derived from endogenous modulators
The question of transmitter storage and release from the synaptic
vesicles has been amply debated in the vas deferens neuro-effector
junction as well as in the brain. Although the hypothesis of chemical
transmission has expanded for almost 80 years, details of its cellular
and molecular operation are still far from clear. The debate is focused
on whether co-transmitters are stored in a same synaptic vesicle and
hence are released from a common sympathetic synaptic vesicle, or
whether separate vesicles pools are available for each transmitter with
separate transmitter selectivity. In the latter case, the vesicles are assumed to be mobilized by separate and distinct synaptic vesicle pools.
Knight et al. (2003) has summarized the current proposals indicating
4 potential scenarios for differential vesicle secretion from the vas
deferens: i) NE and ATP are stored and are released from the same vesicle; ii) NE and ATP are stored in distinct vesicles and are released separately; iii) NE and ATP may be stored and secreted from different sets of
varicosities; and iv) NE and ATP may be stored and released from the
same vesicle but in different proportions from different vesicles. These
possibilities are actively being pursued worldwide. Although attempts
to purify and characterize synaptic vesicles from the tissue nerve
endings have been largely unsuccessful, the most compelling evidence
to differentiate between this alternate hypotheses originate from studies using modulators of the exocytic release process.
The support for the proposal that NE and ATP are largely stored in
separate vesicles in sympathetic nerve endings derives mainly from
experimental results from studies which ligands such as angiotensin II,
prostaglandins (Ellis and Burnstock, 1989), CGRP (Donoso et al.,
2012), endothelin-3 (Mutafova-Yambolieva and Westfall, 1995) and
α2- or β-adrenoceptor agonists (Gonçalves et al., 1996; Donoso et al.,
2002). Another line of evidence derives from time course experiments
detailing presynaptic NE and ATP overflow. Temporal dissociation of
ATP and NE release is another concept that has reinforced the notion
that these sympathetic co-transmitters originate largely from two different vesicle populations (Todorov et al., 1994; Donoso et al., 2012).
NPY co-stored with NE, are co-released from large dense core vesicles
of sympathetic nerves of the bovine vas deferens (De Potter et al.,
1988; Torres et al., 1992). Altogether these two pieces of evidence are
consistent with the interpretation that the sympathetic co-transmitters
are likely stored and released from separate vesicle populations as
based on differential release studies.
4.5. Vesicular transport mechanisms
Transmitter transporters are transmembrane proteins of increasing
biological and therapeutic interest in view of the paramount relevance
consigned to the recycling transmitter process. Within the purines,
two families of ADO transporters have been well characterized due
to the widespread clinical use of nucleosides in cancer chemotherapy.
Equilibrative and concentrative purine nucleoside transporters have
been cloned; selective drug inhibitors for these transporters are available; however due to carrier specificity, ATP is not a substrate of ADO
transporters. Although selective ATP transporters were postulated for
a long time, the proteins responsible for ATP vesicular storage are still
elusive. Within the past five years the group headed by Moriyama at
Okayama University, Japan, identified the first vesicular nucleotide
transporter (VNUT, Sawada et al., 2008; Iwatsuki et al., 2009). This
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L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
protein belongs to the SLC17 phosphate transporters family and bears
strong structural homology with glutamate transporters. As other
transporters, VNUT is a twelve transmembrane domain protein with
N- and C-terminal ends localized towards the intracellular cytoplasm.
The VNUT is specific for the transport of ATP, ADP, UTP and GTP while
adenosine and adenine were inactive. VNUT uses membrane potential
as its driving force and requires chloride for transport activity
(Sawada et al., 2008). Non-hydrolysable ATP analogs such as AMPPNP, γS-ATP or di-adenosine triphosphate were reported among the
compounds examined that inhibited VNUT activity. The ATP transport
was also blocked by two inhibitors of vesicular glutamate transporters
such as Evans Blue and diisothiocyanotostilbene-2,2′disulfonate
(Sawada et al., 2008; Miyagi et al., 2011). Functional assays to demonstrate the physiological implications of VNUT in sympathetic neurotransmission, and particularly in the vas deferens, are absent. Little is
known as yet whether VNUT is also involved in the recycling of ATP
by the presynaptic terminal or whether a separate transporter is
involved as with bioamines.
In contrast to VNUT, the vesicular monoamine transporter (VMAT)
as well as the plasma membrane dopamine, noradrenaline-adrenaline
transporters (DAT, NET) have been well characterized; studies are available in the vas deferens. The synaptic vesicles are filled with NE, an
action mediated by VMAT activity. Reserpine and tetrabenazine are
classical high affinity VMAT inhibitors; as stated in the introductory section, the pharmacology of cocaine, which blocks NET was first examined
by Huković (1961) in the guinea pig vas deferens and later by many
other investigators searching for the mechanism of action of the famous
tricyclic antidepressants in the periphery and the central nervous system (see reviews by Iversen, 1965, 1971). Multiple investigations have
further detailed the pharmacodynamics of VMAT inhibitors on the NE
release process (Bauerfeind et al., 1995). Interestingly, reserpine blocks
VMAT without supposedly interfering with ATP transport, an issue that
likely will need to be re-assessed. In reserpine-treated rats, vas deferens
neurotransmission was not suppressed, an action thought to depend
upon remaining nerve-evoked purinergic components (HuidobroToro, 1985) a hypothesis that needs to be reassessed using VNUT
blockers. Regarding the plasma membrane presynaptic NET, its pharmacology is well characterized since tricyclic antidepressants such as
imipramine and its derivatives and newer generation drugs including
venlafaxine (Pifl et al., 2005), mazindol (Millan et al., 2001), bupropion
(Stahl et al., 2004) and related compounds, are partially selective for the
NET but do not block VMAT activity to the same extent. See Fig. 2 lower
panel illustrating putative topographical localization of these transporters in the sympathetic varicosity.
The synaptic vesicles are mildly acidic due mainly to a v-ATPase
activity, which hydrolyses ATP promoting the entrance of protons to
the vesicles (see diagram in Fig. 2 lower panel). v-ATPase activity creates a proton gradient used by VNUT to transport ATP into the vesicle
(Tabares and Betz, 2010). This pump has been described in the vas
deferens (Brown and Breton, 2000), and lately it was suggested to participate in vesicular co-transmitter release (El-far and Seagar, 2011).
Batteries of v-ATPase inhibitors are available. These compounds will
help to assess its role in sympathetic co-transmission (Huss and
Wieczorek, 2009); results within the years to come should clarify this
issue in the vas deferens and other sympathetically innervated tissues.
junction, since both ATP and NE exert both pre and postjunctional effects
at the neuro-effector junction. In addition, NPY also is a well-recognized
negative presynaptic modulator (Torres et al., 1992; Westfall, 2004),
demonstrating the complexity and redundant regulatory mechanisms
that might operate simultaneously in the neuro-effector junction. In addition to the co-transmitters, a number of endogenous locally produced
compounds, also known as autacoids, have been characterized as modulators of sympathetic responses acting either at pre or postjunctional sites
as already presented. Other conspicuous physiological regulators in this
neuro-effector junction include adrenaline through β2-adrenoceptors
(Todorov et al., 2001) and local tissue prostanoids. Presynaptic inhibitory
cannabinoid receptors have also been documented (Devane et al., 1992;
Pertwee et al., 1995; Christopoulus et al., 2001). Other putative modulator
agents were presented previously in the context of their roles as differential modulators of co-transmitter release. Apart from these, the
postjunctional membrane of the neuro-effector junction is also regulated
by chemicals, either transmitters or modulators, arising from cells in
the immediate vicinity of the neuro-effector junction. Among other
postjunctional modulators, dopamine acting through D2 and/or D4 receptors (Morishita and Katsuragi, 1998; 1999), serotonin (Yoshida and Kuga,
1986) or endothelin-1 (Donoso et al., 1992) needs to be added to the
listing. Since there is dispute regarding differential mechanisms operating
in the epididymal versus the prostatic portions of the rat vas deferens, the
pharmacology of these receptors needs to be carefully detailed.
4.7. Ectonucleotide-triphosphohydrolases (ENTPDase), ectoATPases, and
related enzymes
As expected, and as a proof of synaptic efficiency, sympathetic cotransmitters have a relatively short biological half-life at the vas deferens
neuro-effector junction. Extracellular ATP is broken down by a collection
of enzymes, generally termed ectoATPases, because of their extracellular
setting. Great progress has been observed in the past two decades regarding the characterization of these enzymes, some of which were crystallized, providing structural elements that help to account for their
kinetic properties (Zimmermann et al., 2012). Unfortunately, no high affinity and selective inhibitors are commercially available as yet; a fact
that has hindered comprehensive studies to reveal their physiological
relevance and role in purinergic signaling in this and other tissues.
The vas deferens contains ecto enzymes such as e-NTPDase1, 3, and 8
enzymes, which are at variance from other NTPDases since the former is
localized in the extracellular membrane from either pre or postjunctional
sites. The latter enzymes are intracellular or even some of them are
stored in synaptic vesicles (NTPDases 5, 6) and released together with
vesicular stored ATP (Mihaylova-Todorova et al., 2001; 2002), further
supporting the rapid hydrolysis of the nucleotide to avoid extensive
P2X1R desensitization. It is generally assumed that extracellular ATP
has an exceedingly short half-life due to abundant ectoATPses. In addition to these enzymes, the vas deferens also contains 5′nucleotidase activity which generates ADO from ATP. The vas deferens also expresses
alkaline phosphatases which hydrolyze intra and extracellular nucleotides, including ATP, as will be detailed later. Figs. 2 and 3 show schematically these enzymes localized either in the pre or post junctional
membranes. In addition, part of them may be micro regionalized into
lipid rafts as illustrated diagrammatically in Fig. 3.
5. The postjunctional smooth muscle membrane, site of
effector mechanisms
4.6. Neuromodulation
The inhibitory presynaptic role of NE is largely mediated by α2A- or
α2D-adrenoceptors (Trendelenburg et al., 1999; Lehtimäki et al., 2008).
In addition ATP either through P2Y or P2XR activation also inhibits and/
or facilitates sympathetic transmission (Gonçalves et al., 1996; Queiroz
et al., 2003). ADO also plays a pivotal role in neuromodulation; the
nucleoside cannot be considered any longer an inactive byproduct of
ATP metabolism. It is formed by the sequential action of NTPDase and
5′-nucleotidase activity. This adds further interest to this neuro-effector
The most relevant feature of the smooth muscle cell surface in the
near vicinity of the nerve ending is the expression of the postjunctional
transmitter receptors. As previously cited, the motor activity of the vas
deferens is under the dual control of ATP and NE, see kymograph tracings presented in Fig. 1, illustrating the phasic and tonic components
elicited by electrical field stimulation (EFS). While the phasic component is predominantly mediated by ATP acting in this tissue through
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P2X1Rs, NE is mainly related to the tonic motor element, due predominantly to α1A-adrenoceptors plus a minor contribution of α1Dadrenoceptors and α2A/D-adrenoceptors (Deng et al., 1996; Cleary
et al., 2002, 2003). It is important to recall that the P2X1R was originally
cloned from this tissue (Valera et al., 1994). As a consequence of P2X1R
activity, the purinoceptor channels open during few milliseconds promoting the entrance of sodium and calcium which depolarize the
smooth muscle membrane and spreads, likely through gap junctions,
over the entire smooth muscle surface consistent with a functional muscle syncytium (Burnstock and Verkhratsky, 2010). In addition, the activation of α-adrenoceptors, elicits indirectly the release of intracellular
stored calcium as a consequence of phospholipase C activity. In addition,
the biochemical mechanisms derived from protein kinase C activity
caused by diacylglycerol activation further assist the complexity of the
contractile machinery induced tissue tension. It is not at all clear whether the postjunctional membrane expresses other P2X channels, like
the P2X4R or modulator P2YR. This issue remains as yet unresolved
properly. For an updated review of the intracellular signaling pathways
involved, see Koslov and Andersson (2013).
The release of ATP from sympathetic nerve varicosities plays an important motor role in the EFS-mediated vas deferens contractions.
While most studies have been performed using rodent vas deferens, recent work using human tissue biopsies also highlight the role of ATP as a
main motor transmitter at least in the longitudinal muscle layers of the
human ductus (Amobi et al., 2012; Donoso et al., 2014). As to whether
ATP also participates in the motor activity of the circular layer of the
human tissue is not known, since in contrast to the longitudinal muscle
layers, exogenous ATP applications to human tissue rings, cause rather
erratic or inconsistent contractions as opposed to the robust contractions observed in longitudinal tissue strips (Amobi et al., 2012). In the
rat vas deferens, the ATP-evoked motor component (phasic) is larger
in the tissue segment closer to the prostatic end in contrast to the rather
smaller contractions attained in the proximity of the testis epididymis.
The kymograph illustrated in Fig. 1 shows that the prostatic portion of
the rat vas deferens, has a prominent purinergic phasic component,
which although also present in the epididymal end, it is not as robust.
Independent of these minor differences, ATP acts in a concerted manner
together with NE all along the tissue. In sum, and consonant with the
concept of co-transmission, the vas deferens neuro-effector junction
operates with a dual mechanism that combines a rapid inotropic
response with a slightly slower metabotropic intracellular metabolic
cascade accounting for the compounded motor responses observed in
the EFS recordings.
Prolonged or repeated applications of ATP or structural analogs such
as α,β-methylene ATP (m-ATP, characterized as a slowly hydrolysable
ATP analog), exhibit within seconds to minutes markedly reduced
receptor-gated currents or smooth muscle contractile responses to
ATP or mATP in the vas deferens bioassay. This phenomenon is termed
desensitization; most purinergic receptors desensitize, the rates depend
on the receptor subtype and ligand concentrations. The P2X1R and
P2X3R being the most prone to desensitize while the P2X7R is the
least affected (Coddou et al., 2011) and constitutes a special case within
the group; the rest of the P2XRs exhibit varying rates of desensitization.
The molecular basis of this process remains largely unknown, but it is
likely that desensitization reflects ligand-induced long lasting receptor
perturbations (Coddou et al., 2011); we are under the hope that structural principles will assist the better understanding of the molecular
basis of desensitization. In the vas deferens, desensitization of the
P2X1R occurs rapidly either following repeated exposures to purinergic
agonists, or particularly following a single 1–10 μM m-ATP addition for
5 min or less. In a recent publication, Donoso et al. (2014) used m-ATP
as a prototype of a selective and potent P2X1R agonist to desensitize
the human P2X1R present in biopsies derived from human vas deferens
material; authors concluded that the P2X1R is critical for human tissue
contractility, particularly in the longitudinal smooth muscle layer, but
not in the circular layer of human vas deferens samples.
15
In addition, the smooth muscle membrane is enriched with
ectoATPases, a set of membrane enzymes that rapidly hydrolyze ATP
into ADP and its metabolites. While ADP has about 100-fold lesser affinity and efficacy as P2X1R agonists, AMP and ADO are inactive (Coddou
et al., 2011); notwithstanding, ADO has affinity for A receptors
(Burnstock, 2007), part of which are postjunctional and may lead to
smooth muscle relaxation. In addition, subsets of presynaptic adenosine
A2AR and A2BR, modulate the release of NE/ATP from the nerve terminal (Donoso et al., unpublished observations, see Figs. 2 and 3). The
smooth muscle membrane also expresses an extra neuronal reuptake
2 transporter for NE, which works as a relatively unspecific organic cationic transporter. The physiology of this transporter is not clear; it has
lesser affinity for most catecholamines as compared to NET, but accounts for an extra neuronal transmitter uptake mechanism. Fig. 2 illustrates the tissue neuro-effector junction emphasizing the overflow of
transmitters and the ATPase activity of several enzymes.
As with the presynaptic nerve ending, the postjunctional membrane
also expresses receptors for a multiplicity of endogenous compounds
including NPY and other peptides, some of which are important modulators of sympathetic transmission. Although their physiological
role and diversity as postjunctional modulator peptides escape our current understanding, the role of angiotensin II, bradykinin (Acevedo
et al., 1990), calcitonin gene related peptide (Donoso et al., in
preparation) among others, remains to be cleared. These receptors
might serve as targets for auto/paracrine autacoids, which might play
a significant role as local modulators of the tissue contractility, particularly under pathological conditions.
We have not ignored that upon muscular contraction, the smooth
muscles per se may also contribute to some extent to a fraction of the
ATP released by hitherto unknown mechanism(s). Our estimates in the
rat vas deferens indicate that the smooth muscles per se may contribute
at most with about 20–25% of the total extracellular ATP released
(Donoso et al., 2012), a finding compatible with the previous findings
of Von Kugelgen and Starke (1991b) and Vizi et al. (1992). A similar finding was also reported in skeletal muscles (Buvinic et al., 2009), implying
that the contractile process of either skeletal or smooth muscles must
trigger by itself the release of a measurable amount of extracellular
ATP. The machinery activated in this phenomenon is not known, it
could involve vesicular or non-vesicular ATP release from unidentified
ATP pools.
Recent studies on the cell biology of smooth muscles consistently indicate that its plasma membrane is not homogeneous. It appears to be
composed of multiple microdomains enriched in glycosphingolipids
and cholesterol, among other conspicuous membrane lipids. These
membrane subdomains are referred to as lipid rafts and are characterized as dynamic signaling platforms which are in dynamic assembling
and disassembly process. Lipid raft domains are enriched in G proteins
and proteins such as flotilins, caveolins which are used as markers of
these membrane subdomains (Norambuena et al., 2008). Some P2XRs
are associated to raft domains; in particular the P2X1R (Allsopp et al.,
2010; 2011). In human studies using vas deferens biopsies, the P2X1R
was found localized into membrane rafts (Donoso et al., 2014). In view
that some α-adrenoceptors, such as the α1A have also been identified
in lipid rafts (Morris et al., 2008), it is plausible to assume that other
adrenoceptor subtypes might also share this particular membrane distribution. Fig. 3 illustrates schematically that ENTDPase, the 5′NT, as well as
the P2X1R are likely all regionalized into lipid rafts. As to whether VNUT,
or a related nucleotide transporter, is also a lipid raft protein, remains to
be determined. This common micro domain association is a privileged
subcellular localization particularly suited and fully compatible with
cross talk mechanisms that might be responsible for the integral physiological response as it occurs upon sympathetic stimulation. When such
responses are mimicked by joint exogenous application of both transmitters simultaneously, we observed marked synergisms of the motor responses compatible with the common lipid raft localization of both
receptors (Huidobro-Toro and Parada, 1988).
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L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
Upon sympathetic nerve discharges, we may reasonably assume
that a depolarization wave spreads along the smooth muscle cell surface
to cause a coordinated tissue contraction. Not every smooth muscle cell
receives directly a quantum of co-transmitters, but as mentioned, the
depolarization wave spreads over the tissue surface. The coordinated
smooth muscle activity reveals the tissue acts as a functional syncytium.
For this to occur, all the smooth muscle cells must be functionally coordinated through ionic channels (Burnstock and Verkhratsky, 2010).
Prominent among them, connexin/pannexin channels communicate
adjacent smooth muscle cells (Pelegrin and Surprenant, 2006); when
two such hemichannels couple face-to-face two cells, they form functional gap junctions (Sáez et al., 2005). Several channel blockers have
been examined to experimentally ascertain this hypothesis and demonstrate that this tissue operates as a synchronized syncytium (Burnstock
and Verkhratsky, 2010). In support of this contention, heptanol acting as
a non-selective gap junction inhibitor, decreased vas deferens EFS
evoked excitatory junction potentials, likely causing the uncoupling of
smooth muscle cells (Venkateswarlu et al., 1999), supporting the functional role of gap junctions in vas deferens contractility. Notwithstanding, the use of more selective connexin/pannexin blocking agents is
required to confirm this proposal.
6. The structural biology of signaling elements in purinergic
sympathetic transmission
6.1. Structure of ligand gated ionic channels
Within the past few years, based on the report of the crystal structure of zebra fish P2X4R (Kawate et al., 2009) new structural information is available to understand, at the molecular level, the function of
the ATP receptor channel involved in the purinergic component of the
tissue motor responses. Moreover, the structural resolution of the secondary up to quaternary resolution of the truncated Danio rerio
(ΔP2X4R) structure in its apo (without ATP) or holo-state (with ATP
in its binding site) was recently published (Hattori and Gouaux,
2012). The additional structural information offers a key and unprecedented opportunity to understand molecular details of receptor
functioning and assist the design of novel, tailor-made, ligands to characterize the multiple purinergic receptors. Moreover, with the report of
the crystal structure of the ectoenzymes that degrades ATP and based
on crystallized receptors belonging to the G protein coupled family of
receptor proteins, new insights allow a structural basis of sympathetic
co-transmission as we have never achieved before. Therefore, based
on crystal information we intend to provide a refreshed view on sympathetic co-transmission at the molecular/atomic level.
As mentioned, P2XR channels are formed by only three subunits,
forming a trimer which has strong interactions between subunits; in
contrast to the nicotinic or GABA/glycine receptors which form a
pentamer, or glutamate, NMDA or AMPA receptors that merge forming
a tetramer, P2XRs were the first ligand-gated ionic channel described of
only three subunits. If the three subunits are identical, P2XRs are termed
homomeric, while if composed of two different subunits such as P2X2/3,
P2X1/5 or P2X4/6, the P2XRs are heteromeric. No P2XR composed of
three-different subunits has been described. Regarding P2XR topology,
best information derives from the crystallized P2X4R first described in
the apo and later in the holo state with 3 ATP molecules bound. The
crystallized zebra fish P2X4R is incomplete due to technical limitations
that favor the crystallization technique. In this particular case, the
P2X4R was purposely truncated at the N-terminal and C-terminal segments, since the exclusion of the more flexible cytoplasmic chains favored the crystallization process. Notwithstanding this important
caveat, it is still possible to infer that the cytoplasmic N- and Cterminal P2XR ends stabilize the receptor in the membrane (Hattori
and Gouaux, 2012), emphasizing particularly the role of membrane
phosphoinositides in the stability of the membrane receptor (Bernier
et al., 2008). Each subunit consists of a single aprox 350 residue long
sequence, the exception being the P2X7R which has the largest intracellular C-terminus. Each subunit has two transmembrane domains (TM1
and TM2), a large extracellular loop that encodes the “head”, where
the ATP binding site is localized (orthosteric site). The trimer structure
forms a big extracellular bulk domain that contacts the membrane
plane through three “columns”, each of them formed by two loops
which link the TM1 and the TM2. The P2XR monomer, presents a general topology common to all the P2XRs. The general topology of the receptor trimer is shown in Fig. 2A, a cartoon drawn at the same scale as the
GPCR shown for comparative properties in the cell membrane plane.
More specific details of the P2XR receptor topology are shown in
Fig. 4. This particular diagram was modeled for the P2X1R. Note how
the three subunits compose the channel, particular emphasis is played
on the receptor head which contains the orthosteric or ATP binding
site (defined as the jaws of the receptor) and the TM domains; the Nand C-terminal portions were modeled to scale since they were not derived from the crystal structure of the basic P2X4R.
The gating mechanism involves a poorly defined conformational
change between the “head” of the extracellular domain and the TM2 domain; the critical role of an aromatic residue cluster conformed by
Tyr55-Phe326-Phe49 was reported to play a role in the conformational
change leading to channel pore opening (Rokic et al., 2013). In addition,
Gln56, Asp264 and Asn262 plus the cysteine's that form the 5th disulfide bridge were also proposed as a fundamental part of the P2XR gating
mechanism linking the P2XR “head” with the TM2 domains involved in
channel opening (Rokic et al., 2013). The functional P2XR trimer comprises 6 alpha helixes which are linked to the TM domain, with the 3
TM2 regions contributing to the channel gate. Each TM2 has a projection
to the extracellular loop by a long beta sheet that includes residues
which are part of the ATP binding pocket. The opening of the channel
is due mainly to the rotation of the TM2 region (Hattori and Gouaux,
2012). Moreover, in the particular case of the P2X1R, transmission electron microscopy clearly shows lateral fenestrations essential for ion entrance (Roberts et al., 2012). In sum, although the field has experienced
much progress delineating common features of the P2X1R topology
with other P2XRs, it is not possible as yet to draw a general structural
conformation specific for each P2XR characterizing their essential
properties.
At present we lack real proofs of the P2XR topology within the
membrane environment including the N- and C-terminal tails. Notwithstanding, the state of the art modeling allows discussing some relevant
information. The N- and C-terminus have variable lengths among the
P2XRs indicating low conservation sequences. Those of the P2X1R are
relatively short, while the C-terminal tail of the P2X7R is especially
long (Coddou et al., 2011). In addition, almost all the C-terminal tails
have a conserved traffic motif implying that this sequence is related
possibly to plasma membrane routing (Chaumont et al., 2004).
Independent of the current limitations discussed, homology modeling allows using a known atomic disposition, derived from the zebra
fish P2X4R crystal (Hattori and Gouaux, 2012), as a template to create
a structural model of a non-crystallized channel which bears more
than 40% sequence identity with the crystallized protein (Eswar et al.,
2007). This model is presented in Fig. 4 for the P2X1R. Based on this premise, we inferred a P2X1R model and deduced information from other
P2XRs especially when their main physiological properties are conserved as well as that of critical residues required for its functioning.
Based on such premises, we conjecture the ATP binding pocket in the
“head” of the extracellular domain of the P2X1R, in an interface formed
by two adjacent subunit interfaces; some of the residues pertain to one
of the subunit, others to the adjacent. The importance of these residues
was first demonstrated by alanine-scanning mutations in the large extracellular loop, where a single change drastically decreased the ATPgated currents. Among these critical charged residues (Lys69, Lys70,
Phe185, Tre186, Asn290, Phe291, Arg292 and Lys309), play a pivotal
role in the development of the ATP-gated currents (all the residues
are, and will be referred according to the Rattus norvegicus P2X1R
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L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
numbering, Uniprot code P47824, following Browne et al. (2010) and
Evans (2010). Moreover, based on the stable trimeric composition of
P2XRs, as was first demonstrated for the P2X1 and the P2X3R (Nicke
et al., 1998), it is possible to assume that full opening of all the P2X channel pore occurs following receptor occupation with three ATP molecules, an information that correlates well with electrophysiological
studies conducted in bullfrog sensory neurons, which show the requirement of three ATP molecules for full ATP-gated currents (Bean et al.,
1990). This hypothesis was recently corroborated by elegant experiments with concatenated P2X2R subunits which in addition have one
or more amino acids mutated by site directed mutagenesis of the primary amino acid sequence. In full agreement with this proposal, single
P2X1R conductance experiments indicate that full opening of the receptor channel occurs when three ATP binds the functional channel; proportional less current flows when fewer ATP molecules are bound per
full receptor constitution (Stelmashenko et al., 2012).
In agreement with homology models, the ATP binding site is localized in the P2XR head in close vicinity of the head which for the
17
P2X2R has been demonstrated to trap ATP as “jaws”. It is possible to record small currents by the tightening of the “jaws” even without ATP
addition (Jiang et al., 2012). The head encompasses three disulfide
bridges which compact this domain topology, and is importantly involved in P2X1R activation and desensitization (Kawate et al., 2009;
Lörinczi et al., 2012). As an extension of this notion, the positive charged
residues between the disulfide bridges of the head region are determinant for the binding of NF449, a purported selective P2X1R antagonist
(El-Ajouz et al., 2012). These charged residues are proper of the
P2X1R and therefore could be a lead for developing potent and selective
new P2X1R ligands. Fig. 2 upper panel shows main topological issues of
the P2XRs showing the association of subunits to conform the functional
receptor composed of three subunits, necessary for the active receptor.
Fig. 4 shows structural elements of the head, the ATP binding site in
analogy to the jaws and the conformational changes induced by ATP
binding. Pharmacological studies performed with any of these artificially mutated receptors showed ATP concentration–response curves
which suggested either an alteration in the ligand potency or its efficacy
Fig. 4. General P2X1R topological features. The P2X1R is the functional ATP gating channel of the vas deferens motor activity. This receptor is composed of three subunits; it can be homo or
heteromeric. The ATP binding site is located in the “jaws” of the receptor, formed by two adjacent subunits. Part of this binding site is composed by a long β-sheet that extends to the second
transmembrane (shown in dark blue). The cytoplasmic C-terminal shows the putative PIP2 binding (allosteric regular site). The other part of the jaw is composed by a β-sheet with conserved lysines (green); this continues to the first transmembrane which possesses a short cytoplasmic domain that has been identified as part of the cholesterol binding domain. Also the
N-terminal has been described as part of the cytoskeleton contact; both intracellular tails facilitate the HSP90 interaction with P2X1R. This trimer channel has a cationic entrance at the
extracellular membrane position in the so called lateral fenestrations, which are wider in the holo P2XR state. Finally the communications of the conformational changes induced by ATP
(red ball) binding and pore gating, imply conformation changes through conserved residues that make a hinge domain. ATP represented as a red ball, recognizes its binding site at a locus
termed the “jaws” of the receptor. Note the schematic conformation changes induced by receptor occupation, compatible with the changes in the TM region allowing cation fluxes an indication of channel conductance activation.
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18
L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
(Ennion et al., 2000), but did not reveal further information of the role of
these residues in the P2XR gating mechanism. However, based on the
homology modeling of the P2X1R, it became possible to identify the residues involved in the ligand binding and separate those involved in the
ATP-induced conformational changes leading to the opening of the
channel pore (Allsopp et al., 2011).
Regarding the pore and channel selectivity, the P2X1R has major
calcium conductance (Evans et al., 1996), a property that favors vas
deferens muscular depolarization; this finding is entirely compatible
with its co-transmitter role. Interestingly, the access of cations to the
P2XR channel pore differs strikingly from other ligand gated ionic channels. Solid structural evidence, based on homology modeling, further
gives support to the proposal that the cation flow occurs mainly through
lateral fenestrations which are edged by the columns which connects
the extracellular bulk to the TM1 domain rather than from a central
tunnel pore as with inotropic receptors (Kawate et al., 2009). Lateral
fenestrations have a favorable electrostatic potential and a suitable
size for the pass of positive charged ions (Kawate et al., 2011;
Samways et al., 2011). See diagram of the receptor schematized in
Fig. 4; note the trimeric P2X conformation and the receptor in its ATP
occupied and unoccupied conformations. Some of the amino acid
residues involved in the gating mechanism were discovered using site
directed mutagenesis in receptors expressed in heterologous biological
systems much earlier than the studies derived from the crystals. Such
is clearly the case for the P2X2R, highlighting the studies of Jiang et al.
(2001).
Alternative P2XR splicing is a common feature among P2X2R; these
variants have been used to further characterize structure activity relationships. These natural mutants show significant differences in channel
properties (Khadra et al., 2012). The P2X1R is no exception to this rule;
P2X1/P2X2 receptor chimeras show that the first 16 residues of the Nterminal have a role in the regulation of the efficacy for the P2X1R,
while the C-terminal has a role in the regulation of the time-course
and recovery from the desensitized state (Allsopp et al., 2013). Recent
data indicates that the intracellular tails interact with signaling pathway
molecules like protein kinase C (Ennion and Evans, 2002; Liu et al.,
2003; Roberts et al., 2012). In addition, in HEK293 cells, the amplitude
of P2X1R ATP-gated currents has been somehow associated to the cell
cytoskeleton, since actin disruption by cytochalasin D, resulted in markedly reduced currents (Lalo et al., 2011); this interaction apparently is
related to residues in the cytoplasmic N-terminal residues. In this context, another relevant intracellular element is the HSP90 which, has
also been described as an interacting P2X1R protein. P2X1/P2X2R
chimeras and pharmacological studies with geldanamycin suggest that
the P2X1R trafficking is regulated by HSP90 via interactions through
N- and C-terminal residues (Lalo et al., 2012). This finding could be relevant for the vas deferens physiology considering the leading transmitter role of P2X1R in a tissue with abundance of actin fibers. Fig. 4 shows
the HSP90 putative binding and cytoskeleton interaction in a general
P2X1R representation, indicating the likely interaction of the receptor
with scaffolding intracellular proteins.
A common feature of many ionic channels, which is no exception to
P2XRs is the finding that upon continual ligand application to the receptor, the magnitude of the current recorded becomes progressively reduced. This phenomenon is referred as desensitization. Within the
P2XRs, the P2X1R is known to be the most prone to desensitization
followed by the P2X3R and less by the P2X4R. The P2X7R is notoriously
resistant to desensitization independent of the time is exposed to ATpor
related agonists. Two major hypotheses can be proposed regarding
P2X1R desensitization: either the receptor is rapidly but temporarily
modified by phosphorylation or a related covalent structural modification, or the receptor remains in a “desensitized” conformation following
ligand-induced activation. Recovery from desensitization depends in
one case in protein removal of the covalent modifier, or as in the second
case, it requires a time for protein relaxation to its natural state. In the
latter case, no covalent modification of the receptor is required for
desensitization. Critical to choose between these alternatives is the crystallization of the receptor in its desensitized state. In favor of the latter
possibility, the P2X4R has an intermediate rate of desensitization,
which although 100-fold less than the P2X1R, it is however, still important. We hope that this issue may be resolved in the years to come, using
the D. rerio P2X4R crystal as a template for receptor crystallization
following mATP desensitization experiments.
6.2. Allosterism as a regulatory mode of P2XRs at the structural level
Allosteric regulation is a well-known and key regulatory mechanism
of enzymes which can also be extended to ion channels and among
them, to P2XR channels. Allosterism profoundly affects ion channel
kinetics (Coddou et al., 2011; Jacobson et al., 2011). Transition metals
such as zinc and copper as well as protons have been thoroughly investigated as allosteric P2XR regulators; these elements act as either
positive or negative allosteric regulators depending on the P2XR subtype (Wildman et al., 2002; Huidobro-Toro et al., 2008). As the P2X1R
plays a key role in vas deferens signaling, novel physiological models
should integrate allosteric information derived from heterologous
models to the physiological relevance of these modulators in tissue
bioassays or in studies with receptors reconstituted in several isolated
cell systems.
Vesicular co-transmitter release entails the secretion of synaptic
vesicle content including protons which may mildly acidify the neuroeffector space, as purposely illustrated in Figs. 2 and 3. Since the
P2X1R is proton sensitive, and decreases the ATP-gated current amplitude in acidic medium (pKa 6,3) (Wildman et al., 2002), it is plausible
to infer that the P2X1R amplitude may be partially attenuated and account, in part, for desensitization following sustained ATP-induced bioassay contractions. In addition, Zn2+ decreases the ATP potency and is
additive with the pH effect (Wildman et al., 1999). Divalent trace metals
are also involved as regulators in central neurons (Maske, 1955), although their role in vas deferens transmission remains to be systematically examined. The modulator role of protons can be extended to P2XR
located in presynaptic membranes also exposed to allosterism. Protons
potentiate the P2X2R with pKa 7.3 (Clyne et al., 2002) and showed complex effects on the P2X3R as a modulator. Acidic media increased ligand
efficiency but decreased its potency, so the effect finally depends on the
concentration (Gerevich et al., 2007). Considering that the effective ATP
concentration in the vas deferens neuro effector synapsis has not been
determined, it is difficult to predict the true effect of the pH on the
ATP-gated currents in the bioassay. If both parameters are potentiated
by low pH, we deem possible to interpret that the vesicular release
causes a fine allosteric modulation in the P2XR involving a decrease of
the postsynaptic and a potentiation of the presynaptic action. In view
that Zn2 + is a positive allosteric modulator of P2X2Rs (Clyne et al.,
2002; Lorca et al., 2011), this could support the idea of an allosteric
modulation in the vesicular release/muscle contraction process. Moreover, Lorca et al. (2005) identified common amino acid residues in the
P2X2R responsible for the positive allosteric modulation elicited by
either zinc or copper in this receptor.
Other putative allosteric modulators such as cholesterol, an essential
membrane constituent, are critical for the generation of proper ATPgated currents. P2XRs chimeras suggest that the cholesterol binding
site is restricted to the N-terminal intracellular tail; the cholesterol
modulator effect was not observed in P2X2 and P2X3R (Allsopp et al.,
2010), an indication of receptor specificity among P2XRs. This data correlates with the decrease of ATP-evoked contractions in vas deferens
treated with methyl β-cyclodextrin used to decrease tissue membrane
cholesterol (Norambuena et al., 2008). Another exciting issue regarding
cholesterol physiology relates to the possible micro regionalization of
the P2X1R in cholesterol rich lipid-rafts. Although lipid rafts are claimed
to be artifacts by some investigators, studies are emerging in whole tissues. Bennett et al. (1998) showed P2X1R clusters near the sympathetic
varicosities, suggesting the role of rafts in smooth muscles, a finding
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further supported by pharmacological and structural data. Modifying
cytoskeleton stability alters cholesterol depletion; the structural interpretation of this phenomenon requires more studies, but suggests interplay between membrane cholesterol and cytoskeleton in P2X1R
regulation (Lalo et al., 2011). Likewise phosphoinositides, such as PIP2,
which are lipids of the internal membrane bilayer, essential for P2XR activity and modulation (Bernier et al., 2012); these lipids bind positively
charged P2X motifs in the N- and C-carboxy terminus. The binding
amino acid sequence was initially identified for the P2X1R (Bernier
et al., 2008a), and latter extended for P2X2R (Fujiwara and Kubo,
2006), P2X3R (Mo et al., 2009), P2X4R (Bernier et al., 2008b) and the
P2X7Rs (Zhao et al., 2007). Fig. 4 shows schematically that both cholesterol and PIP2 putative binding sites in the P2X1R, supporting the role of
these endogenous constituents as allosteric modulators of the P2X1R.
The P2X1/5 receptor heteromer is mainly due to P2X1R modulation,
since the P2X5R does not bind PIP2 (Ase et al., 2010). In these studies,
phosphoinositide depletion elicited with wortmannin or LY294002,
was rescued by PIP2 administration, adding physiological relevance to
this observation. Moreover, the C-terminal segment conforms a PIP2
binding site in a dual motif (Bernier et al., 2012). This observation agrees
with the finding that wortmannin application in the rat vas deferens decreased the ATP evoked contractions (Donoso et al., unpublished observations). Moreover, this regulatory mechanism does not appear to apply
to all P2XRs, since no change was found in the P2X2R and P2X3R ATPgated currents in cholesterol depleted cells suggesting that this lipid
does not play a main role as allosteric modulator in these channel functions (Allsopp et al., 2010).
6.3. G-protein coupled receptors (GPCRs) in sympathetic signaling;
involvement of adenosine (ADO) and P2Y receptors
GPCRs comprise a large family of receptors that includes NE and related bioamine transmitters, a variety of hormones, sensory stimuli such
as most odorants, tastants, light, pheromones among others. Probably
this is the most extended family of membrane receptors which can be
activated by a vast array of ligands including both physical and chemical
stimuli, from light to pheromones, from unicellular organisms to
humans. The relative importance of this versatile family of proteins is
reflected in the fact that it is thought to comprise almost 5% of the
human genome. Both sympathetic co-transmitters have as target
GPCRs; as mentioned, NE acts on postjunctional α1A adrenoceptors
and presynaptically through the several α2-adrenoceptors, while
ATP acts on presynaptic P2Y receptors and NPY through neuropeptide
Y-Y2 receptors as modulators of sympathetic co-transmitter release,
all receptor members of the GPCR family (see Figs. 2 and 3). Moreover,
ADP/ATP and adenosine have a regulatory role on sympathetic cotransmission. In view that the adenosine A2A receptor was within the
first crystallized GPCR, several years after rhodopsin (Katritch et al.,
2013), and since by homology it is possible to model the several P2Y receptors, which as described play a role as presynaptic modulators of
sympathetic transmission. Therefore, based either on the A2AR crystallized structure or β-adrenoceptor crystals, we deem exciting to review
this area of progress and comment on its relevant input to autonomic
pharmacology. As anticipated, structural facts offer novel possibilities
to design clinical useful drugs (Hohoff et al., 2009; Fu and Longhurst,
2010; Sangsiri et al., 2013).
The GPCR common structure consists of a varying size N-terminal
extracellular end, seven TM domains, connected by three extracellular
loops with the corresponding three intracellular loops, plus a relatively
large sized intracellular C-terminal loop (Katritch et al., 2013; Heng
et al., 2013, for a cartoon of the general receptor topology see Figs. 2
upper framed panel and 5). This receptor family has a common mechanism to transduce the ligand-receptor induced conformational changes
through the multiple trimeric G protein complexes formed by three
separate α, β, and γ subunits which dissociate and activate cell membrane effector proteins, from enzymes to channels. More than 20
19
different trimeric G proteins have been characterized; each is composed
of varying subunits. The main classes of trimeric G proteins, based on
their effector target enzymes include (Gs, Gi, Gq11). Upon activation
by GPCR ligand interaction, the G proteins dissociate into Gα-subunit
and Gβγ-subunit following the GDP/GTP exchange process. Both the
Gα- as well as the Gβγ-subunit activates different effectors according
the tissue and the physiologic response (see review by Heng et al.,
2013). In the particular case of the α1A-adrenoceptors, the smooth
muscle contractile response is linked to Gq11 protein, which targets on
phospholipase C. NE binds to a pocket formed in the TM domains
(Katritch et al., 2013); a stimulus which induces a conformational
change which promotes GDP/GTP exchange leading finally to GTP hydrolysis (Yao et al., 2009) and G protein activation. A schematic representation of the A2AR based on its crystal structure plus its
accompanying G protein complex is shown in Fig. 5; the classical cartoon showing the 7 transmembrane regions (serpentine receptors)
plus the modeled receptor with ADO in its binding site (orthosteric
site). In the same figure, the A2AR, as a prototype of a GPCR, such as
the P2Y1R or P2Y2R is represented with a ligand at the orthosteric site
and an allosteric modulator. Note the topological structure of the
seven TM domains of the receptor upon its interaction with the subunits of trimeric protein G upon ligand accommodation at its binding
site and the difference in its general topology upon binding of an allosteric modulator.
The kinetic model for GPCR structure–function relationship derived
by Kenakin (2004, see scheme presented in Fig. 5) is suitable to study
receptor theory and postulate the ligand-activated receptor states: the
inactive, but also the endogenously activated states and the putative interactions of inactive receptors with the G protein as schematically illustrated in Fig. 5. The vas deferens as a bioassay is an exciting biological
model to systematically examine and compare the simulated structural
conformations and eventual modifications elicited by different ligands,
including agonist, antagonist and most recently inverse agonist drugs.
In the latter group, for example ZM241385, accounts for the pharmacological properties at a defined receptor, at the light of the inverse
agonism described for this compound (see illustration in the lower
part of Fig. 5). ZM241385 is apparently an inverse ADO A2A agonist reported to modulate sympathetic vesicular release (Queiroz et al., 2002).
In the same way, the novel concept of bias agonism has changed the
paradigm of receptor theory; since ligands exert functional selectivity
according to the mode of ligand binding as a result of allosteric interactions (Wootten et al., 2013). No case of biased agonism has been described yet for the A2R or the P2YR subtypes (Jacobson et al., 2011),
but this is a fast moving area within this field.
The human A2AR topology was analyzed following X-ray crystallography at the 2.6 Å resolution (Xu et al., 2011). The receptor was crystallized with either occupancy of an inverse agonist such as ZM241385, or
UK-432097, a recognized antagonist. Differences in the X-ray crystallography support differential conformational changes by different ligands,
as was anticipated by receptor theory. It is important to recall that as
with the case of the β-adrenoceptor crystal structure, it was necessary
to alter the receptor intracellular domain by introducing lysozyme
(T4L) to favor the crystallization process, a common feature to GPCR
and ionotropic as discussed for the P2X4R. Nevertheless, a clear difference in the topology of A2AR occupied by agonist or antagonist was
found especially in conformations of the TM V and VII domains. The
TM VII marked the most difference when comparing the A2AR to the
rhodopsin receptor (Xu et al., 2011). Based on the A2AR crystals it was
possible to model the molecular docking for different ligands like caffeine and xanthines with the aim of improving the currently available
adenosine receptor ligands (Doré et al., 2011). The binding pocket
shape determines changes when agonists or inverse agonists bind;
this could be an explanation for different affinities between these
drugs for the A2AR (Bennett et al., 2013). The concept of inverse
agonism has gained interest; therapeutic opportunities are emerging
considering their valuable clinical profile and pharmacological
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20
L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
Fig. 5. General GPCR features G based on the A2AR topology. a) Schematic diagram of the A2AR as a representative class A GCPR in the classical representation (left), a more accurate representation based on the crystal structure (middle) which shows the orthosteric binding site of ADO and the relative size of the trimeric G protein. The right scheme indicates the putative
binding site of different allosteric modulators like cholesterol and sodium, the binding site of the inverse agonist Fab2838, the binding site of the regulatory proteins like USP4 and α-actin
involved in the receptor traffic and the putative ARNO binding which may explain a non-cannonic signaling. b) The cubic ternary complex is a good model that shows the equilibrium
between the ligand (L), the receptor (R) and the G protein (G); it's a suitable model to understand the existence of inverse agonism, because it explains that when a molecule, such as
the ZM241385 or Fab2838 in the case of A2AR, reduces the proportion of basal receptor activity in the G protein (R*G and LR*G) then that ligand behaves as an inverse agonist.
potential. Molecular bio modeling may allow the design of new inverse
agonists; for example, ZM241385 considered initially an A2AR antagonist (Poucher et al., 1995) it is currently accepted to be an A2AR inverse
agonist (Ng et al., 2013) and an A2BR inverse agonist (Gao et al., 2001; Li
et al., 2007). For the latter subtype, DPCPX, and MRS1706 also act as inverse agonists (Li et al., 2008). Moreover, allosteric inverse agonist
A2AR-antibody Fab fragment (Fab2838) was recently described (Hino
et al., 2012). The A2BR has not yet been crystallized; however, by homology modeling using the A2AR as a template, it is possible to make
a model to infer structural information (Thimm et al., 2013). By analogy,
since the P2YRs have not been crystallized, structural insights for the
P2Y1R and the P2Y2R are based on the rhodopsin receptor crystal
(Ivanov et al., 2006), (Ivanov et al., 2007) and the β1-adrenoceptor
was used as a temple for the P2Y12R (Chen et al., 2011), where successful molecular docking for ADP and related synthetic compounds has
been achieved. Novel ligands designed according to these principles
will soon be available, a long desired goal in the field.
GPCRs are also susceptible to allosteric modulation by small molecules. For example, cholesterol has been predicted to have an allosteric
binding site based on the A2AR crystal (Lee and Lyman, 2012). Sodium
ions have been shown to alter the ligand binding in A2AR (Liu et al.,
2012). Moreover, allosteric regulation could encompass and be extended to include the G proteins which can be modified by membrane lipids
(Wedegaertner et al., 1995) and mono ADP-ribosylation (Dani et al.,
2011), implying variations in the receptor intracellular signaling cascades. One peculiar characteristic in the A2AR is its long cytoplasmic
C-terminal (120 residues) that makes possible multiple protein like
with α-actinin and ubiquitin-specific-4 (USP4) which mediate the receptor traffic, and ARF-nucleotide binding site opener (ARNO) which
can explain the alternative signaling of A2AR by heterotrimeric G protein (Zezula and Freissmut, 2008).
Finally, the current GPCR paradigm considers receptor dimerization,
as has been confirmed by FRET and BRET techniques showing that the
A2AR dimerizes in HEK293 cells with several other GPCRs (Canals
et al., 2004). On the other hand, keeping with our interest in sympathetic
co-transmission and its modulation, only the P2Y1R has been detected in
heterologous micro domains using FRET (Choi et al., 2008). Due to the
A2AR crystal, structural information of the interface and the changes
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L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
induced by the dimerization are valuable to design selective drugs
(Fanelli and Felline, 2011). In the same way, receptor heterodimerization
is also considered rather common within GPCRs (Prinster et al., 2005)
and has been described particularly for the A1R-P2Y1R partners
(Yoshika et al., 2002). In the vas deferens, as in other sympathetic biological models, the α1A-adrenoceptor has not been ascertained whether it
oligomerizes; however, a complex regulatory mechanism might emerge
since the A2AR modulates presynaptic vesicular release of sympathetic
co-transmitters and may eventually dimerize with presynaptic P2YRs;
in addition some ARs are known to desensitize as was reported for
the A1AR (Nie et al., 1997). While P2Y1Rs have not been described in
the vas deferens, both the P2Y1R and P2Y12R have been reported in richly innervated sympathetic tissues (Quintas et al., 2009). In addition, the
P2Y12R and/or P2Y13R has also been detected in the vas deferens
(Queiroz et al., 2003) so the likelihood of a possible heterodimer
interaction cannot a priori be discarded. See schematic illustration of
Fig. 2 showing presynaptic regulatory mechanisms on a sympathetic
varicosity.
This holistic GPCR paradigm offers new structural and functional information supporting the view that the vas deferens in spite of its complexity is considered a simple physiological and relevant model
particularly suited as a bioassay for sympathetic drugs, as was reported
more than 50 years ago. In addition, structural topology of the P2X1R as
well as relevant GPCRs as discussed offer exciting opportunities to
design new clinically useful drugs for diseases involving autonomic
pathophysiological dysfunctions or dysautonomies (Hohoff et al.,
2009; Fu and Longhurst, 2010; Sangsiri et al., 2013).
6.4. Ecto enzymes in purinergic signaling
As indicated previously, the short half-life of extracellular nucleotides is tightly controlled by multiple ectoenzymes, a collection of extracellular enzymes present either in the pre or postjunctional membrane
of the tissue neuro-effector junction. The main role of these enzymes is
to hydrolyze ATP and related nucleotides consistent with their role in
co-transmission, avoiding the prolonged permanence of nucleotides in
the neuro-effector junction causing eventually desensitization of
purinoceptors. These enzymes together with the receptors for purines
and the nucleotide transporters constitute what we refer as the
“purinergic triad” of transmission. Some of these enzymes have been
crystallized, in some cases truncated enzyme species are available (as
also was the case for the P2XRs or GPCRs), offering novel molecular
insights of the mechanisms of catalysis. Figs. 2 and 3 depict the presence
of several of the enzymes in the vas deferens neuro-effector junction,
while Fig. 6 details common structural elements including their
multiple subunit structure and glycosylated derivatives of phosphatidylinositol as GPI anchors (GPI). Some of these enzymes are now
known to be released together with transmitters (Westfall et al.,
2002), posing a new paradigm for a synapse or a neuro-effector junction, since the finding of nucleotidase release together with cotransmitters must imply the necessity of fast ATP hydrolysis at this
site. Schematic representations of some of these enzymes, based on
their topological characteristics derived from their crystal structure
are presented in Fig. 6.
6.5. Ectonucleotide-triphosphohydrolase, ENTPDases (CD 39)
This family of enzymes comprises 8 forms, some of which are membrane bound, others are soluble. E-NTPDases 1, 2, 3 and 8 are membrane
bound extracellular enzymes (Robson et al., 2006), while the rest are
soluble enzymes acting in the cytosol. Interestingly for autonomic transmission, variants 5 and 6, are packed in synaptic vesicles and are released to the extracellular space (Westfall et al., 2002), expediting ATP
hydrolysis as a mean to increase ATP hydrolysis. It is postulated that
fast ATP metabolism decreases the rate of purinoceptor desensitization
and in consequence, lead to the endurance of sympathetic motor
21
transmission in this tissue. Structurally these enzymes are composed
of at least 2 subunits with a large extracellular and intracellular domain
linked through TM domains including posttranscriptional modifications
such as palmitoylation with intracellular membrane oriented lipids
(Fig. 6). In the quest to unravel structural determinants of enzyme activity, a truncated NTPDase, lacking the two TM domains, was crystallized
and analyzed by X ray diffraction. In spite of these caveats, the study revealed two major topological features: 1) the substrate binding pocket
for ATP or analogs such as AMPPNP; this site is localized in a cleft between extracellular domains and 2) a divalent cation site for metal–nucleotide coordination, necessary for nucleotide hydrolysis (Zebisch and
Sträter, 2008). Upon substrate binding to ENTPDase 1, the catalytic site
suffers a rotational movement of 7.4° as compared with ENTPDase 2
(Zebisch et al., 2012). Moreover, recent crystals from the Toxoplasma
gondii soluble ENTPDase 1 and ENTPDase 3 showed a similar rotational
(Krug et al., 2012; 2013); however the proposed mechanism did not
ponder the influence of the TM domains (Fig. 6), since these domains
were truncated to facilitate enzyme crystallization. Moreover, the
ENTPDase crystal of Legionella pneumophila in holo conformation with
ARL67156 bound was also published. This compound is a synthetic nucleotide analog with inhibitor properties (Vivian et al., 2010) found selective for ENTPDases 1 and 3 (Lévesque et al., 2007). The crystal
structure of this enzyme revealed that the ARL67156 occupied the
same nucleotide binding site as AMPPNP, but since the inhibitor is
bulkier, it has less direct contact with the enzyme but a larger indirect
interaction with surrounding water molecules (Vivian et al., 2010).
Active ENTPDase 1 is a dimer; the oligomer was not observed when
the TM truncated ENTPDase 1 variant was examined, suggesting a new
role for the TM domains since TM helixes interact in a very flexible way
altering its enzymatic activity (Grinthal and Guidotti, 2004). Soluble
ENTPDase 1 lacking the TM domains, was 10 fold less active than the
full enzyme (Wang et al., 1998), supporting the importance of the TM
domains in enzyme activity, an observation consonant with the data
of Grinthal and Guidotti (2004). Moreover, membrane cholesterol depletion using methylβ cyclodextrin reduced enzyme activity, which
was reestablished upon cholesterol addition (Papanikolaou et al.,
2005), emphasizing the regulatory role of lipids in protein interactions
at the membrane level and/or possibly in the proper enzyme conformation. At present it is still debated whether cholesterol modulates
inducing a structural conformational change or promotes the enzyme
association with related membrane proteins (Papanikolaou et al.,
2011). In addition to the cholesterol interaction, ENTPDase1 is
palmitoylated (see Fig. 6), which acts as a caveolae target (Koziak
et al., 2000). Notwithstanding, ENTPDase1 is not exclusively associated
to caveolae, according to the studies on polarized cells (Papanikolaou
et al., 2011). The intracellular enzyme N-terminal has a potential intracellular phosphorylation site (Lemmens et al., 2000), which could also
contribute to the lower activity shown by the truncated enzyme forms
(Wang et al., 1998; Papanikolaou et al., 2011). Since ENTPDase1 is localized in raft microdomains, in the vicinity of P2YRs (Papanikolaou et al.,
2011), it is plausible to suggest a fine enzyme tuning mechanism, since
P2Y1Rs are preferentially activated by ADP. This cellular distribution
could be related to a novel regulatory mechanism of the sympathetic
terminal, where in addition, P2Y12 and/or elicit significant presynaptic
modulation (Queiroz et al., 2003). Much more is known about the biology of ENTPDase 1 than the rest of these enzymes, and at this time it
might be used as a working model.
Based on isothermal titration calorimetric technique, rapid kinetic
enzyme studies were conducted (Krug et al., 2012; 2013; Zebisch
et al., 2012). This method plus enzyme crystallography clarified and differentiated the inhibitor effect of polyoxometals on ENTPDases 1 and 2.
While the poly tungstate salt (POM-1, Na6 [H2W12O40]) did not generate stable crystals, the ammonium heptamolybdate (AHM, NH4)6
[Mo7O24] bound to two lysine's involved in the metal cluster–protein
complex localized in the interdomain cleft of the nucleotide binding
site.
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22
L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
Fig. 6. Schematic representation of ectonucleotidases involved in vas deferens purinergic signaling. The ectonucleotidases are functional dimers, a) the membrane bound NTPDases 1, 2, 3
and 8 posses two membrane α-helices in the TM domain. This domain is critical for the dimerization to occur. In addition the TM domain may be palmitoylated and is regulated by
cholesterol, this favors the NTPDase localization into lipid rafts. The nucleotide hydrolysis occurs in the cleft of the two domains of the extracellular region, which produces a movement
between the domains that surround the nucleotide. b) The 5′eNT is a functional dimer bound by the C-terminal domain with the other monomer and also to the membrane through a GPIanchor as depicted schematically. This enzyme can be soluble or membrane bound, in both cases it functions as a dimeric enzyme. The catalytic process produces a major movement
between in the monomers. c) The alkaline phosphatases are also dimers bound to the membrane by a GPI-anchor and can be soluble or membrane bound. In this case, the dimer is
bound by the N-terminal domain and it presents a wide variety of inhibitors, for example L-Phen, which binds near the active site, but also can bind in an allosteric site to regulate the
catalytic enzyme activity.
6.6. 5′ecto nucleotidase, 5′eNT, (CD 73)
5′eNTs are homodimers which hydrolyze nucleotide monophosphates to nucleosides such as ADO; these enzymes are either soluble or membrane bound by glycosylated derivatives of glycosylated
phosphatidylinostol (GPI anchors, as shown schematically in Fig. 6).
GPI are hydrophobic anchors inserted into the lipid bilayer
(Hunsucker et al., 2005). Both subunits interact by non-covalent
bonds (Martínez-Martínez et al., 1998), the catalytic center binds
Zn2+ required for catalysis (Knapp et al., 2012). A characteristic of this
enzyme is its inhibition by ATP/ADP (Grondal and Zimmermann,
1987). The human truncated 5′eNT crystal structure with ADO and
AMPCP bound (Heuts et al., 2012), confirms the dimer structure
where each subunit has two major domains and, like the ENTPDses,
the nucleotide binding site is located at the cleft between domains.
The homodimer interphase is linked to the C-domain, where the GPI anchor and the substrate binding site is localized, leading the N-terminal
free to move 114° (Knapp et al., 2012). Based on the Escherichia coli
5′NT and on the characterized antraquinone binding site, screening of
ligand-based inhibitors was advanced before the human crystal structure became available (Ripphausen et al., 2012). Recently, based on
the crystal of the human 5′eNT a virtual screening based on the
AMPCP inhibitor site is available (Furtmann and Bajorath, 2013).
6.7. Alkaline phosphatase, APs
Like other ATPases, APs are two-subunits or dimer enzymes
(Hoylaerts et al., 1997; Le Du et al., 2001); according to structural analysis APs can form homo and heterodimers (Le Du and Millan, 2002).
Structural biology supports that the GPI-membrane bound human placental alkaline phosphatase inserts into lipid raft domains (Saslowsky
et al., 2002; Giocondi et al., 2008); moreover, physicochemical techniques show the enzyme in unilamellar vesicles (Kahya et al., 2005;
Giocondi et al., 2007). Crystals of the human form reveal that each monomer has a distinct active site with two Zn2+ and one Mg2+ surrounding
the catalytic site (Le Du et al., 2001). In addition, the monomers are tightly bound explaining why the catalytic properties of each monomer are
controlled by the conformation of the second subunit (Hoylaerts et al.,
1997). APs can be GPI anchored through the C-terminal end
(Kozlenkov et al., 2002); when released from the cell membrane soluble
APs can be found with or without the GPI tail (Anh et al., 2001), see
scheme shown in Fig. 6c for details.
APs hydrolyze nonspecifically phosphate groups in molecules including nucleic acids; in the particular case of ATP, APs hydrolyze the nucleotide sequentially until ADO (Ohkubo et al., 2000); following a noncooperative allosteric mechanism (Hoylaerts et al., 1997). Fig. 6c shows
diagrammatically the enzyme active site and an allosteric site in the vicinity of the active enzyme site. Classical AP inhibitors include L-amino
acids such as: L-Phe (shown as L-Phen in Fig. 6), L-Trp, L-homoarginine,
and L-Leu (Fishman and Sie, 1971; Doellgast et al., 1977) and levamisole
(Van Belle, 1976). Based on X-ray diffraction of human placental AP crystals, the enzyme inhibition mechanism was re-examined (Hoylaerts
et al., 1997; Kozlenkov et al., 2004). Recent tailor made selective ligands
such as the arylsulfonamides are available (Dahl et al., 2009) which have
assisted a better characterization of the enzyme activity.
7. Towards an integral view of the purinergic component of
sympathetic co-transmission
Based on the notion that the P2X1R as well the α1A-adrenoceptors
are apparently confined to lipid raft domains of the vas deferens smooth
muscle membranes, it is possible to infer aspects of the functional significance of this particular receptor distribution for sympathetic cotransmission. Considering that the P2X1R is an excitatory ion channel,
and since the activation of the α-adrenoceptor is coupled to the release
of at least two distinct intracellular signals, the generation of a fast excitatory depolarization plus a slower metabolic element occurring in the
immediate vicinity are fully compatible with the kymograph recordings
showing an initial purinergic phasic contribution followed by the slower
tonic contribution (Fig. 1). The notion that these signals are possibly
generated in raft domains, enriched in G proteins, arguments that
these platforms are critical for signaling events related to sympathetic
co-transmission in the vas deferens. The significance of lipid rafts as specialized signaling sites is rapidly gaining support; at present we only
have some dispersed puzzle pieces. The finding of Donoso et al.
(2014) showing lipid raft distribution for the P2X1R in the human tissue
is encouraging and supports this contention. Moreover, rat vas deferens
preparations exposed to agents that reduce membrane cholesterol content, resulted in impaired purinergic responses (Donoso et al., 2014).
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In addition, the modulator role of NPY on sympathetic cotransmission cannot be overlooked. The role of this peptide has been
better characterized in the vascular system, where the peptide has
a constrictor effect of its own (Racchi et al., 1997; Boric et al., 1995).
In the rat vas deferens NPY does not have a postjunctional effect rather
it exerts a dominant presynaptic negative modulator role on cotransmitter's release. Bitran et al. (1991) reported that the peptide potency to block the tissue contractions elicited by nerve-evoked stimulation was circa 20 nM; the inhibition; in agreement with Huidobro-Toro
et al. (1985), was more marked in the prostatic end of the vas deferens.
The inhibition is sensitized by reserpine treatment, particularly in the
prostatic end of the rat tissue (Huidobro-Toro, 1985) and is dependent
on animal age and testosterone, since in castrated rats the effect is reduced (Bitran et al., 1991). The presynaptic modulatory effect of NPY
is due to inhibition of NE release via a cAMP independent mechanism
(Bitran et al., 1996); no studies have been conducted at present to assess
whether NPY also affects ATP release from this tissue. Altogether, these
exciting conclusions highlight the notion that NPY in the vas deferens is
a negative presynaptic modulator, an effect presumably mediated
through presynaptic neuropeptide Y2 receptor activation, a receptor
mechanism distinct from postjunctional neuropeptide Y1 receptors,
which in contrast with vascular smooth muscles, are not observed in
the rat vas deferens smooth muscles (see diagram in Fig. 2).
8. Novel role of the epithelium in sympathetic excitability
Only within the past 10 years, researchers have addressed whether
the epithelium cell lining participates in vas deferens smooth muscle reactivity. This idea offers obvious resemblances to the classical studies
conducted almost 50 years ago using the classical isolated helicoidal
vascular strips to study vascular reactivity, where the paramount role
of the endothelium was disclosed and lead to the discovery of NO and
the pivotal role of the endothelium in vascular wall regulation
(Furchgott and Zawadzki, 1980; Moncada and Higgs, 2006). Nowadays,
the role of the vascular endothelium as a source of numerous physiologically relevant vasoactive compounds with either dilator or constrictor
properties is well recognized (Moncada and Higgs, 2006; Bohm and
Pernow, 2007). Among the most notable endothelial compounds apart
from NO, prostacyclin, the as yet unresolved hyperpolarizing factor
and the multiple endothelins, considered the most potent and persistent vasoconstrictors known, all play a role in vascular wall homeostasis
(Bohm and Pernow, 2007). Within the past two decades, endothelial
dysfunction is a major clinical concern and a determinant pathophysiological condition of a collection of vascular diseases.
It has not escaped our attention whether in analogy with the endothelium, the vas deferens epithelium lining plus its lamina propria and
its complex network of cells may modulate the contractile smooth muscle responses in this tissue. Pharmacologists headed by Prof A.
Jurkiewicz at the Federal University in Sao Paulo first reported that clonidine elicits the release of an endogenous modulator from the rat vas
deferens that reduces the clonidine-induced increase in tissue motor
tone (Okpalaugo et al., 2002). The nature of the chemical released by
clonidine has not been identified; however, they made an ancillary experiment where they observed that in tissues without the epithelial
layer, reactivity was significantly restored upon addition of an extract
from the removed epithelial cell layer. This observation allowed the proposal that the epithelium might release an inhibitory agent that blocked
the clonidine-induced increase in motor tone (Okpalaugo et al., 2002).
In the search of a putative inhibitor, Ruan et al. (2008) reported that
the exogenous application of ATP to rat vas deferens bioassays elicited
the release of a purported epithelial eicosanoid since the purinergic
motor response was reduced in tissue incubation with indomethacin,
a classical cyclooxygenase inhibitor. These findings suggested a novel
regulatory mechanism, mediated apparently by prostaglandin E2 secreted from the epithelium in response to neuronal ATP released; the
23
proposed epithelial PGE2, hyperpolarizes smooth muscle cells via the
activation of PGE2-receptors in the smooth muscles.
Our laboratory has also been lately interested in these observations
and found that prostatic preparations from rat vas deferens where the
epithelium was manually removed, become more sensitive to the inhibitory action of exogenous ATP applications, supporting the role of an inhibitory chemical released by the epithelium or surrounding mucosal/
sub mucosal cells (Huidobro-Toro et al., 2013). Moreover, we consistently observed that removal of the rat vas deferens epithelium
displaced to the right, in a non-competitive manner, the NE, but not
the ATP, concentration–response curve (Huidobro-Toro et al., 2013).
This finding is compatible with the idea that the epithelium releases a
chemical, which in this case, reduced the smooth muscle reactivity particularly of NE as a prototype adrenergic agonist. Therefore, it is plausible as in the case of the endothelium, the epithelium cell layer of the rat
vas deferens secrete more than one chemical to modulate smooth muscle reactivity, some are likely inhibitors but other agents might also
stimulate the tissue motor activity; the latter type of compounds have
not been described as yet.
Based on these experimental findings, we have not ignored that the
principle may be extended to different tissue epitheliums and their
adjacent lamina propia cells. Such might be the case of the intestines,
bronchi, bladder and other hollow organs, where the epithelium and
surrounding mucous layers and plexus also play a role as modulators
of the tissue motor responses under physiological and importantly during pathophysiological conditions. In fact, the bladder urothelium expresses pressure-sensitive mechanoreceptors which release ATP to
start the bladder voiding reflex, an effect mediated by P2X2/3 receptors
(Mulryan et al., 2000). Likewise, the respiratory epithelium detects bitter and acid substances (Krasteva et al., 2011; Krasteva and Kummer,
2012) which significantly modify the frequency of airway epithelial
cilia movements. Altogether this collection of exciting new data indicates that the epithelium of several tissues participates in cell communication via secretion of multiple chemicals. In this regard, the vas
deferens might serve as a versatile experimental model to test novel
working hypothesis far beyond its classical role as a bioassay or a
model system to disentangle co-transmission.
9. Clinical perspectives
The paramount role of purines and particularly, ATP, in the motor activity of the vas deferens might be considered as a pillar to understand
the rational basis of male reproductive tract diseases. In this regard,
the contractile responses of the vas deferens elicited by sympathetic
nerve stimulation were reduced by over 60% in P2X1R transgenic mice
deficient in P2X1R. This phenotype was associated to a 90% decrease
in male fertility an effect not accounted by a decreased in gamete production (Mulryan et al., 2000). Therefore, the P2X1R may be associated
to multiple causes of human infertility which in the end might be related one way or another to a weakened purine motor component. Within
other variables, a lesser expression of the P2X1R, lack of ATP coordination with the co-transmitter role of NE, failure to spread adequately
the depolarizing wave between cells, or to the erroneous distribution
of the ATP and α-adrenoceptors in the purported lipid rafts, all might reflect deficiencies in sperm transport in reproductive pathologies. These
conditions might finally result in inadequate tissue contractility and
therefore, in reduced sperm migration. No important P2X1R splice variants are known at present which may account for an abnormal receptor
and therefore in a reduced co-transmission. In addition, α2A/D presynaptic adrenoceptor activity might also impinge the control of tissue contractility, since transgenic mice lacking these receptors result in a major
loss of prejunctional α2-adrenoceptor activity (Cleary et al., 2002).
Two pharmacological treatments have relevant clinical consequences as possible male contraceptives, although we are aware that
the strength of the purinergic component in rodents is stronger than
in humans. i) The use of a supposedly selective α1A-adrenoceptor
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24
L.C. Navarrete et al. / Autonomic Neuroscience: Basic and Clinical 185 (2014) 8–28
antagonists, prescribed in the treatment of humans suffering from benign prostatic hyperplasia, is known to elicit deficits in ejaculation as a
significant human side effect. Studies with silodosin showed a complete
lack of ejaculation in young human volunteers due to lack of seminal
emission (Kawabe et al., 2006; Kobayashi et al., 2008; Shimizu et al.,
2010) due to reversible failures in vas deferens motor activity upon cessation of drug administration. ii) The reduction of the contractility following phosphodiesterase inhibition by drugs such as rolipram,
milrinone or sildenafil effectively blocked NE-induced contractions,
suggesting that these inhibitors also reduce ejaculation (Birowo et al.,
2010). In this regard, blockade of the P2X1R by suramin, or newer and
more selective P2X1R might also reduce ejaculation and is a plausible
initial target for a male contraceptive strategy.
Vasectomy is a male contraceptive procedure related to the physical
obstruction of sperm passage along the vas deferens ductus. At times,
urologists are urged to revert this condition. Studies have been performed both in man and animals demonstrating the practicality of this
procedure (Alexander, 1979; Dixon et al., 1987). Human studies show
that even after fifteen years of surgery, the innervation of the epididymal portion of the tissue is severely compromised (Dixon et al., 1998),
an indication that nerve terminals recover slowly and only to a limited
extent after this procedure. As expected for a denervated organ,
prolonged NE supersensitivity was observed; unfortunately the ATP
motor responses were not examined (DeGaris and Pennefather,
1987a). Closer to the clinical setting, the same investigators examined
whether anastomosis of epididymal or prostatic halves of the rat vas
deferens after vasectomy restored animal fertility and the tissue content
of NE. Two months after the surgery, while NE levels increased to 40% of
control values, fertility reassumed (DeGaris and Pennefather, 1987b).
Therefore it is concluded that allowing sufficient time, anastomosis
can reassume fertility as has been frequently observed in human clinic.
At the light of the fundamental role of ATP as a motor component of
the vas deferens contractility, it appears reasonable to propose that
P2X1R antagonists might be explored as a possible male contraceptive
mechanism, as has been advanced by Burnstock and Verkhratsky
(2010). The pharmaceutical industry might be interested in developing
novel P2X1R antagonists for this defined purpose, in addition to other
effects of the P2X1R in other smooth muscles, as in the vascular tree.
10. Concluding remarks
The prominent sympathetic network of nerve endings in the rodent
vas deferens has been vastly explored for decades as an experimental
model to study the biochemistry, physiology and the pharmacology of
autonomic co-transmission and sympathetic acting drugs. Notwithstanding, the key contributions that emerged from these studies, including the cloning of the P2X1R from the tissue (Valera et al., 1994),
introduced the physiological relevance of purine transmission in this
tissue from rodents to humans, establishing the cellular basis for sympathetic co-transmission. The seminal observation that transgenic mice
lacking P2X1R expression evoke only about half of the tissue tension
elicited by sympathetic nerve stimulation further highlighted the functional role of co-transmission in the tissue motor responses and set the
scenario to examine the role of ATP in sympathetic reflexes. The insight
of structural biology has added a novel and powerful approach assisting
the understanding of P2X1R channel functioning revealing novel and
unsuspected aspects of this particular channel topology. Combining
this bioassay with electrophysiological techniques plus the principles
of structural biology, has given us a fresh view of a preparation used
for over half a century from which we presume there is still much to
learn, like the plausibility of the reciprocal modulation of the epithelium
or mucosal plexus and the tissue contractility.
Albeit a plethora of cell biology techniques has facilitated current understanding of the tissue physiology, there are still many unresolved
questions that hopefully will be cleared in the years to come. One of
them refers to the fast desensitization of the P2X1R and the molecular
events that regulate this process as well as the physiological implications
of this phenomenon. Another challenging issue refers to the release of
ectoATPases together with ATP and NE from the synaptic vesicles and
the fast degradation of the ATP in this neuro-effector junction as well
as the finding that not all varicosities discharge co-transmitter's with
every nerve terminal depolarization. The influence of sensory nerve endings and the reciprocal crosstalk between motor sympathetic efferent
pathways and sensory terminals, like the CGRP-containing sensory neuron and sympathetic efferens, is another novel issue that will lead to exciting research opportunities. Likewise, the paramount role of the tissue
epithelium in the ductus motor action also will attract novel research
and further the use of this bioassay in the prosecution of these studies.
The time has come ripe to use this simple bioassay provided with cell biology and molecular biology tools to exploit and further unravel the cellular basis of sympathetic co-transmission in the physiological context of
this tissue. Principles of structural biology will also assist to examine cotransmission at the molecular and atomic level highlighting a novel view
of medicine examined at the molecular level.
Acknowledgments
This study was supported by Millennium Nucleus of Structural
Biology (NuBEs), Grant P10-035-F; additional funding derived from
FONDECYT Grants 1110672 and 1141132.
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