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1 of 30 in PresS. Am J Physiol Gastrointest Liver Physiol (February 22, 2007). doi:10.1152/ajpgi.00450.2006
Articles
1
Inhibitory Neuromuscular Transmission Mediated by the P2Y1
Purinergic Receptor in Guinea-Pig Small Intestine
Guo-Du Wang, Xi-Yu Wang, Hong-Zhen Hu¶, Sumei Liu, Na Gao§, Xiucai Fang*, Yun
Xia, and Jackie D. Wood
Department of Physiology and Cell Biology
The Ohio State University College of Medicine, Columbus, Ohio 43210
Running title: Purinergic P2Y1 Neuromuscular Transmission
Present address: *Division of Gastroenterology
Peking Union Medical College Hospital
Beijing, Peoples’ Republic of China
¶Scripps Research Institute
La Jolla, CA, USA
§ Senomyx, Inc.
La Jolla, CA 92037
Correspondence:
Jackie D. Wood, Ph.D.
Department of Physiology and Cell Biology
The Ohio State University,
College of Medicine and Public Health,
304 Hamilton Hall, 1645 Neil Avenue,
Columbus, OH 43210,
USA
Tel. 1-614-292-5449
Fax 1-614-292-4888
Email: [email protected]
Copyright © 2007 by the American Physiological Society.
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ABSTRACT
ATP is a putative inhibitory neurotransmitter responsible for inhibitory junction
potentials at neuromuscular junctions (IJPs) in the intestine. This study tested the
hypothesis that the purinergic P2Y1 receptor subtype mediates the IJPs. IJPs were
evoked by focal electrical stimulation in the myenteric plexus and recorded with “sharp”
intracellular microelectrodes in the circular muscle coat. Stimulation evoked three
categories of IJPs: (1) Purely purinergic IJPs were suppressed by the selective P2Y1
purinergic receptor antagonist, MRS2179. Purely purinergic IJPs comprised 26% of the
IJPs. (2) Partially purinergic IJPs (72% of the IJPs) consisted of a component that was
abolished by MRS2179 and a second unaffected component. The MRS2179-insensitive
component was suppressed or abolished by inhibition of formation of nitric oxide by LNAME in some, but not all IJPs. An unidentified neurotransmitter, different from nitric
oxide, mediated the second component in these cases. (3) Non-purinergic IJPs were a
small third category (4%) of IJPs, which were abolished by L-Name and unaffected by
MRS2179. Exogenous application of ATP evoked IJP-like hyperpolarizing responses,
which were blocked by MRS2179. Application of apamin, which suppresse opening of
small conductance Ca2+-operated K+ channels in the muscle, decreased the amplitude of
the purinergic IJPs and the amplitude of IJP-like responses to ATP. The results support
ATP as a neurotransmitter for IJPs in the intestine and are consistent with the hypothesis
that the P2Y1 purinergic receptor subtype mediates the action of ATP.
Key Words: ATP; neurotransmission; enteric nervous system, intestinal motility, smooth
muscle
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Introduction
Inhibition of the circular musculature ahead of the advancing bolus is an essential
component of peristaltic propulsion in the small and large intestine. This component of
the organization of propulsive motility is organized by the enteric nervous system (ENS)
and reflects elevated activity in the inhibitory motor innervation of the musculature.
Evidence in 1963 first implicated ATP as one of the inhibitory neurotransmitters at
neuromuscular junctions in the intestine (9, 10). This role for ATP has been confirmed
repeatedly in subsequent years (10, 11, 12).
Inhibitory junction potentials (IJPs) were first reported for the guinea-pig taenia coli
by Burnstock et al. in 1963 (9) and analyzed in more detail in 1966 (4). Excitation of
ENS inhibitory motor neurons by transmural electrical field stimulation or by application
of nicotinic receptor agonists is now commonly known to evoke IJPs, which are readily
detected with intracellular electrophysiological recording methods, in intestinal circular
muscle. In human, guinea-pig, dog and mouse preparations the IJPs are generally
biphasic and consist of an initial large amplitude and rapidly-activating component (fast
IJP) followed by a smaller and more slowly-activating component (slow IJP). In human,
guinea-pig and mouse preparations the slow IJP is abolished by treatments that block the
synthesis of nitric oxide and is mimicked by exogenous application of nitric oxide (3, 22,
33, 36,), which is evidence that the late slow IPSP reflects the release of nitric oxide from
inhibitory motor neurons. Inhibition of nitric oxide formation does not alter the initial fast
IJP in human, guinea-pig or mouse intestinal circular muscle (25, 36, 44). This suggests
that the fast IJP is not mediated by release of nitric oxide.
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In guinea-pig small intestinal circular muscle, the fast IJP and the fast IJP-like
action of ATP are suppressed by apamin, suramin, PPADS, reactive blue-2 and
desensitization of post receptor signal transduction by ADP S (16, 22, 30, 44). Reactive
blue-2, which is a P2 receptor antagonist, suppresses hyperpolarizing responses evoked
by the stable ATP analog, , -methylene ATP, in strips of circular muscle from rat
caecum (30). On the other hand, , -methylene ATP does not bind to P2Y1 receptors
cloned from chick brain (41), which suggests that a second P2Y receptor might be
present in rat intestine. In guinea-pig taenia coli, purine nucleotides evoke muscle
relaxation with a potency order of diadenosine polyphosphate P1,P3-diadenosine
triphosphate Ap3A = diadenosine polyphosphate P1,P4-diadenosine tetraphosphate Ap4A
> ATP > diadenosine polyphosphate P1,P4-diadenosine tetraphosphate Ap4A >
diadenosine polyphosphate P1,P5-diadenosine pentaphosphate Ap5A and these actions are
suppressed by suramin with a pA2 value of ~5, which is suggestive of involvement of the
P2Y1 receptor subtype (23). Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid
(PPADS) also acts as an antagonist at the P2Y1 receptor with potency similar to reactive
blue 2 and suramin (28, 29). In guinea-pig taenia coli, PPADS shifts the concentrationresponse curve for stimulus-evoked inhibitory junction potentials and IJP-like actions of
ATP to the left, which suggests that P2Y1 receptors are expressed in this intestinal muscle
(42).
Neurally-evoked excitatory junction potentials (EJPs), which appear to be
purinergic, have been reported for the guinea-pig taenia coli (34, 46). The EJPs are
unmasked by inhibition of the IJPs by apamin and are suppressed by desensitization with
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, -methylene ATP, and by PPADS and suramin. On the other hand, the EJPs are
unaffected by selective P2Y1 antagonist, MRS-2179.
The hyperpolarizing component of the fast IJP and the fast IJP-like action of
ATP reflect activation of outward membrane current that is carried by small-conductance
Ca2+-activated K+ channels (39, 26). The post receptor signal transduction cascade for
this action of ATP in myocytes obtained from guinea-pig taenia coli involves stimulation
of phospholipase C, release of inositol trisphosphate and elevation of free Ca2+ inside the
smooth muscle fibers (27). On the other hand, in the mouse colon, apamin-sensitive
hyperpolarization induced by activation of P2Y receptors is reported to be mediated
primarily by stimulation of adenylate cyclase and release of Ca2+ from intracellular
ryanodine-dependent stores (47). Suppression of both the fast IJP and the fast IJP-like
action of ATP by apamin reflects inhibition of opening of the Ca2+-activated K+ channels
in the muscle membranes (1, 38). Suramin, PPADS and reactive blue-2 are marginally
selective antagonists at the P2 purinergic receptor subtype and suppression of both the
fast IJP and the fast IJP-like action of ATP by these drugs implicates involvement of this
receptor category in the generation of the fast IPSP and the fast IPSP-like action of ATP
(14, 17).
Lack of availability, until recently, of selective antagonists for P2 purinergic
receptor subtypes has impeded progress toward unequivocal identification of the receptor
responsible for the fast IJP and the fast IJP-like action of ATP. Recent evidence reported
by M. Jiménez and co-workers in Barcelona strongly implicates the P2Y1 as the
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purinergic receptor responsible for the fast IJP and fast IJP-like action of ATP for the
circular muscle coat of human distal and sigmoid colon (21).
The most potent and selective P2Y1-receptor antagonist available to date is
MRS2179, which is the bisphosphate derivative 2’-deoxy-N 6 -methyladenosine-3’, 5’bisphosphate (13, 31). It is a competitive antagonist at the turkey P2Y1 receptor with a
pA2 -value of 6.99. Moreover, it is ineffective at the human P2Y2 , the human P2Y4 and
the rat P2Y6 receptor (4). MRS2179 has an apparent pKB value of 6.75 at the human
P2Y1 receptor and a pA2 value of 6.18 for inhibition of the mimicry of purinergic slow
excitatory postsynaptic potentials by ATP in neurons in the guinea-pig small intestinal
submucosal plexus (24).
The general aim of our study was to test the hypothesis that the fast IJP and the
fast IJP-like action of exogenously applied ATP in guinea-pig small intestinal circular
muscle reflect stimulation of the P2Y1 purinergic receptor. A preliminary report has been
published in abstract form (40).
Materials and Methods
Adult male Hartley-strain guinea-pigs (300-350 g) were stunned by a sharp blow
to the head and exsanguinated from the cervical vessels according to protocols approved
by the Ohio State University Laboratory Animal Care and Use Committee and United
States Department of Agriculture Veterinary Inspectors. Whole-mount preparations were
obtained from mid-jejunum and ileum. The segments were microdissected for
electrophysiological recording as described earlier (20, 45).
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Conventional intracellular electrophysiological recording methods with “sharp”
glass microelectrodes were used to record stimulus-evoked IJPs in the circular muscle
coat of mucosa-free preparations that had strips of longitudinal muscle removed to
expose the myenteric plexus. The preparations were pinned to Sylgard resin at the
bottom of a 2.0-ml recording chamber that was perfused at a rate of 10-15 ml min-1 with
Krebs solution warmed to 37 °C and gassed with 95% O2 / 5% CO2 to buffer at pH 7.37.4. The composition of the Krebs solution was (mM): 120 NaCl, 6 KCl, 2.5 CaCl2, 1.2
MgCl2, 1.35 NaH2PO4, 14.4 NaHCO3, and11.5 glucose. Nifedipine and scopolamine (1
µM) were added to the Krebs solution to suppress muscle movements during
electrophysiological recording. The microelectrodes were filled with 2 M KCl or 4 M
potassium acetate and had resistances of 80-120 M . The preamplifier (M-767, World
Precision Instruments, Sarasota, FL) was equipped with a bridge circuit for intraneuronal
injection of electrical current. Constant current rectangular pulses were driven by a Grass
SD9 stimulator (Grass Instrument Division, Astro-Med, Inc., Warwick, RI). Electrometer
output was amplified and observed on oscilloscopes (Tektronics 3012, Tektronics,
Beaverton, OR) and Astro-Med thermal recorders (Astro-Med, Inc., Warwick, RI) and
saved on digital recording tape. Neuro-muscle junction potentials were evoked by focal
electrical stimulation of interganglionic fiber tracts in the myenteric plexus with bipolar
insulated tungsten stimulating electrodes placed perpendicular to the longitudinal axis of
the preparation and connected through stimulus-isolation units (Grass SIN5) to Grass S48
stimulators (Grass Instrument Division, Astro-Med, Inc., Warwick, RI). Stimulus
parameters were single pulses with durations of 2 ms and amplitudes of 2 mA.
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Pharmacological agents were applied by addition to the bathing solution. Agents
used were: ATP, L-NAME (N -Nitro-L-arginine methyl ester hydrochloride),
scopolamine, nifedipine, apamin, vasoactive intestinal peptide (VIP), Tetrodotoxin
(TTX) and VIP6-28 each of which were obtained from Sigma (St. Louis, MO). MRS2179
(2’-Deoxy-N6-methyadenosine 3’, 5’-bisphosphate tetraammonium salt), TNP-ATP
(2’,3’-O-(2,4,6-Trinitrophenyl)adenosine-5’-triphosphate) and suramin were obtained
from Tocris Bioscience (Ellisville, MO). PPADS (pyridoxal phosphate-6-azo(benzene2,4-disulfonic acid) was purchased from RBI (Natick, MA).
Data analysis
Data are presented as means ± S.E.M. with n values referring to numbers of muscle cells.
Continuous curves for concentration- response relationships were constructed with the
following least-squares fitting routine using Sigmaplot software (SPSS Inc., Chicago,
IL): V = Vmax/[1 + (EC50/C)nH]. Where V is the observed membrane potential response,
Vmax is the maximal response, C is the corresponding drug concentration, EC50 is the
concentration that induces the half -maximal response, and nH is the apparent Hill
coefficient. Student's t test was used to determine significance with P < 0.05 considered
to be significant.
Results
Focal electrical stimulation applied to ganglia or interganglionic fiber tracts in the
myenteric plexus evoked IJPs of variable amplitude and duration and excitatory junction
potentials EJPs in the circular muscle (Figs. 1, 2, 3, 4). EJPs were not always apparent.
When apparent, they were seen as transient membrane depolarization beyond the resting
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potential, which occurred at the end of the IJPs (Fig. 3). We identified three kinds of
IJPs, which will be referred to as purely purinergic, partially purinergic and nonpurinergic. Purely purinergic IJPs were identified as those abolished by the selective
P2Y1 purinergic receptor antagonist, MRS2179 (Fig. 2). Partially purinergic IJPs were
composed of a MRS2179-sensitive component and a non-MRS2179-sensitive
component. The non-MRS2179-sensitive component was sometimes, but not always,
suppressed by inhibition of nitric oxide formation. Table 1 provides data for the
proportion of IJPs in the study that were purely purinergic, partially purinergic or non
purinergic in the jejunum and ileum. All kinds of stimulus-evoked junction potentials
were suppressed or abolished by TTX.
Purely Purinergic IJPs
Purely purinergic IJPs made-up 26% of the IJPs recorded in the study (Table 1).
These IJPs were abolished by a sufficient concentration of MRS2179 (Figs. 2AB). In
lower concentrations, MRS2179 acted in concentration-dependent manner, with an IC50
of 0.19 ± 0.01 µM, to suppress the amplitude and duration of the purely purinergic IJPs
(Figs. 1B, 2AB). Apamin, which is an agent that suppresses opening of small
conductance Ca2+-activated K+ channels in intestinal smooth muscle (39), also reduced
the amplitude of purely purinergic IJPs concentration-dependently with an IC50 of 0.03 ±
0.01 µM (Figs. 1C, 2AB). Suramin, a non-selective antagonist at P2 receptor subtypes,
also suppressed or abolished the purely purinergic IJPs (Figs. 2AB). Putative inhibition of
P2X1 and P2X3 purinergic receptors with the selective high-affinity antagonist, TNP-ATP
(37, 32) did not alter the purely purinergic IJPs (Fig. 2AB). The purely purinergic IJPs
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were likewise unaffected by a selective VIP receptor antagonist (VIP6-28) or by inhibition
of nitric oxide synthase by L-NAME (Figs. 2AB).
Partially Purinergic IJPs
Partially purinergic IJPs made-up 70% of the IJPs recorded in the study (Table
1). These IJPs were suppressed, but not abolished by MRS2179 (Figs. 1, 4, 5). Of 72
partially purinergic IJPs, 33% showed a hyperpolarizing component in the presence of
MRS2179 that was suppressed after addition of L-NAME to the bathing medium (Fig. 4).
This component activated more slowly and had smaller amplitude relative to the
MRS2179-sensitive component. Neither component occurred when a combination of
sufficient concentrations of MRS2179 and L-Name was present in the bathing solution
(Fig. 4). These IJPs fit into a two-component subcategory of partially purinergic IJPs,
comprised of a purinergic (fast) and nitrergic (slow) component.
Neither putative blockade of small conductance Ca2+-activated K+ channels by
apamin, blockade of P2X receptors by TNP-ATP, nor VIP receptor blockade by VIP6-28
suppressed the small-L-NAME-sensitive component of the IJP (Fig. 4AB).
The remainder of the partially purinergic IJPs also included the second small
hyperpolarizing component; however, this component was resistant to suppression by a
combination of MRS2179 and L-NAME. Neither putative blockade of small conductance
Ca2+-activated K+ channels by apamin, blockade of P2X receptors by TNP-ATP, nor VIP
receptor blockade by VIP6-28 suppressed the small-L-NAME-sensitive component of the
IJP (Fig. 5AB).
Non-purinergic IJPs
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Non-purinergic IJPs made-up 4% of the IJPs recorded in the study (Table 1). The
non-purinergic IJPs were suppressed or abolished by L-NAME (Figs. 3AB). Unlike the
purely purinergic IJPs, the non-purinergic IJPs were unaffected by the presence of
apamin, TNP-ATP, MRS21792 or VIP6-28 in the bathing solution (Figs. 3AB).
IJP-like Action of ATP
IJP-like membrane hyperpolarization was evoked by ATP (10-30 µM) in 49 of 56
preparations (Fig. 6). These hyperpolarizing responses to exogenously applied ATP were
concentration-dependent, reversible and mimicked the IJPs in the “purely purinergic”
category. The EC50 for the IJP-like action of ATP was 6.6 ± 1.3 µM (Fig. 7). The IJP-like
responses to 10-µM ATP were suppressed by: 1) 92±15% in the presence of 10-µM
MRS2179 in 14 preparations; 2) 95±12% in the presence of 1-µM apamin in 6
preparations; 3) 76±8% in the presence of 20-µM PPADS in 9 preparations. The IJP-like
responses, evoked by ATP, were not influenced by 10-µM TNP-ATP in 14 preparations,
1-µM TTX in 25 preparations or 200-µM L-NAME in 18 preparations. Exposure to VIP
(100 nM -1 µM) did not evoke hyperpolarizing responses in any of 19 preparations
(Figure 6).
Discussion
The properties of neuromuscular junction potentials evoked by focal electrical
stimulation in the myenteric plexus in our study were generally similar to those reported
for several earlier studies in which the potentials were evoked by transmural electrical
field stimulation (4, 16, 21, 25, 33, 36, 38, 44). The waveforms of the IJPs evoked by
transmural electrical field stimulation in these studies were described as consisting of an
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initial larger amplitude, rapidly-activating hyperpolarizing component (fast IJP) and a
smaller and longer-lasting hyperpolarizing component (slow IJP). We found that both
components were also evoked by focal electrical stimulation of neurons in the myenteric
plexus, where the cell bodies of motor neurons to the circular muscle coat reside (7, 8).
Nevertheless, the slow IJP was often obscured by the larger fast IJP and was uncovered
after blockade of the fast IJP by MRS2179.
Focal electrical stimulation in the myenteric plexus also activated excitatory
motor neurons to the circular muscle and this was reflected by stimulus-evoked EJPs.
These EJPs occurred in the presence of scopolamine and were therefore non-cholinergic.
Because the simultaneously-evoked IJPs usually overpowered the EJPs, the longerlasting EJPs were seen generally at the end of the IJPs.
The amplitudes of the three kinds of neuromuscular junction potentials were
variable and did not lend themselves to quantitative analysis. Several factors might
account for the variability. One factor might be the number of motor neurons brought to
firing threshold by the stimulus and a direct association between the number of activated
neurons and the amplitude of the evoked IJP. A second factor might be the frequency of
evoked firing of the motor neurons because the amount of neurotransmitter released at
the neuromuscular junctions occurs in direct relation to the firing frequency. A third
factor relates to the possibility for the junction potentials to be occurring in muscle fibers
at distances removed from the fiber impaled by the microelectrode. The smooth muscle
behaves as a functional electrical syncytium due to electrical coupling between individual
fibers. Consequently, junction potentials can spread electrotonically with passive
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decrement from neuromuscular junctions at distant muscle fibers to the fiber from which
an electrical record is obtained (2, 6).
Purely Purinergic IJPs
The purely purinergic IJPs in our study appear to be the same as the fast IJPs in
the reports of others (16, 21, 22, 25, 33, 36, 44). Occurrence of a purely purinergic IJP is
suggestive of a population of inhibitory motor neurons in the myenteric plexus, which is
exclusively purinergic. The potent suppression of the purely purinergic IJPs by
MRS2179, considered together with the high selectivity and affinity of MRS2179 for the
P2Y1 purinergic receptor subtype, strongly suggests that the purinergic component of
IJPs (i.e., fast IJPs) in guinea-pig small intestinal circular muscle is mediated by the P2Y1
receptor subtype. This conclusion was reinforced by our finding that exogenous ATP
mimicked the purely purinergic (i.e., fast IJP) and that the IJP-like action of ATP was
suppressed by MRS2179.
Partially Purinergic IJPs
The IJPs, which we called partially purinergic, probably corresponded to the slow
IJPs that have been reported by others. The partially purinergic IJPs might reflect the
simultaneous stimulation of one or more inhibitory motor neurons that release ATP and
one or more that release another inhibitory neurotransmitter. On the other hand, there
might be a population of inhibitory motor neurons in the myenteric plexus that co-release
ATP together with a second non-purinergic neurotransmitter. Our results do not
differentiate between these two possibilities. Nevertheless, our results show suppression
of the second component of some of the partially purinergic IJPs by L-NAME, which
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suggests nitric oxide as the inhibitory neurotransmitter. The second component of the
partially purinergic IJPs was not always suppressed by L-NAME and therefore not
mediated by nitric oxide. We tested only one possible antagonist (i.e., VIP6-28) for the
second L-NAME insensitive component and found no significant suppression of this
component by the VIP receptor antagonist.
Non-Purinergic IJPs
The incidence of stimulus-evoked IJPs in our non-purinergic category was only
4%. When non-purinergic IJPs occurred, they were always suppressed or abolished by
L-NAME, which suggests that they were purely nitrergic.
Purinergic Myenteric Neurons
At least, two functionally distinct populations of purinergic neurons are present in
the myenteric plexus of guinea-pig small intestine. One population consists of the
inhibitory motor neurons to the circular muscle that were emphasized in the present
study. A second purinergic population connects synaptically with secretomotor neurons
in the submucosal plexus (24). ATP is the neurotransmitter released at P2Y1 excitatory
postsynaptic receptors on the secretomotor neurons by these projections from the
myenteric plexus (18, 24). Focal electrical stimulation in the myenteric plexus activates
the purinergic projections and evokes slow synaptic excitation in a population of
submucosal secretomotor neurons. Purinergic secretomotor activation in turn stimulates
secretion of Na+, Cl-, HCO3- and H2O from the mucosal secretory glands (18, 19).
Elevated glandular secretion is this case is expected to increase the liquidity of the
luminal contents.
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Inhibitory motor neurons in the myenteric plexus project their axons in the aboral
direction to the circular muscle coat along the longitudinal axis of the intestine (7).
Activation of these projections during peristaltic propulsive motility relaxes the circular
muscle ahead of the advancing luminal contents (8, 43). Accumulating evidence suggests
that programed firing of purinergic inhibitory motor neurons and their release of ATP at
P2Y1 receptors, expressed by the circular muscle, are significant enteric
neurophysiological functions in generation of propulsive motility. There is evidence also
which suggests that programed firing of purinergic neurons in the myenteric plexus and
the release of ATP at P2Y1 receptors, expressed on secretomotor neurons, evoke mucosal
secretion (15, 18, 19). An important unanswered question is whether the myenteric
purinergic projections to the circular muscle and to submucosal secretomotor neurons
stem from one and the same neuronal cell body. Do purinergic neurons in the myenteric
plexus individually project to the muscle and secretomotor neurons or do single
purinergic neurons project axons that bifurcate to innervate both the musculature and
secretomotor neurons? If the latter were the case, then firing of these neurons would
simultaneously inhibit the circular muscle and stimulate secretion in the same segment of
intestine. A single purinergic neuronal connection to descending inhibition of the
circular muscle and stimulation of secretion is consistent with observations that stroking
of the intestinal mucosa reflexly evokes both descending inhibition of the circular muscle
and mucosal secretion (15, 35).
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ACKNOWLEDGMENT
National Institutes of Health grants RO1 DK 37238, RO1 DK 068258 to J.D.
Wood and KO8 DK60468 to Y. Xia, a Pharmaceutical Manufacturers of America
Foundation Postdoctoral Fellowship to S. Liu and an overseas study grant to X. Fang
from Peking Union Medical College Hospital in Beijing, China supported this work.
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Figures and Legends
Figure 1. MRS2179 and apamin suppressed the partially purinergic IJP.
(A) MRS2179 suppressed fast component of the IJP in a concentration-dependent manner
in the guinea-pig ileum. The IJPs were evoked by focal application of a single electrical
stimulus pulse to a ganglion in the myenteric plexus. (B) Concentration-response relation
for suppression of fast IJPs by MRS2179 in 12 preparations. The IC50 for suppression of
the IJPs by MRS2179 was 0.2 µM±0.3 µM. (C) Concentration-response relation for
suppression of fast IJPs by apamin in 17 preparations. The IC50 for suppression of the
IJPs by apamin was 0.03±0.01 µM.
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Figure 2. Pharmacological analysis of purely purinergic IJPs. (A) Purely purinergic IJPs
were abolished by 2-µM MRS2179 and by 0.5-µM apamin and by neural blockade with
TTX. Neither 0.5-µM VIP6-28, 10-µM TNP-ATP nor 200-µM L-NAME suppressed the
IJPs. (B) Quantitative data for actions of pharmacological agents on purely purinergic
(fast) IJPs. Numbers of preparations are given in parentheses. Concentrations were:
MRS2179 (2 µM); apamin (0.5 µM); TNP-ATP (10 µM); TTX (1 µM); VIP6-28 (0.5 µM);
L-NAME (200 µM); suramin (2 µM).
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Figure 3. Pharmacological analysis of non-purinergic IJPs. (A) Non-purinergic IJPs were
abolished by 200-µM L-NAME and by neural blockade with TTX. Neither 10-µM
MRS2179 nor 1-µM apamin suppressed the IJPs. (B) Quantitative data for actions of
pharmacological agents on non-purinergic (slow) IJPs. Numbers of preparations are
given in parentheses. Concentrations were: MRS2179 (2 µM); apamin (1 µM); TNP-ATP
(10 µM); TTX (1 µM); VIP6-28 (0.5 µM); L-NAME (200 µM).
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Figure 4. Pharmacological analysis of partially-purinergic IJPs, which had a L-NAMEsensitive non-purinergic (slow) component. (A) Partially purinergic IJPs were suppressed
by 10-µM MRS2179, 200-µM L-NAME and by neural blockade with TTX. MRS2179
or apamin selectively suppressed the purely purinergic (i.e., fast) component of the IJPs.
L-NAME selectively suppressed the non-purinergic (i.e., slow) component of the IJPs.
Co-application of 10-µM MRS2179 and 200-µM L-NAME abolished both components
of the IJPs (i.e., fast and slow components). Neither 2-µM VIP6-28 nor 10-µM TNP-ATP
suppressed the IJPs. (B) Quantitative data for actions of pharmacological agents on
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partially-purinergic (slow) IJPs, which had a L-NAME-sensitive non-purinergic (slow)
component.. Numbers of preparations are given in parentheses. Concentrations were:
MRS2179 (10 µM); apamin (1 µM); TNP-ATP (10 µM); TTX (1 µM); VIP6-28 (0.5 µM);
L-NAME (200 µM); suramin (2 µM).
Figure 5. Pharmacological analysis of partially-purinergic IJPs, which had a L-NAMEinsensitive non-purinergic (slow) component. (A) Partially purinergic IJPs were
suppressed by 10-µM MRS2179, 1-µM apamin and by neural blockade with TTX.
MRS2179 or apamin selectively suppressed the purely purinergic (i.e., fast) component
of the IJPs. Co-application of 200-µM L-NAME and 10-µM MRS2179 suppressed the
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purinergic (i.e., fast) component of the IJPs, but not the non-purinergic (i.e., slow)
component. Neither 2-µM VIP6-28 nor 10-µM TNP-ATP suppressed the IJPs. (B)
Quantitative data for actions of pharmacological agents on partially-purinergic (slow)
IJPs, which had a L-NAME-insensitive non-purinergic (slow) component. Numbers of
preparations are given in parentheses. Concentrations were: MRS2179 (10 µM); apamin
(1 µM); TNP-ATP (10 µM); TTX (1 µM); VIP6-28 (2.0 µM); L-NAME (200 µM);
suramin (2 µM).
Figure 6. Hyperpolarizing responses to exogenous application of ATP mimicked
stimulus-evoked purinergic IJPs. (A) Application of 10-µM ATP in the bathing solution
evoked IJP-like responses (B) Application of 200-µM L-NAME did not change the
resting membrane potential. (C) Application of 0.5-µM VIP did not change the resting
membrane potential. (D) The presence of 10-µM MRS2179 in the bathing medium
suppressed the hyperpolarizing action of 10-µM ATP. (E) The presence of 1-µM TTX in
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the bathing medium did not suppress the hyperpolarizing action of 10-µM ATP. (F) TNPATP (1-µM) in the bathing medium did not suppress the hyperpolarizing action of 10µM ATP. (G) Apamin (1-µM) in the bathing medium suppressed the hyperpolarizing
action of 10-µM ATP. (F) PPADS (1-µM) in the bathing medium suppressed the
hyperpolarizing action of 10-µM ATP.
Figure 7. Concentration-response curve for ATP-evoked membrane hyperpolarization in
the circular muscle coat of guinea-pig small intestine.
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Tables