Am J Physiol Heart Circ Physiol 290: H1818 –H1825, 2006.
First published December 9, 2005; doi:10.1152/ajpheart.01062.2005.
Nicotinamide adenine dinucleotide is released from sympathetic nerve
terminals via a botulinum neurotoxin A-mediated mechanism in canine
mesenteric artery
Lisa M. Smyth, Leanne T. Breen, and Violeta N. Mutafova-Yambolieva
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
Submitted 7 October 2005; accepted in final form 4 December 2005
norepinephrine; neurotransmitter release; cotransmission; adenosine
5⬘-triphosphate
THE SPLANCHNIC CIRCULATION comprises a substantial portion of
the cardiac output and blood volume and is generally assumed
to play an important part in overall cardiovascular homeostasis,
in addition to providing adequate perfusion in the gastrointestinal system. Disturbances in the neural control of mesenteric
arteries in particular might contribute to the pathogenesis of
hypertension and local vascular dysfunctions. In fact, any
condition that is associated with sympathetic nervous system
hyperactivity (i.e., stress, strenuous exercise, and hemorrhage)
may manifest disproportionate vasoconstrictive responses in
Address for reprint requests and other correspondence: V. N. MutafovaYambolieva, Dept. of Physiology and Cell Biology, Anderson Medical Bldg./
MS 352, Univ. of Nevada School of Medicine, Reno, NV 89557-0271 (e-mail:
[email protected]).
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the splanchnic circulation. Therefore, a great deal of effort has
been devoted to understanding the mechanisms of autonomic
vascular control in the mesentery. The perivascular nerves of
the mesenteric vasculature in various species consist primarily
of sympathetic postganglionic nerve terminals, in which norepinephrine (NE), ATP, and neuropeptide Y (NPY) are the
established cotransmitters (5, 7, 9, 10, 20, 33, 34) . In addition,
various neuropeptides including CGRP, vasoactive intestinal
peptide (VIP), and substance P (SP), primarily released from
sensory motorneurons, also contribute to the neural control of
the mesenteric circulation (13, 15, 17, 19, 22, 30). The substances that are released by action potentials serve as neurotransmitters, neuromodulators, or both (7, 8, 22). The precise
combinations of neurotransmitter and neuromodulator substances in vasomotor pathways vary with species (12). Canine
mesenteric arteries, in particular, have been shown to corelease
NE and ATP in a tetrodotoxin (TTX)- and guanethidinesensitive manner (4, 5, 28). The tissue superfusates collected
during stimulation of peripheral nerve terminals also contain
the ATP metabolites ADP, AMP, and adenosine. Most recently, we found that short-pulse electrical field stimulation
(EFS) of the canine mesenteric artery in vitro evokes the
release of ␤-nicotinamide adenine dinucleotide (␤-NAD) along
with NE, ATP, ADP, AMP, and adenosine (35). The release of
␤-NAD depends on neural activity and is not a result of smooth
muscle contraction. Exogenous ␤-NAD reduces the EFSevoked release of NE (35), suggesting that ␤-NAD may represent a novel extracellular player at the vascular neuroeffector
junction in the mesenteric circulation. The source(s) and mechanisms of release of this novel extracellular factor, however,
remain to be elucidated. The present study was designed,
therefore, to tackle this issue by investigating the role of
sympathetic neurotransmitters, neuropeptides, and synaptosomal-associated protein of 25 kDa (SNAP-25)-mediated exocytosis in the evoked release of ␤-NAD in the canine isolated
mesenteric artery devoid of endothelium. In most instances, the
release of NE, ATP, and ␤-NAD was evaluated in parallel. Our
results suggest that ␤-NAD is released on stimulation of
postganglionic sympathetic nerve terminals and its release
partially depends on SNAP-25-mediated exocytosis. These
results introduce ␤-NAD as a putative neurotransmitter or
neuromodulator in the mesenteric circulation.
MATERIALS AND METHODS
Tissue preparations. Sixty-four mongrel dogs of either sex (averaging 15 kg) were obtained from vendors licensed by the U.S.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/06 $8.00 Copyright © 2006 the American Physiological Society
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Smyth, Lisa M., Leanne T. Breen, and Violeta N. MutafovaYambolieva. Nicotinamide adenine dinucleotide is released from
sympathetic nerve terminals via a botulinum neurotoxin A-mediated
mechanism in canine mesenteric artery. Am J Physiol Heart Circ
Physiol 290: H1818 –H1825, 2006. First published December 9, 2005;
doi:10.1152/ajpheart.01062.2005.—Using high-performance liquid
chromatography techniques with fluorescence and electrochemical
detection, we found that ␤-nicotinamide adenine dinucleotide (␤NAD) is released in response to electrical field stimulation (4 –16 Hz,
0.3 ms, 15 V, 120 s) along with ATP and norepinephrine (NE) in the
canine isolated mesenteric arteries. The release of ␤-NAD increases
with number of pulses/stimulation frequencies. Immunohistochemistry analysis showed dense distribution of tyrosine hydroxylase-like
immunoreactivity (TH-LI) and sparse distribution of TH-LI-negative
nerve processes, suggesting that these blood vessels are primarily
under sympathetic nervous system control with some contribution of
other (e.g., sensory) neurons. Exogenous NE (3 ␮mol/l), ␣,␤-methylene ATP (1 ␮mol/l), neuropeptide Y (NPY, 0.1 ␮mol/l), CGRP (0.1
␮mol/l), vasoactive intestinal peptide (VIP, 0.1 ␮mol/l), and substance P (SP, 0.1 ␮mol/l) had no effect on the basal release of ␤-NAD,
suggesting that the overflow of ␤-NAD is evoked by neither the
sympathetic neurotransmitters NE, ATP, and NPY, nor the neuropeptides CGRP, VIP, and SP. Botulinum neurotoxin A (BoNTA, 0.1
␮mol/l) abolished the evoked release of NE, ATP, and ␤-NAD at 4
Hz, suggesting that at low levels of neural activity, release of these
neurotransmitters results from N-ethylmaleimide-sensitive factor attachment protein receptor/synaptosomal-associated protein of 25 kDamediated exocytosis. At 16 Hz, however, the evoked release of NE,
ATP, and ␤-NAD was reduced by BoNTA by ⬃90, 60, and 80%,
respectively, suggesting that at higher levels of neural activity,
␤-NAD is likely to be released from different populations of synaptic
vesicles or different populations of nerve terminals (i.e., sympathetic
and sensory terminals).
RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
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B. Flow rate was 1 ml/min and run time was 20 min. Column
temperature was ambient, whereas the autosampler temperature was
4°C. The fluorescent detector was set to record signals at an excitation
wavelength of 230 nm and emission wavelength of 420 nm, which are
the optimum conditions for detection of etheno-derivatives of nucleotides and nucleosides as shown previously (3). The amounts of
nucleotides in each sample were calculated from calibration curves of
nucleotide standards run simultaneously with every set of unknown
samples. Results were normalized for sample volume and tissue
weight, and the overflow of nucleotides was expressed in femtomoles
per milligram of tissue.
HPLC assay of NE. The overflow of NE was assayed as described
previously (28). Briefly, 115-␮l aliquots from the samples were
acidified with 3 ␮l of 1 M perchloric acid to pH 2.6 and injected (70
␮l) into an isocratic HP1100 HPLC system equipped with an
HP1049A electrochemical detector (Agilent Technologies) and a
MD-150 column (ESA, Chelmsford, MA). The mobile phase for
separation consisted of the following (in mmol/l): 50 NaH2PO4, 0.2
EDTA, 3.0 l-heptanesulfonic acid, 10 LiCl, and methanol 3% vol/vol
in deionized water (pH 2.6). The HPLC systems were controlled and
data collected by a HP Kayak XA computer equipped with HP
ChemStation (A.06.03) software (Agilent Technologies). The
amounts of NE in each sample were calculated from calibration
curves of NE standards run simultaneously with every set of unknown
samples. Results were normalized for sample volume and tissue
weight, and the overflow of NE was expressed in femtomoles per
milligram of tissue.
Immunohistochemistry. Immunohistochemical analysis was performed on segments of second- and third-order branches of the
superior and inferior mesenteric artery. Whole mount preparations
were prepared after removal of the surrounding adipose and connective tissue. Segments of artery were opened longitudinally and pinned
to the base of a dish filled with Sylgard elastomer (Dow Corning,
Midland, MI) with the adventitial side facing upward. Tissues were
fixed in paraformaldehyde (4% wt/vol in 0.1 M phosphate buffer for
20 min at 4°C). After fixation, preparations were washed for 3– 4 h in
PBS (0.01 M, pH 7.4). Tissues were incubated at 4°C overnight in 1%
BSA to reduce nonspecific antibody binding. This solution also
contained 0.5% Triton X-100 to permeabilize the tissue for easier
antibody penetration. For cryostat studies, segments of second- and
third-order branches of the canine superior and inferior mesenteric
artery were perfused with Krebs solution before being fixed with
paraformaldehyde for 30 min at 4°C. After fixation, tissues were
washed with PBS and processed through a sucrose gradient (5, 10, and
15% sucrose-PBS; 15 min each) before being immersed in 20%
sucrose-PBS overnight. The following day, the tissue was cut into
⬃2-mm segments and embedded in Tissue-Tek (Miles, Elkhart, IN)
before being quickly frozen in liquid nitrogen. Cryostat sections were
cut at 8- to 10-␮m thicknesses by using a Leica CM3050 cryostat and
collected on coated glass slides (Surgipath, Richmond, IL). Sections
were preincubated with 1% BSA-PBS for 1 h before being incubated
with antibodies.
For examination of SNAP-25-like immunoreactivity (SNAP-25LI) in whole mount preparations, tissues were incubated for 24 – 48 h
at 4°C with a monoclonal antibody raised against SNAP-25 protein
(1:100; catalog number 111011, Synaptic Systems, Göttingen, Germany). SNAP-25-LI was detected by Alexa 594 secondary antibody
(1:1,000 in PBS, rabbit anti-mouse, 1 h, room temperature; Molecular
Probes, Carlsbad, CA, red fluorescence).
For double-label immunostaining of Protein Gene Product (PGP
9.5) and tyrosine hydroxylase (TH) in both whole mount preparations
and cryostat sections, tissues were first incubated with rabbit PGP 9.5
primary antibody (1:1,000 in PBS, 24 – 48 h at 4°C, catalog number
RA-95101, Ultraclone, Isle of Wight, UK). The tissues were subsequently washed in PBS for several hours. PGP 9.5-LI was detected by
Alexa 594 secondary antibody (1:1,000 in PBS, goat anti-rabbit, 1 h,
room temperature, Molecular Probes, red fluorescence). The tissues
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Department of Agriculture and used for this study. The use of the
laboratory animals for these experiments was approved by the University of Nevada Animal Care and Use Committee. The animals were
euthanized with pentobarbitone sodium (100 mg/kg iv). The abdomens were opened, and segments of second- and third-order branches
of the superior and inferior mesenteric artery (0.5–1 mm in diameter)
were dissected out and bathed in oxygenated Krebs solution containing (in mM) 118.5 NaCl, 4.2 KCl, 1.2 MgCl2, 23.8 NaHCO3, 1.2
KH2PO4, 11.0 dextrose, and 1.8 CaCl2 (pH 7.4). Vessels were
denuded of their endothelium by rubbing with a rough-surface needle
and slowly perfusing with distilled water. This procedure successfully
removes endothelium while maintaining smooth muscle contractility
intact (27).
Overflow experiments. The tissue segments were placed in 200-␮l
water-jacket Brandel superfusion chambers as described previously
(3–5, 29, 39). Briefly, after 45 min equilibration, the tissues were
subjected to a 15-s “conditioning” stimulation with a train of square
wave pulses of 0.1-ms duration and a frequency of 4 Hz. Thirty
minutes after the conditioning stimulation, the preparations were
subjected to EFS for 120 s with a train of suprathreshold pulses of 0.3
ms at 4 or 16 Hz. These parameters were chosen after verification that
NE overflow is abolished by either TTX (0.3–1 ␮mol/l) or ␻-conotoxin GVIA (5 nmol/l). Samples of the superfusion solution were
collected before the electrical stimulation (resting overflow) and
during the electrical stimulation (nerve-evoked overflow) in ice-cold
Eppendorf tubes. Samples were analyzed for ATP and ␤-NAD content
by using HPLC techniques with fluorescence detection as described
previously (3, 5, 28, 35). Aliquots of some superfusate samples were
processed for NE assay by HPLC techniques with electrochemical
detection as described previously (28).
To test the possibility that sympathetic neurotransmitters could
evoke ␤-NAD overflow, tissues were superfused for 15 min with
either NE (3 ␮mol/l), ␣,␤-methylene ATP (␣,␤-MeATP, 1 ␮mol/l), or
NPY (0.1 ␮mol/l), and the basal overflow of ␤-NAD was evaluated in
the absence and presence of each substance. To test the possibility that
neuropeptides could evoke ␤-NAD overflow, tissues were superfused
with either CGRP (0.1 ␮mol/l), VIP (0.1 ␮mol/l), or SP (0.1 ␮mol/l)
for 5 min, and the content of ␤-NAD in the superfusate samples in the
absence and presence of a neuropeptide was evaluated. In another
series of experiments, arteries were incubated for 5.5 h at 37°C in a
sealed vial containing 250 ␮l of RPMI-1640 solution, pH 7.4. For
botulinum neurotoxin A (BoNTA) treatment, BoNTA was added to
the 250 ␮l of RPMI-1640 solution to make a final concentration of
100 nmol/l. Vessels were washed three times (5 min each) in Krebs
solution without BoNTA before being placed in Brandel superfusion
chambers, and overflow experiments were performed to analyze the
content of NE, ATP, and ␤-NAD in the tissue superfusates collected
before and during EFS at 4 or 16 Hz (0.3 ms, 15 V, for 120 s).
Sample preparation. A modified method of Levitt et al. (16) was
employed to detect 1,N6-etheno-derivatives of the endogenous nucleotides present in the tissue superfusates. Briefly, 100 ␮l of a citrate
phosphate buffer (pH 4.0) were added to 200 ␮l of the superfusate
sample in plastic Eppendorf tubes (Fisher Scientific). 2-Chloroacetaldehyde (10 ␮l) was added to the samples in a fume hood; the samples
were then heated for 40 min at 80°C in a dry bath incubator (Fisher
Scientific). With the use of this procedure, endogenous ATP is
derivatized to 1,N6-etheno-ATP (eATP), whereas endogenous
␤-NAD is derivatized to 1,N6-etheno-ADP-ribose (eADPR) as
described previously (3, 35). The reaction was stopped by placing
the samples on ice.
HPLC assay of etheno-nucleotides and etheno-nucleosides. The
HP1100 liquid chromatography module system (Agilent Technologies, Wilmington, DE) was used throughout this study and has been
previously described (3). The mobile phase consisted of 0.1 mol/l
KH2PO4 (pH 6.0) as eluant A; eluant B contained 35% methanol and
65% eluant A. Gradient elution was employed according to the
following linear program: time 0, 0% eluant B; 18 min, 100% eluant
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RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
NPY was purchased from Bachem Biosciences (King of Prussia, PA).
CGRP was purchased from Tocris Cookson (Ellisville, MO). BoNTA
was purchased from List Biological Laboratories (Campbell, CA). All
drugs were initially dissolved in double-distilled water, except
BoNTA, which was dissolved in BSA (1 mg/ml).
Statistics. Data are presented as means ⫾ SE. Means were compared by one-way ANOVA (Graph Pad Prism version 3, Graph Pad
Software). A probability value of P ⬍ 0.05 was considered significant.
RESULTS
Canine mesenteric arteries are densely innervated by sympathetic nerves. Whole mount preparation of mesenteric artery
labeled for PGP 9.5-LI, a pan-neuronal marker, revealed a
dense network of nerve fibers within the arterial wall (Fig. 1A).
Arteries labeled for TH-LI, the rate-limiting enzyme involved
in the synthesis of NE, also revealed a network of neural fibers
with a density similar to PGP 9.5-LI (Fig. 1B). Double labeling
for PGP 9.5-LI and TH-LI revealed that the majority of the
nerve fibers that contained PGP 9.5-LI also contained TH-LI
(Fig. 1C); a small population of nerve fibers that contained
PGP 9.5-LI did not contain TH-LI. Cryostat sections of mesenteric artery revealed that the PGP 9.5-LI and TH-LI were
located within a dense layer at the level of the adventitia (Fig.
1, D–F). The majority of the nerve fibers showed colocalization of PGP 9.5-LI and TH-LI.
Fig. 1. Whole mount and cryostat sections of canine mesenteric artery labeled with Protein Gene Product (PGP 9.5)-like immunoreactivity (PGP 9.5-LI) and
tyrosine hydroxylase-like immunoreactivity (TH-LI). A: distribution of PGP 9.5-LI nerve fibers (arrows; Alexa 594) in whole mount preparation of artery. B:
same area of artery labeled for TH-LI (arrows; Alexa 488). PGP 9.5-LI and TH-LI were colocalized in the same nerve network (C, yellow pixels). Cryostat section
of a mesenteric artery was labeled for PGP-LI (D, arrows) and TH-LI (E, arrows). Distribution of PGP 9.5-LI and TH-LI in cryostat sections (F) was similar
to that in whole mounts and was found on adventitial side (a) of the vessel. Only a few noncolocalized (red) spots that were labeled with PGP 9.5-LI are noted
(*). These findings suggest that arteries are primarily innervated with sympathetic nerves. Lumen (l), endothelium (e), and smooth muscle (sm) of vessel segment
are illustrated. Scale bar in F applies to all panels.
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were then washed overnight in PBS with subsequent addition of sheep
TH primary antibody (1:400 in PBS for 24 – 48 h at 4°C, catalog
number A-2027; Signal Transduction Products, San Clemente, CA).
The tissues were then washed again overnight in PBS at 4°C, and
TH-LI was detected by Alexa 488 secondary antibody (1:1,000 in
PBS, donkey anti-sheep, 1 h, room temperature, Molecular Probes,
green fluorescence).
For double-label immunostaining of TH and SNAP-25 in cryostat
preparations, tissues were first incubated with sheep TH primary
antibody as specified in the previous paragraph, followed by donkey
anti-sheep Alexa 488 secondary antibody for 1 h at room temperature.
The preparations were then washed in PBS for several hours before
being incubated with a monoclonal SNAP-25 primary antibody (1:
100; Synaptic Systems). SNAP-25-LI was detected by Alexa 594
secondary antibody (1:1,000 in PBS, rabbit anti-mouse, 1 h, room
temperature).
Control preparations were prepared by omitting either primary or
secondary antibodies from the incubation solutions. After being
washed with PBS, specimens were mounted on slides with Vectashield (Vector Laboratories, Burlingame, CA). Tissues were examined with a Zeiss LSM 510 Meta confocal microscope (Zeiss) with an
excitation wavelength appropriate for Alexa 488 or Alexa 594. Confocal micrographs of Z-series scans of 10 –15 optical sections through
a depth of 3– 40 ␮m were taken. Final images were constructed with
Zeiss LSM Meta software and reconstructed with the use of Adobe
Photoshop 7.0.
Chemicals. Norepinephrine hydrochloride, ␤-NAD, ␣,␤-MeATP,
and SP were all purchased from Sigma Chemical (St. Louis, MO).
RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
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Fig. 3. Exogenous neurotransmitters and neuropeptides do not evoke ␤-NAD
overflow in canine mesenteric arteries. Exogenous application of norepinephrine (NE; 3 ␮mol/l), ␣,␤-methylene ATP (␣,␤-MeATP, 1 ␮mol/l), or neuropeptide Y (NPY, 0.1 ␮mol/l) (A) or CGRP, vasoactive intestinal peptide
(VIP), and substance P (SP) (all 0.1 ␮mol/l) (B) does not significantly change
basal overflow of ␤-NAD. Data represent means ⫾ SE from 4 – 6 experiments.
Fig. 2. Electrical field stimulation (EFS; 0.3 ms, 15 V, 120 s) evokes frequencydependent overflow of ␤-NAD in canine isolated mesenteric artery. Original
chromatograms are shown from HPLC-FLD analysis of ␤-NAD in tissues
superfusates collected before (PS, prestimulation) and during nerve stimulation
at 4 and 16 Hz (0.3 ms, 120 s). Aliquots were derivatized with 2-chloroacetaldehyde (80°C, pH 4, 40 min) to 1,N6-etheno (e)-nucleotides (i.e., eATP,
eADP, eAMP) and 1,N6-etheno-adenosine (eADO). At both frequencies of
stimulation, eADP-ribose (eADPR) with elution time of ⬃11.2 min is also
observed. eADPR reflects overflow of ␤-NAD (35). Amounts of all purines are
increased with stimulation frequency.
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Sympathetic neurotransmitters NE, ATP, and NPY do not
evoke overflow of ␤-NAD. To investigate whether the classic
sympathetic neurotransmitters NE, ATP, and NPY were responsible for the release of ␤-NAD during electrical stimulation, these transmitters or their analogues were exogenously
applied to the mesenteric arteries. Figure 3A shows results
from canine isolated mesenteric arteries incubated with either
NE (3 ␮mol/l), ␣,␤-MeATP (1 ␮mol/l), or NPY (0.1 ␮mol/l).
The basal overflow of ␤-NAD in arteries incubated with NE,
␣,␤-MeATP, or NPY did not differ significantly from control
tissues, suggesting that the overflow of ␤-NAD is not induced
by NE, ATP, or NPY and hence is not mediated by activation
of ␣-adrenoceptors, purinergic receptors, or NPY receptors.
Neuropeptides CGRP, VIP, and SP do not evoke overflow of
␤-NAD. To investigate whether neuropeptides were responsible for the release of ␤-NAD during electrical stimulation,
CGRP, VIP, and SP were exogenously applied to the mesenteric arteries. CGRP (0.1 ␮mol/l), VIP (0.1 ␮mol/l), or SP (0.1
␮mol/l) did not evoke release of ␤-NAD in the superfusate
samples (Fig. 3B), suggesting that these neuropeptides (which
may have been released during EFS) could not account for the
release of ␤-NAD in this blood vessel.
Inhibition of SNAP-25 by BoNTA reduces the EFS-evoked
release of ␤-NAD. We then carried out experiments to investigate whether disruption of SNAP-25 after treatment in vitro
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Release of ␤-NAD increases with the number of stimuli.
Figure 2 shows typical chromatograms from superfusate samples collected before and during EFS of canine mesenteric
artery subjected to EFS with 0.3-ms pulse width for 120 s.
Thus the tissues were subjected to 480 and 1,920 pulses at 4 Hz
and 16 Hz, respectively. In samples subjected to ethenoderivatization and analyzed with a fluorescence detector, peaks
that correspond to eATP (elution time ⬃9.9 min), 1,N6-ethenoADP (eADP, elution time ⬃10.8 min), eADPR (elution time
⬃11.2 min), 1,N6-etheno-AMP (eAMP, elution time ⬃12.6
min), and 1,N6-etheno-adenosine (eADO, elution time ⬃16.6
min) were observed. As shown before (3), eATP, eADP,
eAMP, and eADO correspond to ATP, ADP, AMP, and ADO,
respectively. However, the eADPR peak reflects primarily the
content of ␤-NAD in the superfusate samples (35). Therefore,
the peak at 11.2 min was referred to as ␤-NAD throughout this
study. The overflow of ␤-NAD evoked by 4 and 16 Hz was
2.8 ⫾ 1.1 (n ⫽ 5) and 16.8 ⫾ 2.4 (n ⫽ 8) fmol/mg tissue,
respectively. We have previously shown that the release of
␤-NAD at 16 Hz in the canine mesenteric artery was significantly reduced by either TTX (0.3–1 ␮M) or guanethidine (3
␮mol/l) (35), suggesting that either ␤-NAD was released from
sympathetic nerve terminals or factors that are released from
these terminals evoke the release of ␤-NAD.
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RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
DISCUSSION
As aforementioned, the purinergic/adrenergic cotransmission is particularly important for the neural control of the
splanchnic circulation (e.g., Refs. 4, 5, 9, 28, 33, 37). Most
recently, we reported that in addition to NE, ATP, and the ATP
metabolites ADP, AMP, and adenosine, the tissue superfusates
collected during stimulation of postganglionic nerve terminals
in canine mesenteric artery also contain ␤-NAD along with
small amounts of the ␤-NAD metabolites ADPR and cyclic
ADPR (35). Exogenous ␤-NAD (100 nmol/l) inhibits the
evoked release of NE in this blood vessel (35), although the
full range of pre- and postjunctional actions of extracellular
␤-NAD at the vascular neuroeffector junction remains to be
elucidated. Our previous work (35) showed that activation of
sympathetic nerve terminals is critical for the release of
␤-NAD during stimulation of this blood vessel. On the basis of
these observations, we hypothesized that either ␤-NAD is
released from sympathetic nerve terminals or factors released
on stimulation of these terminals evoke the overflow of ␤-NAD
from neuronal or nonneuronal sources. The present study was
designed, therefore, to expand on these initial observations and
investigate whether the release of ␤-NAD is evoked by the
three major sympathetic neurotransmitters NE, ATP, and NPY.
In addition, we investigated whether neuropeptides that may be
released from sensory nerve afferents or autonomic nerve
terminals (e.g., CGRP, SP, and VIP) would induce the release
of ␤-NAD. Finally, we investigated whether the evoked release
of ␤-NAD is through stimulus-dependent exocytosis of synaptic vesicles associated with SNAP-25.
Consistent with previous investigations in peripheral blood
vessels, double-labeling immunofluorescence and confocal microscopy revealed that the majority of nerve terminals at the
adventitial layer of the canine mesenteric artery contain TH-LI
and hence are adrenergic/sympathetic in nature. A small portion of the PGP 9.5-positive nerve fibers was negative for
TH-LI, suggesting that a small population of other nerve types
(possibly sensory afferents) (22) is also present in this blood
vessel. Exogenous NE, NPY, and ␣,␤-MeATP did not increase
the basal amounts of ␤-NAD in the superfusate samples.
Fig. 4. Whole mount and cryostat sections
of canine mesenteric artery labeled with synaptosomal-associated protein of 25 kDa
(SNAP-25)-like immunoreactivity (SNAP25-LI) or TH-LI. SNAP-25-LI shows network-like distribution (A, arrows, Alexa
594). TH-LI (B, arrows, Alexa 488) and
SNAP-25-LI (C, arrows, Alexa 594) were
found on the adventitial side (a) of vessel.
Double label revealed identical distribution
of TH-LI and SNAP-25-LI (D, yellow pixels). Lumen (l), endothelium (e), and smooth
muscle (sm) of vessel segment are illustrated. Scale bar in D applies to B, C, and D.
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with BoNTA (100 nmol/l) will affect the EFS-evoked release
of ␤-NAD. Indeed, SNAP-25-LI showed neural network-like
distribution in a whole mount preparation of canine mesenteric
artery (Fig. 4A). Cryostat sections revealed that SNAP-25 was
located within the adventitial layer of the mesenteric artery
(Fig. 4B) and was largely colocalized with TH-LI (Fig. 4, C
and D) in this blood vessel. Incubation of artery segments with
RPMI-1640 for 5.5 h did not significantly affect the EFSevoked release of NE, ATP, and ␤-NAD. However, incubation
of artery segments with BoNTA (100 nmol/l for 5.5 h) caused
significant reduction of the EFS (16 Hz, 0.3 ms, 120 s)-evoked
release of ␤-NAD and ATP (Fig. 5, A, D, and F) as well as of
NE (Fig. 5, B and C) in this blood vessel (n ⫽ 6; P ⬍ 0.05),
suggesting that disruption of SNAP-25 by BoNTA significantly affects the EFS-evoked release of NE, ATP, and
␤-NAD. Thus, in BoNTA-treated tissue segments, the evoked
release of NE, ATP, and ␤-NAD was reduced by ⬃90, 60, and
80%, respectively. The evoked release of NE, ATP, and
␤-NAD at 4 Hz of EFS (0.3 ms, 120 s) was abolished after
BoNTA treatment from 5.9 ⫾ 0.8, 1.0 ⫾ 0.1, and 2.8 ⫾ 1.1
fmol/mg tissue NE, ATP, and ␤-NAD, respectively, to undetectable amounts of the three compounds (n ⫽ 3).
RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
H1823
Likewise, exogenous CGRP, VIP, or SP (factors that are
commonly released from sensory fibers) did not evoke ␤-NAD
overflow. Therefore, in the canine mesenteric artery, the release of ␤-NAD is not induced by the action of the classic
sympathetic cotransmitters NE, ATP, and NPY or by the action
of the neuropeptides CGRP, VIP, and SP.
The EFS-evoked release of ␤-NAD in the canine mesenteric
artery increases with the number of stimuli (the present study),
as does the EFS-evoked release of NE (5, 28, 35) and of ATP
(5, 35). The release of all three, NE, ATP, and ␤-NAD, is
significantly reduced by TTX and guanethidine. Furthermore,
the evoked release of both NE and ␤-NAD is significantly
reduced by 6-hydroxydopamine and the selective blocker of
neuronal Cav2.2 (N-type) voltage-operated Ca2⫹ channels
(VOCC) ␻-conotoxin GVIA (35). We conclude, therefore, that
AJP-Heart Circ Physiol • VOL
in the canine mesenteric artery, ␤-NAD is primarily released
from postganglionic sympathetic nerve terminals via a mechanism that depends on the influx of Ca2⫹ through neuronal
VOCC.
The VOCC of the nerve terminal are intimately related to
one group of proteins, the soluble N-ethylmaleimide-sensitive
factor attachment protein receptors (SNAREs), which are involved in the process of synaptic vesicle mobilization (11, 18,
36) in response to the rise in intracellular Ca2⫹ after an action
potential to generate transmitter release (18, 32). The target
membrane-associated SNARE protein SNAP-25 in particular
is associated with the synaptic plasma membrane and is believed to be primarily involved in exocytosis from small
vesicles but not large vesicles (24). SNAREs, including SNAP25, may not be expressed uniformly in all populations of
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Fig. 5. Botulinum neurotoxin A (BoNTA) significantly reduced nerve-evoked release of NE, ATP, and ␤-NAD in canine mesenteric arteries. A: original
chromatograms showing release of ATP (detected as eATP) and ␤-NAD (detected as eADPR) in control (top) and BoNTA (100 nmol/l)-treated tissues (bottom)
in response to EFS at 16 Hz, 0.3 ms, for 120 s. B: original chromatograms showing release of NE in control (top) and BoNTA (100 nmol/l)-treated tissues
(bottom) in response to EFS at 16 Hz, 0.3 ms, for 120 s. EFS-evoked release of NE (C), ATP (D), and ␤-NAD (E) is significantly reduced in BoNTA-treated
tissues. Data represent means ⫾ SE from 6 experiments. *Significant differences from controls; P ⬍ 0.05.
H1824
RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-60031.
AJP-Heart Circ Physiol • VOL
REFERENCES
1. Bark IC, Hahn KM, Ryabinin AE, and Wilson MC. Differential
expression of SNAP-25 protein isoforms during divergent vesicle fusion
events of neural development. Proc Natl Acad Sci USA 92: 1510 –1514,
1995.
2. Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, De Camilli P,
Sudhof TC, Niemann H, and Jahn R. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365: 160 –163, 1993.
3. Bobalova J, Bobal P, and Mutafova-Yambolieva VN. High-performance liquid chromatographic technique for detection of a fluorescent
analogue of ADP-ribose in isolated blood vessel preparations. Anal Biochem 305: 269 –276, 2002.
4. Bobalova J and Mutafova-Yambolieva VN. Co-release of endogenous
ATP and noradrenaline from guinea-pig mesenteric veins exceeds corelease from mesenteric arteries. Clin Exp Pharmacol Physiol 28: 397–
401, 2001.
5. Bobalova J and Mutafova-Yambolieva VN. Presynaptic alpha2-adrenoceptor-mediated modulation of adenosine 5⬘ triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein. J Auton
Pharmacol 21: 47–55, 2001.
6. Brock JA and Cunnane T. Electrophysiology of neuroeffector transmission in smooth muscle. In: Autonomic Neuroeffector Mechanisms, edited
by Burnstock G and Hoyle CHV. Chur, Switzerland: Harwood, 1992, p.
121–213.
7. Burnstock G. The fifth Heymans memorial lecture-Ghent, February 17,
1990. Co-transmission. Arch Int Pharmacodyn Ther 304: 7–33, 1990.
8. Burnstock G. Cotransmission. Curr Opin Pharmacol 4: 47–52, 2004.
9. Donoso MV, Steiner M, and Huidobro-Toro JP. BIBP 3226, suramin
and prazosin identify neuropeptide Y, adenosine 5⬘-triphosphate and
noradrenaline as sympathetic cotransmitters in the rat arterial mesenteric
bed. J Pharmacol Exp Ther 282: 691– 698, 1997.
10. Dunn WR, Brock JA, and Hardy TA. Electrochemical and electrophysiological characterization of neurotransmitter release from sympathetic
nerves supplying rat mesenteric arteries. Br J Pharmacol 128: 174 –180,
1999.
11. Gerst JE. SNAREs and SNARE regulators in membrane fusion and
exocytosis. Cell Mol Life Sci 55: 707–734, 1999.
12. Gibbins IL and Morris JL. Pathway specific expression of neuropeptides
and autonomic control of the vasculature. Regul Pept 93: 93–107, 2000.
13. Hattori Y, Nagashima M, Endo Y, and Kanno M. Glibenclamide does
not block arterial relaxation caused by vasoactive intestinal polypeptide.
Eur J Pharmacol 213: 147–150, 1992.
14. Ishikawa H, Mitsui Y, Yoshitomi T, Mashimo K, Aoki S, Mukuno K,
and Shimizu K. Presynaptic effects of botulinum toxin type A on the
neuronally evoked response of albino and pigmented rabbit iris sphincter
and dilator muscles. Jpn J Ophthalmol 44: 106 –109, 2000.
15. Kawasaki H, Nuki C, Saito A, and Takasaki K. Adrenergic modulation
of calcitonin gene-related peptide (CGRP)-containing nerve-mediated vasodilation in the rat mesenteric resistance vessel. Brain Res 506: 287–290,
1990.
16. Levitt B, Head RJ, and Westfall DP. High-pressure liquid chromatographic-fluorometric detection of adenosine and adenine nucleotides:
application to endogenous content and electrically induced release of
adenyl purines in guinea pig vas deferens. Anal Biochem 137: 93–100,
1984.
17. Li J and Wang DH. Development of angiotensin II-induced hypertension: role of CGRP and its receptor. J Hypertens 23: 113–118, 2005.
18. Lin RC and Scheller RH. Mechanisms of synaptic vesicle exocytosis.
Annu Rev Cell Dev Biol 16: 19 – 49, 2000.
19. Luff SE, Young SB, and McLachlan EM. Ultrastructure of substance
P-immunoreactive terminals and their relation to vascular smooth muscle
cells of rat small mesenteric arteries. J Comp Neurol 416: 277–290, 2000.
20. Lundberg JM and Hokfelt T. Multiple co-existence of peptides and
classical transmitters in peripheral autonomic and sensory neurons–functional and pharmacological implications. Prog Brain Res 68: 241–262,
1986.
21. MacKenzie I, Burnstock G, and Dolly JO. The effects of purified
botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission. Neuroscience 7: 997–1006, 1982.
22. Morris JL, Gibbins IL, Kadowitz PJ, Herzog H, Kreulen DL, Toda N,
and Claing A. Roles of peptides and other substances in cotransmission
290 • MAY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on July 31, 2017
neurons (1, 26, 31). In the canine mesenteric artery, SNAP-25
appears entirely colocalized with TH-LI, suggesting that
SNAP-25 may be particularly important for the release of
neurotransmitters from the postganglionic sympathetic nerve
terminals in this blood vessel. BoNTA cleaves and inactivates
SNAP-25 (2). NE and ATP are stored in and released from
both small and large synaptic vesicles (6, 38). It has been
proposed that the release of neurotransmitters at low frequencies of stimulation represents exocytosis from small vesicles,
whereas at higher frequencies of stimulation, large vesicles
also contribute to the neurotransmitter release (23).
The regime of BoNTA treatment used in the present study
was based on previous functional studies (14, 21, 23, 24) of
autonomic neurons showing that BoNTA incubated in the
nanomolar range with whole tissues for several hours at 37°C
produced substantial reduction or abolition of neurotransmission from some types of neurons. In the present study, the
release of NE, ATP, and ␤-NAD was abolished by BoNTA at
a low number of electrical stimuli, suggesting that at low levels
of activation, NE, ATP, and ␤-NAD are released preferentially
from a population of synaptic vesicles that are associated with
SNAP-25. This population of synaptic vesicles likely represents the small vesicles (24, 25). At a higher number of
electrical stimuli, however, the evoked release of NE, ATP,
and ␤-NAD was reduced by ⬃90, 60, and 80%, respectively.
Thus, at high levels of activation, ⬃20% of ␤-NAD remained
unaffected by BoNTA. Several possible explanations may be
considered: 1) BoNTA did not completely inhibit the SNAP25-mediated exocytosis; 2) the evoked release of ␤-NAD at 4
Hz was abolished by BoNTA because of the relatively low
number of electrical stimuli, whereas increasing the number of
stimuli resulted only in partial block of ␤-NAD⫹ release; or 3)
the component of ␤-NAD⫹ release that remained unaffected
by BoNTA may originate either from a second population of
synaptic vesicles insensitive to BoNTA (e.g., large vesicles) or
from a population of nerve terminals that do not contain
SNAP-25 (e.g., sensory nerves) (25). Further studies are warranted to define the exact mechanism underlying the small
component of ␤-NAD⫹ release that was insensitive to BoNTA.
In conclusion, this study has demonstrated that ␤-NAD
released on activation of sympathetic nerve terminals in the
canine isolated mesenteric artery is due to direct release of
␤-NAD from nerve terminals rather than evoked by the sympathetic cotransmitters NE, ATP, and NPY or the neuropeptides CGRP, VIP, and SP. The release of ␤-NAD, NE, and
ATP from sympathetic postganglionic nerve terminals at a low
level of nerve activation may result from SNAP-25-associated
synaptic vesicles. At higher levels of neural activity, ␤-NAD
may be released from both BoNTA-sensitive and BoNTAinsensitive stores. At higher levels of nerve activation, extracellular ␤-NAD appears released in greater amounts together
with the excitatory neurotransmitters NE and ATP to perhaps
prevent extreme excitation. Therefore, ␤-NAD constitutes a
novel extracellular factor that may contribute to autonomic
neurovascular control in the mesentery.
RELEASE OF ␤-NAD FROM SYMPATHETIC NERVES VIA EXOCYTOSIS
23.
24.
25.
26.
27.
28.
30.
31.
AJP-Heart Circ Physiol • VOL
32. Seagar M, Leveque C, Charvin N, Marqueze B, Martin-Moutot N,
Boudier JA, Boudier JL, Shoji-Kasai Y, Sato K, and Takahashi M.
Interactions between proteins implicated in exocytosis and voltage-gated
calcium channels. Philos Trans R Soc Lond B Biol Sci 354: 289 –297,
1999.
33. Smyth L, Bobalova J, Ward SM, Keef KD, and Mutafova-Yambolieva
VN. Cotransmission from sympathetic vasoconstrictor neurons: differences in guinea-pig mesenteric artery and vein. Auton Neurosci 86: 18 –29,
2000.
34. Smyth L, Bobalova J, Ward SM, and Mutafova-Yambolieva VN.
Neuropeptide Y is a cotransmitter with norepinephrine in guinea pig
inferior mesenteric vein. Peptides 21: 835– 843, 2000.
35. Smyth LM, Bobalova J, Mendoza MG, Lew C, and Mutafova-Yambolieva VN. Release of beta-nicotinamide adenine dinucleotide upon
stimulation of postganglionic nerve terminals in blood vessels and urinary
bladder. J Biol Chem 279: 48893– 48903, 2004.
36. Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, and Rothman JE. SNAP receptors implicated in
vesicle targeting and fusion. Nature 362: 318 –324, 1993.
37. Starke K, Bultmann R, Bulloch JM, and von Kugelgen I. Noradrenaline-ATP corelease and cotransmission following activation of nicotine
receptors at postganglionic sympathetic axons. J Neural Transm Suppl 34:
93–98, 1991.
38. Stjarne L. Basic mechanisms and local modulation of nerve impulseinduced secretion of neurotransmitters from individual sympathetic nerve
varicosities. Rev Physiol Biochem Pharmacol 112: 1–137, 1989.
39. Todorov LD, Mihaylova-Todorova S, Craviso GL, Bjur RA, and
Westfall DP. Evidence for the differential release of the cotransmitters
ATP and noradrenaline from sympathetic nerves of the guinea-pig vas
deferens. J Physiol 496: 731–748, 1996.
290 • MAY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on July 31, 2017
29.
from vascular autonomic and sensory neurons. Can J Physiol Pharmacol
73: 521–532, 1995.
Morris JL, Jobling P, and Gibbins IL. Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator
neurons. Am J Physiol Heart Circ Physiol 281: H2124 –H2132, 2001.
Morris JL, Jobling P, and Gibbins IL. Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons.
Am J Physiol Heart Circ Physiol 283: H2627–H2635, 2002.
Morris JL, Konig P, Shimizu T, Jobling P, and Gibbins IL. Most
peptide-containing sensory neurons lack proteins for exocytotic release
and vesicular transport of glutamate. J Comp Neurol 483: 1–16, 2005.
Morris JL, Lindberg CE, and Gibbins IL. Different levels of immunoreactivity for synaptosomal-associated protein of 25 kDa in vasoconstrictor and
vasodilator axons of guinea-pigs. Neurosci Lett 294: 167–170, 2000.
Mutafova-Yambolieva VN, Carolan BM, Harden TK, and Keef KD.
Multiple P2Y receptors mediate contraction in guinea pig mesenteric vein.
Gen Pharmacol 34: 127–136, 2000.
Mutafova-Yambolieva VN, Smyth L, and Bobalova J. Involvement of
cyclic AMP-mediated pathway in neural release of noradrenaline in canine
isolated mesenteric artery and vein. Cardiovasc Res 57: 217–224, 2003.
Mutafova-Yambolieva VN and Westfall DP. Inhibitory and facilitatory
presynaptic effects of endothelin on sympathetic cotransmission in the rat
isolated tail artery. Br J Pharmacol 123: 136 –142, 1998.
Ralevic V, Karoon P, and Burnstock G. Long-term sensory denervation
by neonatal capsaicin treatment augments sympathetic neurotransmission
in rat mesenteric arteries by increasing levels of norepinephrine and
selectively enhancing postjunctional actions. J Pharmacol Exp Ther 274:
64 –71, 1995.
Safieddine S and Wenthold RJ. SNARE complex at the ribbon synapses
of cochlear hair cells: analysis of synaptic vesicle- and synaptic membrane-associated proteins. Eur J Neurosci 11: 803– 812, 1999.
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