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J Physiol (2003), 550.2, pp. 493-504
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.041731
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ABSTRACT |
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Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y1 receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y1 purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y1 receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca2+ from intracellular stores.
(Received 17 February 2003; accepted after revision23 April 2003; first published online 13 June 2003)
Corresponding author J. D. Wood: Department of Physiology and Cell Biology, College of Medicine and Public Health, Ohio State University, 303 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210-1218, USA. Email: wood.13{at}osu.edu
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INTRODUCTION |
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Slow synaptic excitation (slow EPSP) in enteric neurones is recorded as a slowly activating depolarization of the membrane potential in specific populations of enteric neurones when neurotransmitters are released experimentally by focal electrical stimulation of presynaptic axons in the myenteric and submucosal plexuses (reviewed by Surprenant, 1989; Wood, 1994; Galligan, 1998; Gershon, 1998). Mediators released to the enteric nervous system in paracrine fashion from non-neuronal cell types (e.g. histamine and cytokines from enteric mast cells) can evoke responses that mimic slow synaptic excitation (Wood, 1992; Liu et al. 2003). Two kinds of slow EPSPs are recorded in enteric neurones. An increase in input resistance is associated with the depolarization and augmented excitability for one kind of slow EPSP. The input resistance decreases or remains unchanged during the depolarization and augmented excitability of the second kind. Slow EPSPs with increased input resistance are found generally in AH-type neurones with multipolar Dogiel Type II morphology. Most evidence suggests that the principal ionic mechanism for this type of slow EPSP is suppression of resting Ca2+-dependent K+ conductance that accounts for the membrane depolarization, increased input resistance, and suppression of the Ca2+ component of the rising phase of the action potential (e.g. Grafe et al. 1980). Signal transduction for the slow EPSP with increased input resistance involves coupling of metabotropic receptors through heterotrimeric G proteins to adenylate cyclase, and elevation of intraneuronal cyclic adenosine monophosphate (Palmer et al. 1986, 1987).
Whereas slow EPSPs characterized by increased input resistance during the depolarizing response predominate in AH-type neurones in the myenteric plexus, slow EPSPs characterized by decreased input resistance are routinely found in S-type uniaxonal neurones in the small and large intestinal submucosal plexus. Likewise, application of putative neurotransmitters and paracrine mediators (e.g. serotonin, ATP and substance P) evoke slowly activating depolarizing responses associated with decreased input resistance in S-type neurones in the submucosal plexus.
This report presents evidence that synaptically released ATP acts at P2Y1 purinergic receptors to evoke slow EPSPs that are characterized by decreased input resistance in the submucosal plexus. The evidence suggests that the signal transduction cascade for the submucosal P2Y1 receptor includes activation of phospholipase C, release of inositol 1,4,5-trisphosphate and elevation of cytosolic free Ca2+, which in turn leads to opening of cation conductance channels. Results of the study show that neurones with their cell bodies in the submucosal plexus, in the myenteric plexus and in prevertebral sympathetic ganglia release ATP as a neurotransmitter to evoke slow EPSPs in submucosal ganglion cells. Parts of the results have been published in abstract form (Hu et al. 2001, 2002).
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METHODS |
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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. Submucosal plexus preparations were obtained from segments of small intestine removed 10 to 20 cm proximal to the ileocaecal junction. The segments were microdissected for electrophysiological recording as described earlier (Frieling et al. 1991a; Zafirov et al. 1993). The preparations were placed in 2 ml chambers and superfused with Krebs solution warmed to 37 °C and gassed with 95 % O2 : 5 % CO2 (pH 7.3-7.4), at a rate of 10-15 ml min-1. The composition of the Krebs solution was (mM): 120 NaCl, 6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.35 NaH2PO4, 14.4 NaHCO3, and 11.5 glucose. Equimolar N-methyl-D-glucamine or choline chloride was substituted for NaCl to reduce the Na+ concentration from 120 to 26 mM in protocols that required depletion of Na+ in the Krebs solution.
Transmembrane electrical potentials were recorded with conventional 'sharp' intracellular microelectrodes filled with 4 % biocytin (Sigma, St Louis, MO, USA) in 2 M KCl or potassium acetate containing 0.05 M Tris-(hydroxy-methyl)-aminomethane buffer (pH 7.4) and having resistances of 80-200 M
. Methods for electrophysiological recording of electrical and synaptic behaviour and procedures for development of the intraneuronal marker biocytin were the same as described elsewhere (Frieling et al. 1991a,b; Zafirov et al. 1993; Gao et al. 2002; Liu et al. 2003). Synaptic potentials were evoked by focal electrical stimulation of interganglionic fibre tracts with electrodes made of 20 µm diameter Teflon-coated platinum wire and connected through a stimulus-isolation unit (Grass SIN5) to a Grass S48 stimulator (Grass Instrument Division, Astro-Med, Inc., Warwick, RI, USA). Square pulses of 100-250 µs duration and variable amplitude were used.
Pharmacological agents were applied either by addition to the bathing solution or by pressure microejection from glass micropipettes (10-15 µm tip diameter) with N2 pulses of controlled duration and pressure. Agents used were: noradrenaline (norepinephrine), phentolamine, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) and 8-cyclopentyl-1,3-dimethylxanthine (CPT) all of which were purchased from RBI (Natick, MA, USA); tetrodotoxin (TTX), biocytin, caffeine, U73122, atropine, cyclopiazonic acid (CPA), N-methyl-D-glucamine, somatostatin, choline chloride, nicardipine, ATP, 2-methio-ATP, ADP, ATP-
-S, uridine 5'-triphosphate (UTP), uridine 5'-diphosphate (UDP), 
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-methylene-ATP, pertussis toxin (PTX), ryanodine, idazoxan, cyanopindolol, scopolamine, hCGRP8-37, 2-methio-ADP, ICS-205930, 5-hydroxytryptamine, U73343 and suramin were purchased from Sigma. TNP-ATP (2'- or 3'-0-(trinitrophenyl) adenosine 5'-triphosphate) was obtained from Molecular Probes (Eugene, OR, USA). ZM241385, MRS2179, MRS1220, SB222200, L732138 and 2-aminoethoxydiphenylborane (2-APB) were purchased from Tocris (Ballwin, MO, USA). Arachidonyl trifluoromethylketone (AACOCF3) was from Oxford Biomedical Research (Oxford, MI, USA). Stock solutions were prepared in Krebs solution and stored at -20 °C. Serial dilutions were prepared fresh daily in Krebs solution.
Data analysis
Data are presented as means ± S.E.M. with n values referring to numbers of neurones. Continuous curves for concentration- response relationships were constructed with the following least-squares fitting routine using Sigmaplot software (SPSS Inc., Chicago, IL, USA):
V = Vmax/[1 + (EC50/C)nH],
where V is the observed membrane depolarization, 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.
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RESULTS |
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Synaptic potentials were evoked in 126 of 136 submucosal neurones. In 87 of the 126 neurones, the synaptic events were composed of superpositioned fast EPSPs, slow inhibitory postsynaptic potentials (slow IPSPs) and slow EPSPs that were reminiscent of the earlier findings reported by Surprenant (1984). All 87 neurones were identified as secretomotor neurones based on expression of immunoreactivity for vasoactive intestinal peptide, S-type electrophysiological behaviour, occurrence of slow noradrenergic IPSPs and uniaxonal morphology (Bornstein et al. 1988). Noradrenergic slow IPSPs were identified by their sensitivity to blockade by 2 receptor antagonists. The 2 receptor antagonist idazoxan (300 nM) abolished the slow IPSPs in all studies except where noted (Fig. 1B).
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Figure 1. Demonstration of three kinds of slow EPSPs The first slow EPSP was a pure purinergic EPSP that was abolished by exposure to the selective P2Y1 receptor antagonist MRS2197, the second was a partially purinergic EPSP that was suppressed only partially by MRS2197 and the third was insensitive to MRS2197. The depolarization phase of the first kind was associated with decreased input resistance, depolarization of the second was associated with either decreased input resistance or no change in resistance and depolarization of the third was associated with increased input resistance. A, pure purinergic slow EPSP in the uniaxonal neurone shown in the inset was abolished by MRS2197 or TTX in the continued presence of the | ||
Exposure to MRS2179, which is a selective antagonist for the P2Y1 receptor (Nandanan et al. 2000), decreased the amplitude of stimulus-evoked slow EPSPs with an IC50 of 0.7 ± 0.1 µM in eight neurones. MRS2179 (10 µM) abolished the slow EPSPs in 63 of 126 neurones (Fig. 1A). Slow EPSPs, which were abolished by 10 µM MRS2179, were considered to be mediated solely by synaptic release of ATP. We will refer to these EPSPs as 'pure purinergic slow EPSPs' for purposes of distinguishing them from 'mixed' EPSPs that were partially suppressed by 10 µM MRS2179 and presumed to be evoked by release of ATP and one or more additional excitatory neurotransmitters (Fig. 1B). Slow noradrenergic inhibitory input was present in 55 of the 63 pure purinergic neurones. Amplitude of the pure purinergic EPSPs was 8.8 ± 0.6 mV and ranged from 3 to 18 mV for 33 neurones when evoked by four stimulus pulses applied at 4 Hz. Duration of the purely purinergic EPSPs was 12.1 ± 0.9 s. Application of the non-selective P2 receptor antagonist suramin (300 µM) reduced the amplitude of pure purinergic slow EPSPs to 39.9 ± 8.3 % of control in six neurones (Fig. 2B). Exposure to 10 µM MRS2179 reduced, but did not abolish, the slow EPSPs in 41 of 126 neurones. MRS2179 (10 µM) suppressed mixed purinergic slow EPSPs by 47.9 ± 5.0 % (Fig. 1B). Both kinds of EPSPs were found exclusively in ganglion cells with uniaxonal morphology and S-type electrophysiology.
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Figure 2. Submucosal slow EPSPs were unaffected by receptor antagonists for putative neurotransmitters for slow EPSPs A, pure purinergic slow EPSP in the uniaxonal neurone shown in the inset was abolished by MRS2179 and unaffected by the muscarinic receptor antagonist, scopolamine, the neurokinin1 receptor antagonist L732138, or the CGRP receptor antagonist hCGRP8-37. B, data showing lack of effect of the NK1 antagonist L732138, the NK3 antagonist SB222200, the 5-HT1/2 antagonist cyanopindolol, the muscarinic antagonist scopolamine, the adenosine A2a antagonist ZM241385 or the adenosine A3 antagonist MRS1220. The adenosine A1 receptor antagonist 8-CPT enhanced the purinergic slow EPSP. Only exposure to suramin or MRS2179 significantly suppressed the submucosal slow EPSPs. Numbers in parentheses refer to numbers of neurones. | ||
Slow IPSPs were evoked in 32 of the 41 neurones that had mixed EPSPs with a purinergic component. Overall, 104 of 126 slow EPSPs recorded in the submucosal plexus had a purinergic component and 87 of 126 received noradrenergic inhibitory input and expressed immunoreactivity for vasoactive intestinal peptide.
Exposure to 10 µM MRS2179 did not suppress the amplitude of stimulus-evoked slow EPSPs in 22 out of 126 neurones in which 10 of the 22 neurones expressed S-type electrophysiology and uniaxonal morphology and 12 of the 22 displayed AH-type electrophysiology and Dogiel Type II morphology (Fig. 1C). These EPSPs were apparently mediated exclusively by non-purinergic neurotransmitter(s). Non-purinergic slow EPSPs were of larger amplitude (16.7 ± 3.5 mV, n = 14) and longer duration (3.7 ± 2.3 min, n = 14) relative to pure or mixed purinergic slow EPSPs. The depolarization phase for the non-purinergic EPSPs in the AH-type neurones was associated with increased input resistance. IPSPs were never found in neurones that received only non-purinergic slow excitatory input.
Substance P, serotonin, acetylcholine and calcitonin gene-related peptide are recognized as putative neurotransmitters for slow synaptic excitation in the enteric nervous system (e.g. Wood & Mayer, 1978; Katayama et al. 1979; Pan & Gershon, 2000). Nevertheless, neither the neurokinin1 (NK1) receptor antagonist L732138 (2 µM) in six neurones, the NK3 receptor antagonist SB222200 (1 µM) in five neurones, the serotonergic antagonist cyanopindolol (3 µM) in five neurones, the muscarinic antagonist scopolamine (3 µM) in seven neurones nor the calcitonin gene-related peptide (CGRP) antagonist hCGRP8-37 (2 µM) in five neurones suppressed the MRS2179-sensitive slow EPSPs (Fig. 2A and B). This suggested that the purinergic component of the slow EPSPs was not mediated by synaptic release of substance P, serotonin, acetylcholine or calcitonin gene-related peptide. Unlike the inhibitory action on pure and mixed purinergic EPSPs, the presence of 10 µM MRS2179 did not affect the slow EPSP-like responses to application of substance P or serotonin.
Exposure to TNP-ATP, which is reported to be a P2X receptor antagonist for P2X1 and P2X3 receptors (Virginio et al. 1998; North & Surprenant, 2000), did not alter ATP-evoked responses in seven neurones. Neither the A2a adenosine receptor antagonist ZM241385 (300 nM) (n = 6) nor the A3 receptor antagonist MRS1220 (3 µM) (n = 8) altered the purinergic slow EPSPs in six and eight neurones, respectively. Amplitude of the purinergic slow EPSPs increased to 123.0 ± 2.9 % of control for eight neurones when the adenosine A1 receptor antagonist 8-CPT (1 µM) was present in the bathing solution (see Fig. 2B). This suggested tonic adenosine A1 receptor-mediated inhibition of the synaptic release of ATP. The slow EPSP-like responses to ATP were unaffected by 300 nM ZM241385 in 13 neurones, 3 µM MRS1220 in 10 neurones or 1 µM 8-CPT in 15 neurones (Fig. 3B).
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Figure 3. Depolarizing responses to exogenous application of ATP mimicked stimulus-evoked purinergic slow EPSPs in the uniaxonal neurone shown in the inset A1, stimulus-evoked slow EPSP was abolished during exposure to either the selective P2Y1 antagonist or to TTX. A2, application of ATP in the bathing solution evoked slow EPSP-like responses that were suppressed in a concentration-dependent manner by MRS2179. B, data showing suppression of ATP-evoked responses by suramin or MRS2179 and lack of effect of the adenosine A1 antagonist 8-CPT, the A2a antagonist ZM241385 or the adenosine A3 antagonist MRS1220. Numbers in parentheses are numbers of neurones. C, concentration-response data for maximum amplitude of depolarization during responses to ATP in the absence and presence of three progressively increasing concentrations of MRS2197. D, Schild analysis, results of which suggest competitive antagonism with a pA2 value of 6.18. Data in C and D were obtained from the same neurone. | ||
Actions of ATP
Effects of application of ATP were studied in neurones that received MRS2179-sensitive slow excitatory synaptic input. ATP was applied by either addition to the bathing solution or by micropressure ejection and was done in the presence of 10 µM PPADS, which served to block P2X receptor-mediated responses. Depolarizing responses to exogenously applied ATP mimicked MRS2179-sensitive slow EPSPs (Fig. 3A).
ATP-evoked depolarizing responses were concentration dependent with an EC50 of 1.5 ± 0.2 µM (n = 25). The purinoceptor agonists 2-methio-ADP, 2-methio-ATP, ADP and ATP--S mimicked the responses to ATP with EC50 values of 82.4 ± 8.1 nM in 11 neurones, 0.8 ± 0.1 µM in 15 neurones, 1.2 ± 0.2 µM in 7 neurones and 2.4 ± 0.3 µM in 9 neurones, respectively. UTP in 5 neurones, UDP in 11 neurones and ,-methylene-ATP in 5 neurones did not evoke depolarizing responses in graded concentrations up to 100 µM. Potency order was 2-methio-ADP > 2-methio-ATP > ADP > ATP > ATP--S, which is consistent with reports for cloned and functionally expressed P2Y1 receptors (von Kugelgen & Wetter, 2000). The depolarizing responses to ATP were suppressed in a concentration-dependent manner by MRS2179 with a pA2 value of 6.18 (Fig. 3A, C and D). Suramin, which is also a purinergic receptor antagonist, behaved like a competitive antagonist for the depolarizing responses to ATP with a pA2 value of 3.46.
We investigated the action of PPADS in view of reports that it is a P2X purinoceptor antagonist that suppresses ATP-activated inward current in cultured guinea-pig myenteric neurones and P2Y1 receptor-mediated responses in human tissues (Barajas-Lopez et al. 1996; Zhou & Galligan, 1996; von Kugelgen & Wetter, 2000). Exposure to 10 µM PPADS did not alter the depolarizing responses evoked by 3 µM ATP. Amplitude of the control responses was 14.0 ± 2.7 mV and in the presence of 10 µM PPADS was 13.7 ± 3.2 mV for 15 neurones. PPADS (10 µM) abolished depolarizing responses evoked by micropressure application of ATP in eight neurones. PPADS (10 µM) did not alter purinergic slow EPSPs. Amplitude of control responses for MRS2179-sensitive slow EPSPs was 8.3 ± 1.3 mV and was unchanged at 8.5 ± 1.4 mV for six neurones in the presence of PPADS.
Ionic mechanisms
Stepwise current clamp of the membrane potential in the depolarizing direction decreased the amplitude of the purinergic slow EPSPs (Fig. 4A). Slow EPSPs and EPSP-like responses to micropressure application of 100 µM ATP in the same neurones (e.g. Fig. 4B and Fig. 5C) had similar reversal potentials of -16 and -12 mV, respectively. Reversal potentials for the purinergic slow EPSPs and ATP-evoked slow EPSP-like responses were -4.1 ± 4.5 mV (n = 8) and -2.6 ± 2.9 mV (n = 11), respectively. The depolarizing phase of the purinergic slow EPSPs was associated with decreased input resistance in 20 of 27 neurones. Input resistance was unchanged in six of 27 neurones and increased in a single neurone. ATP-evoked responses were decreased to 5.2 ± 1.2 % of controls after lowering the extracellular Na+ concentration to 26.2 mM. The effects of lowered Na+ suggest that both the MRS2179-sensitive slow EPSPs and MRS2179-sensitive responses to ATP were due to opening of cation channels, primarily Na+ channels.
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Figure 4. MRS2179-sensitive slow EPSPs and depolarizing responses to ATP became progressive smaller as the membrane potential was progressively current-clamped to increasingly depolarized holding potentials ranging from -95 to -12 mV A, the amplitude of stimulus-evoked slow EPSPs was progressively reduced as the membrane potential was progressively depolarized from -95 to -12 mV. B, the amplitude of depolarizing responses to ATP was progressively reduced as the membrane potential was depolarized from -95 to -12 mV in the same neurone as in A in the presence of 10 µM PPADS. C, extrapolation of voltage-response curves suggested similar reversal potentials of -16 mV for MRS2179-sensitive slow EPSPs and -12 mV for slow EPSP-like responses to ATP in the same neurone. | ||
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Figure 5. Signal transduction for MRS2179-sensitive slow EPSPs involved activation of phospholipase C and generation of IP3 A, An MRS2179- and suramin-sensitive slow EPSP was reversibly blocked by pretreatment with the intracellular IP3 receptor antagonist 2-APB in the uniaxonal neurone shown in the inset. B, data on effects of pharmacological tools on MRS2179-sensitive slow EPSPs. Both the IP3 receptor antagonist 2-APB and the phospholipase C inhibitor U73122 significantly suppressed the amplitude of the EPSPs. Neither ryanodine nor U73343, the inactive analogue of U73122, affected the slow EPSPs. C, data on effects of pharmacological tools on slow EPSP-like responses to ATP. Reduction of the extraneuronal Na+ concentration, blockade of IP3 receptors by 2-APB or inhibition of phospholipase C by U73122 each significantly suppressed the amplitude of responses to ATP. Reduction of extraneuronal Ca2+, the inactive analogue of U73122 or ryanodine did not alter the amplitude of the responses to ATP. Numbers in parentheses in B and C refer to numbers of neurones. | ||
Signal transduction mechanisms
The MRS2179-sensitive responses to ATP were not altered significantly by removal of extracellular Ca2+. Lack of effect of depletion of extraneuronal Ca2+ suggests that activation of Ca2+ conductance does not contribute significantly to the membrane depolarization during MRS2179-sensitive responses to ATP (Fig. 5C). On the other hand, all cloned and functionally defined P2Y receptors are coupled to activation of phospholipase C, synthesis of IP3 and mobilization of Ca2+ from intracellular stores (Schachter et al. 1997; Barajas-Lopez, et al. 2000; Fam, et al. 2000; von Kugelgen & Wetter, 2000). The PLC inhibitor U73122 (10 µM) reduced both the MRS2179-sensitive slow EPSPs and MRS2179-sensitive responses to ATP to 23.6 ± 5.7 % (n = 6) and 20.1 ± 7.3 % (n = 8) of control responses, respectively. On the other hand, U73343 (10 µM), the inactive analogue of U73122, affected neither the slow EPSPs nor ATP-evoked responses. Exposure to the PLA2 inhibitor AACOCF3 (50 µM) suppressed neither the amplitude of MRS2179-sensitive slow EPSPs nor the MRS2179-sensitive responses to ATP in any of the neurones examined (n = 7 for ATP responses and n = 5 for MRS2179-sensitive slow EPSPs). Pretreatment with 100 µM 2-APB, which is a recognized IP3 receptor antagonist (Ma et al. 2001), suppressed the MRS2179-sensitive responses to 3 µM ATP to 22.2 ± 3.4 % of control responses in six neurones (Fig. 5C). Exposure to 2-APB likewise suppressed the MRS2179-sensitive slow EPSPs to 19.3 ± 5.8 % of control responses in seven neurones (Fig. 5A).
We tested a hypothesis that PTX-insensitive G proteins link the P2Y1 receptor to the signal transduction cascade for the MRS2179-sensitive slow EPSP and the MRS2179-sensitive responses to ATP. The submucosal plexus preparations were incubated in PTX (2 µg ml-1, 24 h). In eight PTX-treated preparations, exposure to 3 µM ATP evoked characteristic depolarizing responses in 27 neurones that were not significantly different from those in 22 untreated preparations. MRS2179-sensitive slow EPSPs were similarly unaffected by 24 h incubation in PTX (n = 8). No slow IPSPs were evoked in the PTX-treated preparations. Absence of involvement of PTX-sensitive G proteins was evidence against involvement of the P2Y13 purinergic receptor. The P2Y13 receptor is sensitive to MRS2179, but differs from the P2Y1 receptor in being coupled to PTX-sensitive Gi protein (Zhang et al. 2002). Ryanodine was a tool to test for involvement of Ca2+-induced Ca2+ release channels in signal transduction for the MRS2179-sensitive responses. Ryanodine (10 µM) affected neither the MRS2179-sensitive slow EPSPs nor the MRS2179-sensitive responses to ATP (Fig. 5B and C).
Sources of P2Y1 purinergic inputs to submucosal neurones
The evidence suggested that neurones in the submucosal plexus receive slow excitatory synaptic input mediated by release of ATP and activation of P2Y1 receptors. The question of the source of the purinergic input was left open. Possibilities amenable to testing were input derived from neighbouring neurones in the submucosal plexus, input derived from neurones residing in the myenteric plexus and input from sympathetic postganglionic neurones. We addressed the three possibilities by stimulating neurones in the myenteric and submucosal plexus and sympathetic fibres in the intestinal mesentery to fire while synaptic events in submucosal ganglion cells were recorded with microelectrodes. Focal electrical stimulation applied in the myenteric plexus, and electrical stimulation of sympathetic fibres in the intestinal mesentery were methods used to fire putative presynaptic neurones. Presynaptic neurones were also fired by localized application of 5-HT (Moore & Vanner, 1998).
Local application of 5-HT
In view of evidence that 5-HT is a mediator in secretory reflex pathways in the guinea-pig submucosal plexus (Cooke et al. 1997; Pan & Gershon, 2000), we applied 5-HT by pressure microejection to the surfaces of individual submucosal ganglia while recording with microelectrodes in neighbouring ganglia. We found that 30 ms 'puffs' of 100 µM 5-HT evoked MRS2179-sensitive slow EPSPs in neurones in neighbouring ganglia in trials on four of six neurones (Fig. 6). The maximal amplitude of the depolarization phase of the slow EPSPs ranged from 3 to 5 mV (mean = 4.4 ± 1.2 mV; n = 4). The responses evoked by 'puffs' of 5-HT appeared to be synaptically mediated because exposure to TTX (1 µM) abolished the EPSPs in all four neurones (Fig. 6B). Depolarizing responses evoked by 'puffs' of 5-HT applied directly to the neurones with purinergic EPSPs averaged 11.3 ± 2.5 mV and were unaffected by MRS2179 in five neurones (Fig. 6D). The responses evoked by 'puffs' of 5-HT either directly onto the neurones that received purinergic input or onto neighbouring ganglia were suppressed or abolished by pretreatment with the 5-HT3 serotonergic receptor antagonist ICS-205930 (n = 3). These observations were interpreted to mean that purinergic ganglion cells in the submucosal plexus project to and synapse with neurones in neighbouring regions of the plexus. ATP released at the synapses acted at P2Y1 postsynaptic receptors to elevate excitability in the neurones in neighbouring ganglia. The purinergic neurones that projected to neighbouring ganglia express receptors for 5-HT, one kind of which was a 5-HT3 serotonergic receptor
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Figure 6. Purinergic neurones in the submucosal plexus were stimulated by 5-HT to release ATP at their synapses with neighbouring neurons A, diagram of the preparation. Electrical behaviour was recorded while applying 5-HT by micropressure ejection ('puff') directly onto the ganglion with the recorded ganglion cell (Site 2) and onto ganglia elsewhere in the preparation (Site 1). B a 'puff' of 5-HT applied to one ganglion evoked a MRS2179-sensitive slow EPSP in a neurone located in a second neighbouring ganglion. The 5-HT-evoked slow EPSP was blocked by TTX, which helped to confirm that conduction of action potentials in a presynaptic neurone was necessary for the generation of the slow EPSP. C, photomicrograph of the biocytin-filled neurone from which the records in B-D were obtained. D, 'puffs' of 5-HT directly onto the biocytin-filled neurone in C evoked slow EPSP-like responses that were insensitive to MRS2179. Downward deflections on the voltage traces in B and D are electrotonic potentials evoked by repetitive intraneuronal injection of constant-current hyperpolarizing pulses. | ||
Myenteric plexus input
In the light of evidence reported by Bornstein et al. (1987) that some of the EPSPs recorded in the submucosal plexus of the guinea-pig reflected synaptic input coming from the myenteric plexus, we developed a submucosal plexus preparation with attached myenteric plexus that enabled study of synaptic inputs received by submucosal ganglion cells from neurones in the myenteric plexus (Fig. 7A). Electrical activity was recorded intracellularly in submucosal neurones while applying focal electrical stimulation to ganglia or interganglionic connectives in the myenteric plexus. The stimulating electrodes were placed in orally and aborally directed positions within 1 cm of the recording microelectrode in the submucosal plexus. One or two stimulus pulses applied at orally directed sites in the myenteric plexus evoked fast EPSPs that were followed by slow EPSPs with amplitudes 5.4 ± 2.6 mV in five of eight preparations. The depolarizing phase of the slow EPSPs was associated with decreased input resistance. Exposure to 300 nM TTX, 10 µM MRS 2179 or to 300 µM suramin (n = 5) suppressed the slow EPSPs that were evoked by stimulation at orally directed sites in the myenteric plexus (Fig. 7B). On one occasion, in one of the eight preparations, a slow EPSP with increased input resistance during the depolarizing phase was evoked. Exposure to TTX, but not MRS2179 abolished this particular EPSP. The fast EPSPs in four neurones were abolished by exposure to hexamethonium or TTX but not by MRS 2179.
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Figure 7. Neurones in the submucosal plexus received slow purinergic excitatory synaptic input from neurones in the myenteric plexus A, diagram of the preparation. Electrical behaviour was recorded in neurones in the submucosal plexus while applying focal electrical stimulation to the myenteric plexus in regions either oral or aboral to the recording site in the submucosal plexus. B, focal electrical stimulation in the myenteric plexus located on the oral side of the recording site in the submucosal plexus evoked slow EPSPs that were blocked by TTX or MRS2179. C, focal electrical stimulation in the myenteric plexus located on the aboral side of the recording site in the submucosal plexus evoked slow EPSPs that were blocked by TTX or MRS2179. D, uniaxonal neurone from which the records in B were obtained. E, uniaxonal neurone from which the records in D were obtained. | ||
Focal electrical stimulation at sites in the myenteric plexus aboral to the exposed submucosal plexus, like stimulation at the orally directed side, evoked fast EPSPs followed by slow EPSPs with amplitudes of 4.1 ± 0.6 mV in seven of eight neurones. Exposure to 300 nM TTX abolished both the fast and slow EPSPs. Exposure to 10 µM MRS2179 significantly suppressed or abolished the slow EPSPs without any changes in the fast EPSPs (Fig. 7C).
These findings for the myenteric plexus suggested that purinergic myenteric neurones may project in the oral or aboral directions to supply slow excitatory synaptic input to neurones in the submucosal plexus. ATP released from the myenteric projections at their synapses with the submucosal target neurones acted at P2Y1 purinergic receptors to evoke slow synaptic excitation.
Sympathetic postganglionic neuronal input
Burnstock (2001) reviewed evidence for ATP release as a co-transmitter with noradrenaline from sympathetic postganglionic axons in the intestine. The noradrenaline released from sympathetic fibres in the submucosal plexus acts at 2 adrenoceptors on submucosal neurones to evoke IPSPs (North & Surprenant, 1985; Liu et al. 2003). To test the suggestion that co-release of ATP from sympathetic fibres might evoke slow synaptic excitation in submucosal neurones, we developed an in vitro submucosal plexus preparation that had the mesentery together with the sympathetic innervation attached (Fig. 8A and B). Sympathetic input to the preparation was stimulated by applying focal electrical stimulation to nerve trunks in the mesentery. Electrical stimulation of the mesenteric nerve trunks evoked IPSPs followed by slow EPSPs with amplitudes of 4.3 ± 2.4 mV in 10 of 27 preparations (Fig. 8C). Exposure to 300 nM idazoxan abolished the slow IPSPs and revealed the full extent of the slow EPSP. Blockade of the P2Y1 purinergic receptors by addition of 10 µM MRS2179 in the continued presence of idazoxan abolished the sympathetically mediated slow EPSPs (Fig. 8C).
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Figure 8. Neurones in the submucosal plexus received slow purinergic excitatory synaptic input from sympathetic postganglionic neurones A, photomicrograph of the submucosal preparation with attached mesentery. B, diagram of the preparation. Electrical behaviour was recorded in submucosal neurones while applying electrical stimulation to nerve trunks in the mesentery. C, electrical stimulation of a mesentery nerve evoked slow noradrenergic IPSPs that partially obscured slow EPSPs. Blockade of the IPSP with the | ||
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DISCUSSION |
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The results are consistent with identification of the excitatory P2Y1 purinergic receptor on guinea-pig submucosal neurones as a metabotropic receptor. Coupling of the receptor to its signal transduction cascade did not involve PTX-sensitive G proteins. The receptor differs in this respect from the MRS2179-sensitive P2Y13 receptor, which does couple through a PTX-sensitive Gi protein (Zhang et al. 2002).
P2Y receptors in general are coupled to activation of phospholipase C, synthesis of IP3 and mobilization of intracellular Ca2+ (Schachter et al. 1997; von Kugelgen & Wetter, 2000; Barajas-Lopez et al. 2000; Fam et al. 2000). The P2Y1 receptors expressed by submucosal plexus neurones behave in the same manner, with effects of receptor activation suppressed by inhibitory agents for PLC, but not phospholipase A2. Agents that blocked intraneuronal IP3 receptors effectively suppressed MRS2179-sensitive EPSPs and MRS2179-sensitive responses to ATP. No evidence was obtained for involvement of intraneuronal ryanodine receptors or opening of receptor-coupled Ca2+ channels in the P2Y1 purinergic receptor signalling pathway.
Ionic mechanisms
Two distinctly different kinds of slow EPSPs are involved in neurotransmission at the microcircuit level of enteric neural organization. One is the well-known EPSP that is associated with increased input resistance (i.e. decreased resting K+ conductance) and found mainly in neurones with AH-type electrophysiology and Dogiel Type II multipolar morphology (reviewed by Wood, 1994). Signal transduction for the increased-resistance EPSP involves stimulation of adenylate cyclase, elevation of cAMP and activation of protein kinase A.
The second kind of slow EPSP was the focus of the present study. It is noteworthy for being associated with decreased membrane resistance (i.e. increased conductance) and being a property of neurones with S-type electrophysiological behaviour and uniaxonal morphology. The majority of these neurones in the submucosal plexus are secretomotor neurones. The increased conductance underlying the P2Y1-mediated EPSP was primarily Na+ conductance, as reflected by reduction of the P2Y1-mediated responses by 95 % when extraneuronal Na+ was reduced from 120 to 26 mM. Opening of non-selective cation channels separately or together with decreased K+ conductance along with a primary increase in Na+ conductance was reported to underlie slow synaptic excitation in S-type submucosal neurones (Mihara et al. 1985; Barajas-Lopez et al. 1994). Shen & Surprenant (1993) reported agonist-evoked slow depolarizing responses mediated by suppression of Ca2+-dependent K+ conductance and an inward current due to an increase in a cation-selective (mainly Na+) conductance in unidentified guinea-pig submucosal neurones. In-depth analysis of the currents responsible for the P2Y1-mediated depolarizing responses was beyond the scope of the present study.
ATP neurotransmitter and neuromodulator function
ATP fulfilled Burnstock's (1972) criteria for function as a neurotransmitter and neuromodulator. Identification as a neurotransmitter in the present study was facilitated by the availability of MRS2179 as a selective antagonist. MRS2179 blocked both a specialized form of neurally evoked slow synaptic excitation and the mimicry of the synaptic event by exogenously applied ATP. Selectivity of the neural- and ATP-evoked responses to blockade by MRS2179 identified the receptors as the P2Y1 subtype that was recently cloned from the submucosal plexus (Hu et al. 2002). The P2Y1 receptor was expressed exclusively by neurones with uniaxonal morphology, where it mediated a form of slow synaptic excitation characterized by decreased input resistance during the depolarization phase. Slow synaptic excitation in this population of uniaxonal neurones contrasted with slow EPSPs in AH-Dogiel morphologic Type II enteric neurons, where increased input resistance accompanies the depolarizing phase (Wood & Mayer, 1978).
Purinergic co-transmission
Burnstock (1990) was an early proponent of the concept of co-transmission for which co-release of ATP together with a second neurotransmitter became a prime example. We found evidence for two forms of purinergic co-transmission. One form involved simultaneous release of acetylcholine and ATP from neurones in the myenteric plexus that projected to and synapsed with neurones in the submucosal plexus. The released acetylcholine evoked fast nicotinic EPSPs, while co-released ATP acted at P2Y1 receptors on the same neurones to depolarize the membrane potential and enhance excitability. This was an example of neuromodulation whereby neuronal input-output relations to one neurotransmitter are modulated by the action of a second transmitter. An example occurs in the guinea-pig myenteric plexus, where the amplitudes of fast nicotinic EPSPs that do not reach spike threshold in the absence of slow synaptic input move progressively closer to threshold during the development of a slow EPSP and eventually reach threshold firing level (Wade & Wood, 1988). Aside from the implied functional significance, the evidence for co-release remains equivocal because electrical stimulation may have activated both individual cholinergic and purinergic axons in the same nerve bundle.
Evidence for a second form of co-transmission involved synaptic input to submucosal ganglion cells from sympathetic postganglionic neurones. Co-release of noradrenaline and ATP from sympathetic postganglionic neurones is now an accepted event, especially at blood vessels (Burnstock, 1990). The sympathetic release of noradrenaline in the submucosal plexus was reflected by stimulus-evoked IPSPs that were suppressed by selective 2 adrenoceptor antagonists. Stimulus-evoked release of ATP was reflected by slow EPSPs that were blocked by the selective P2Y1 antagonist MRS2179 in the same neurones. This case of neuromodulation for sympathetic input would work in the opposite way to the example described above for neuromodulatory input from the myenteric plexus. Myenteric co-transmission was synergistic while sympathetic co-transmission in the submucosal plexus was antagonistic. The inhibitory effects of noradrenergic sympathetic input coincident with the excitatory effects of purinergic excitatory input are offsetting events. It may be significant that the noradrenergic inhibitory input is considerably stronger than the purinergic excitatory input, to the extent that it overrides simultaneous excitatory input. The majority of neurones in which sympathetic co-transmission occurs in the submucosal plexus are secretomotor neurones. Our observations that these neurones receive excitatory purinergic input both from the myenteric plexus and other neurones in the submucosal plexus suggest that excitatory purinergic neurotransmission may be involved in the minute-to-minute control of mucosal secretion at the microcircuit level of enteric neural organization. On the other hand, the primary role of sympathetic noradrenergic input is to shunt splanchnic blood to the systemic circulation during stress and/or exercise, and for it to have a powerful role in offsetting excitatory neurotransmission in the enteric microcircuits and ultimately in shutting off secretion is expected.
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Author's present address
J. Ren: Division of Neuroscience, 513 Heritage Medical Research Centre, University of Alberta Edmonton, AB, Canada T6G 2S2.
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