Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X1, P2X2, P2X3 channels, P2Y and A3 metabotropic receptors in neurotransmission.

RATIONALE: The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH: LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS: Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-ß,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPßS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,ß-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS: Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

Dual purinergic synaptic transmission in the human enteric nervous system.

Based on findings in rodents, we sought to test the hypothesis that purinergic modulation of synaptic transmission occurs in the human intestine. Time series analysis of intraneuronal free Ca(2+) levels in submucosal plexus (SMP) from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal fluo-4 AM Ca(2+) imaging. A 3-s fiber tract stimulation (FTS) was used to elicit a synaptic Ca(2+) response. Short-circuit current (I(sc) = chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or electrical field stimulation was used to study I(sc) responses. Ca(2+) imaging was done in 1,222 neurons responding to high-K(+) depolarization from 61 surgical cases. FTS evoked synaptic Ca(2+) responses in 62% of recorded neurons. FTS caused frequency-dependent Ca(2+) responses (0.1-100 Hz). FTS Ca(2+) responses were inhibited by Omega-conotoxin (70%), hexamethonium (50%), TTX, high Mg(2+)/low Ca(2+) (< or = 100%), or capsaicin (25%). A P2Y(1) receptor (P2Y(1)R) antagonist, MRS-2179 or PLC inhibitor U-73122, blocked FTS responses (75-90%). P2Y(1)R-immunoreactivity occurred in 39% of vasoactive intestinal peptide-positive neurons. The selective adenosine A(3) receptor (AdoA(3)R) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IBMECA) caused concentration- and frequency-dependent inhibition of FTS Ca(2+) responses (IC(50) = 8.5 x 10(-8) M). The AdoA(3)R antagonist MRS-1220 augmented such Ca(2+) responses; 2-Cl-IBMECA competed with MRS-1220. Knockdown of AdoA(1)R with 8-cyclopentyl-3-N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1-N-propyl-xanthine did not prevent 2-Cl-IBMECA effects. MRS-1220 caused 31% augmentation of TTX-sensitive distension I(sc) responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in human enteric nervous system (huENS). The P2Y(1)/Galphaq/PLC/inositol 1,3,5-trisphosphate/Ca(2+) signaling pathway, N-type Ca(2+) channels, nicotinic receptors, and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory AdoA(3)R inhibit nucleotide or cholinergic transmission in the huENS.

Mechanically evoked 5-hydroxytryptamine release is mediated by caveolin-associated cholesterol rich membrane domains.

5-Hydroxytryptamine (5-HT) from enterochromaffin cells activates neural reflexes that govern intestinal motility and secretion. Mechanical stimulation of human enterochromaffin cell-derived BON cells activates a G alpha q-signalling pathway coupled to 5-HT release. Molecular mechanisms identifying elements of mechanosensory transduction are unknown. The aim of this study was to determine the role of caveolin and caveolin-associated cholesterol rich microdomains in mechanically stimulated 5-HT release from BON cells. Caveolin-1 transcripts and immunofluorescence were found in BON cells. In the static state, caveolins-1 and -2 co-precipitated with G alpha q in cholesterol rich cell fractions, but not with G alpha s, G alpha i/o and G beta. Mechanical stimulation transiently uncoupled G alpha q from caveolin-1 and increased 5-HT release. Disassembly of caveolin-associated membrane microdomains by filipin or by cholesterol depletion with methyl-beta-cyclodextrin decreased mechanically evoked 5-HT release. These results suggest that caveolin and caveolin-associated cholesterol rich membrane microdomains are key regulators in mechanically evoked 5-HT release from enterochromaffin cells.

Sensory mechanisms: transmitters, modulators and reflexes.

The enteric nervous system in combination with inputs from parasympathetic and sympathetic nerves regulate the contractile, secretory and vasomotor activity of the gastrointestinal track via neural reflexes. Sensory elements which may be present in specialized neurones, enteroendocrine cells or mast cells detect changes in force, chemical composition or even foreign antigens. Sensory elements signal the enteric nervous system to correct these changes by altering contractile activity, secretion and blood flow. Advances have been made in understanding the sensory mechanisms that are involved in 5-hydroxytryptamine (5-HT) release from enterochromaffin cells (EC) or a model for EC cells. These advances relate to roles for ATP and its metabolites ADP and adenosine in mechanotransduction and a role for a sodium glucose cotransporter, a SGLT-like protein, in chemotransduction.

Differential gene expression of adenosine A1, A2a, A2b, and A3 receptors in the human enteric nervous system.

Adenosine receptors (ADORs) in the enteric nervous system may be of importance in the control of motor and secretomotor functions. Gene expression and distribution of neural adenosine A1, A2a, A2b, or A3 receptors (Rs) in the human intestine was investigated using immunochemical, Western blotting, RT-PCR, and short-circuit current (I(sc)) studies. Adenosine A1R, A2aR, A2bR, or A3R mRNAs were differentially expressed in neural and nonneural layers of the jejunum, ileum, colon, and cecum and in HT-29, T-84, T98G, and Bon cell lines. A1R, A2aR, A2bR, and A3R immunoreactivities (IRs) were differentially expressed in PGP 9.5-immunoreactive neurons. A2bR IR occurs exclusively in 50% of submucosal vasoactive intestinal peptide (VIP) neurons (interneurons, secretomotor or motor neurons) in jejunum, but not colon; A2aR is also found in other neurons. A3R IR occurs in 57% of substance P-positive jejunal submucosal neurons (putative intrinsic primary afferent neurons) and less than 10% of VIP neurons. Western blots revealed bands for A3R at 44 kDa, 52 kDa, and 66 kDa. A2aR and A2bR are coexpressed in enteric neurons and epithelial cells. 5'-N-methylcarboxamidoadenosine or carbachol evoked an increase in I(sc). A2bR IR is more prominent than A2aR IR in myenteric neurons, nerve fibers, or glia. A1R is expressed in jejunal myenteric neurons and colonic submucosal neurons. Regional differences also exist in smooth muscle expression of ADOR IR(s). It is concluded that neural and nonneural A1, A2a, A2b, and A3Rs may participate in the regulation of neural reflexes in the human gut. Clear cell and regional differences exist in ADOR gene expression, distribution, localization, and coexpression.

Unlocking mysteries of gut sensory transmission: is adenosine the key?

Endogenous adenosine acts at pre- or postsynaptic A1, A2, or A3 receptors to inhibit synaptic transmission in intrinsic primary afferent/AH neurons, S neurons, and mucosal and motility reflexes. Adenosine provides dual modulation of adenylyl cyclases. Its modulation of sensory transmission may be of therapeutic potential in gut inflammation, ischemia, and constipation.

Changes in intracellular Ca2+ by activation of P2 receptors in submucosal neurons in short-term cultures.

Electrophysiological and Ca2+ microfluorimetric techniques were used to characterize the pharmacological profile of the P2 receptors expressed in submucosal neurons and the changes in intracellular Ca2+ associated with activation of these receptors. ATP caused a fast and slow membrane depolarizations during intracellular recordings. ATP induced a rapid inward current during whole-cell experiments. Receptors mediating the inward current and fast depolarization have the same pharmacological profile and these ATP responses were more sensitive to pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid than Basilen BlueE-3G, and potentiated by suramin. The slow depolarization was not blocked by these P2 receptor antagonists, pertussis toxin, or KT5720 (protein kinase A inhibitor). N-ethylmaleimide or protein kinase C inhibitors (staurosporine and calphostin) blocked this depolarization. ATP induced complex multi-phasic Ca2+ transients in most neurons, classified as fast, slow, or mixed fast/slow responses. In conclusion, the fast and slow Ca2+ responses were mediated by respective activation of P2X and P2Y receptors and were associated with fast and slow depolarizations, respectively.

Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle.

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

FlCRhR/cyclic AMP signaling in myenteric ganglia and calbindin-D28 intrinsic primary afferent neurons involves adenylyl cyclases I, III and IV.

The aims of this study were to improve insight into cAMP signaling in myenteric neurons and glia and identify the adenylyl cyclase (AC) isoforms expressed in myenteric ganglia of the guinea-pig small intestine. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520 nm/580 nm fluorescence emission ratio of the protein kinase A fluorosensor FlCRhR. Forskolin or pituitary adenylyl cyclase activating peptide caused an increase in cAMP levels in cell somas and neurites and elicited a slow EPSP-like response in myenteric AH/Type 2 neurons, whereas the inactive form of forskolin was without these effects. Glia displayed similar cAMP responses. Immunoblot analysis showed that AC I, III and IV were present in myenteric ganglia, with AC I being detected as two bands of 160 kDa and 185 kDa, AC III as two bands near 220 kDa, and AC IV as two bands of greater than 220 kDa. Pretreatment with N-ethylmaleimide and N-glycosidase F revealed an AC IV band at 115 kDa. Preabsorption with specific blocking peptides prevented detection of AC I or AC IV immunoreactive proteins. In ganglia which expressed strong AC IV immunoreactivity, no immunoreactive bands were detected for AC II, AC V/VI, AC VII or AC VIII. The amount of AC isoforms expressed in myenteric ganglia followed the order of AC IV&z.Gt;III>I. Immunofluorescent labeling studies revealed that AC I, AC III and AC IV were variably expressed in myenteric neurons and glia of the duodenum, jejunum and ileum. In the guinea-pig ileum, AC I, III and IV immunoreactivities were respectively present in 26%, 58% and 89% of calbindin-D28-colabeled myenteric neurons. These findings suggest that (1) AC I, AC III and AC IV variably contribute to cAMP signaling in myenteric ganglia, (2) AC I, AC III and AC IV may be differentially expressed in distinct subsets of calbindin-D28 neurons which may represent intrinsic primary afferent myenteric neurons. Our study also provides direct evidence for activation of cAMP-dependent protein kinase.

Activation of neuronal adenosine A1 receptors suppresses secretory reflexes in the guinea pig colon.

The role of adenosine A1 receptors (A1R) in reflex-evoked short-circuit current (Isc) indicative of chloride secretion was studied in the guinea pig colon. The A1R antagonist 8-cyclopentyltheophylline (CPT) enhanced reflex-evoked Isc. Adenosine deaminase and the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine enhanced and reduced reflex-induced Isc, respectively. The A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) inhibited reflex-evoked Isc at nanomolar concentrations, and its action was antagonized by CPT. In the presence of either N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-hydroxytryptamine (5-HT)-mediated pathway or piroxicam to block the prostaglandin-mediated pathway, CCPA reduced the residual reflex-evoked Isc. CCPA reduced the response to a 5-HT pulse without affecting the tetrodotoxin-insensitive Isc responses to carbachol or forskolin. Immunoreactivity for A1R was detected in the membrane (10% of neurons) and cytoplasm (90% of neurons) of neural protein gene product 9.5-immunoreactive (or S-100-negative) submucosal neurons, in glia, and in the muscularis mucosa. A1R immunoreactivity in a majority of neurons remained elevated in the cytoplasm despite preincubation with adenosine deaminase or CPT. A1R immunoreactivity colocalized in synaptophysin-immunoreactive presynaptic varicose nerve terminals. The results indicate that endogenous adenosine binding to high-affinity A1R on submucosal neurons acts as a physiological brake to suppress reflex-evoked Isc indicative of chloride secretion.

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia.

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D28, and s-100. Cultured cells or tissues were incubated with 10 microM BODIPY forskolin for 30 min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D28 (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.

Purinergic Ca2+ signaling in myenteric neurons via P2 purinoceptors.

Fura 2 microfluorimetry was used to test the hypothesis that ATP acts at P1 and P2 purinoceptors to elevate cytosolic free Ca2+ concentrations [Ca2+]i) in calbindin-immunoreactive cultured myenteric neurons from adult guinea pig small intestine. Local "micro-puff" application of ATP or ATP(gamma)S caused an increase in [Ca2+]i in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca2+]i was ATP(gamma)S = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro-N(6)-cyclopentyladenosine. Tetrodotoxin-sensitive synaptic transmission could contribute as much as 25% to the ATP response. The P1 antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked 50% of the peakATP Ca2+ response. P2 antagonists blocked the ATP response: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid > reactive blue 2 > suramin. Suramin enhanced the ATP response in 27.5% of neurons. Some neurons (<15%) displayed distinct multiphasic Ca2+ signatures. About 54% of ATP-responsive neurons expressed calbindin. The data support the following hypotheses: 1) two distinct P2 purinoceptors are linked to the rise in [Ca2+]i in myenteric neurons; 2) purinergic Ca2+ signaling is not restricted to one neuronal phenotype; and 3) intraneuronal Ca2+ is not involved in adenosinergic hyperpolarization in AH/type 2 neurons.

Responsiveness to ATP with an increase in intracellular free Ca2+ is not a distinctive feature of calbindin-D28 immunoreactive neurons in myenteric ganglia.

The aim of this study was to test the hypothesis that ATP elevates cytosolic free Ca2+ levels ([Ca2+]i) in myenteric neurons expressing the Ca2+ binding protein, calbindin-D28. A laser microbeam marked the location of cultured neurons on coverslips and provided unequivocal relocation of ATP-responsive neurons after immunocytochemistry. All myenteric multipolar neurons displayed ATP Ca2+ transients, and 42% also expressed calbindin-D28 reactivity. Statistical analysis of the kinetics and shape of ATP Ca2+ transients revealed no differences between calbindin and non-calbindin neurons. The identity of other responsive neurons is unknown. Less than 8% of ganglion cells with ATP Ca2+ transients were immunopositive for the glial protein S-100. We conclude that one of the actions of ATP in myenteric ganglia is to increase [Ca2+]i which may activate gKCa leading to membrane hyperpolarization in AH, Dogiel Type II neurons expressing calbindin-D28. An efficient buffering mechanism for handling large purinergic Ca2+ loads is a common feature of all types of myenteric ganglion cells.

Electrophysiological subtypes of inhibitory P1 purinoceptors on myenteric neurones of guinea-pig small bowel.

1. Conventional intracellular microelectrode techniques were used to subclassify P1 purinoceptors linked to reduction of cell input resistance, steady-state hyperpolarization of the membrane potential, or inhibition of fast e.p.s.ps, in neurones of microdissected myenteric plexus preparations from guinea-pig ileum. The potencies of P1 purinoceptor agonists were estimated in neurones that were current clamped to a fixed membrane potential. 2. In AH/Type 2 neurones, the A2 agonist, CGS 21680, the A1 agonist, CCPA or the mixed A1-A2 agonist, NECA, suppressed excitability by reducing input resistance (40-50% max.) and causing hyperpolarization (20-25 mV max.). CGS 21680 (0.1-1 microM) enhanced the after-hyperpolarizing potential. 3. From cumulative dose-response data, the potency order for reducing input resistance was CCPA (IC50 = 5.1 +/- 2.2 nM) >>> CGS 21680 (IC50 = 5.6 +/- 2.5 microM). This effect was reversed by the A1 antagonist, CPT (EC50 = 65 +/- 11 nM). 4. In contrast, the potency order for membrane hyperpolarization was CCPA (IC50 = 61 +/- 23 nM) = CGS 21680 (IC50 = 290 +/- 90 nM) > or = NECA (IC50 = 450 +/- 100 nM). Hyperpolarization elicited by CCPA was sensitive to the A1-A2 antagonist, DPSPX. 5. Agonists suppressed fast e.p.s.ps, but not DMPP responses, with an order of CCPA (IC50 = 8.1 +/- 3.0 nM) >>> CGS 21680 (IC30 = 10 +/- 2.9 microM). 6. In conclusion, the excitability of AH/Type 2 neurones is suppressed by activation of high affinity A l receptors that may be linked to a cyclic AMP-dependent pathway, leading to increase in calcium dependent potassium conductance and enhancement of the after-hyperpolarizing potential. Activation of lower affinity non A1 receptors linked to a cyclic AMP-independent pathway reduces excitability and leads mainly to a steady-state hyperpolarization. Adenosine also suppresses nicotinic cholinergic transmission by activating presynaptic high affinity Al receptors.

Enteric glial cells are major contributors to formation of cyclic AMP in myenteric plexus cultures from adult guinea-pig small intestine.

Cultures derived from ganglia isolated from the small intestine of adult guinea-pigs were used to determine relative contribution of neurons and glial cells to stimulation of cAMP formation by forskolin in myenteric ganglia. In untreated cultures (8-12 days), the ratio of glial cells to neurons was 5-fold higher than the ratio in intact myenteric plexus preparations. Treatment with cytosine arabinoside virtually eliminated the glia by the 12th day. Microelectrode recording of excitatory responses to forskolin in AH/Type 2 neurons confirmed the viability of cultured neurons in cytosine arabinoside. Forskolin elevated the cAMP content of cultures and cytosine arabinoside reduced this effect by 80-90%. This suggests that enteric glial cells are the major contributors to cAMP formation in the cultures and that glial cells contribute significantly to elevation of cAMP levels seen in intact myenteric ganglia.

Presynaptic inhibition by adenosine A1 receptors on guinea pig small intestinal myenteric neurons.

BACKGROUND: Adenosine acts at A1 receptors to inhibit the release of most neurotransmitters. This study tested the hypothesis that both exogenous adenosine (ADO) and tonic release of endogenous ADO act at presynaptic A1 receptors to suppress excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in myenteric neurons. METHODS: Intracellular microelectrodes were used to study actions of ADO, the agonists 2-chloro-N6-cyclopentyl ADO, its 1-deaza derivative, 5'-N-ethylcarboxamido ADO, and CGS 21680 or the antagonists 8-cyclopentyl-1,3-dimethylxanthine, its 1,3-dipropyl analog, and 1,3-dipropyl-8-p-sulfophenylxanthine on synaptic behavior in myenteric neurons. RESULTS: Each of the agonists suppressed slow EPSPs in all 35 AH/type 2, 8 of 10 S/type 1, and 7 of 7 nonspiking neurons. ADO also decreased neuronal excitability (n = 63) in AH/type 2 neurons. Agonists suppressed fast nicotinic EPSPs in all 20 S/type 1, 10 nonspiking, and 3 AH/type 2 neurons without having any effect on postsynaptic responses to nicotinic agonists. CCPA was more potent than CGS 21680 in suppressing EPSPs. In 30% of neurons, the only action of antagonists was to block the effect of A1 or A2 agonists on EPSPs. Agonists did not inhibit IPSPs, but unmasked robust slow IPSPs by preventing slow EPSPs. Antagonists acted alone to enhance EPSPs in 70% of neurons. CONCLUSIONS: (1) ADO acts at presynaptic A1 sites to suppress EPSPs in all neurons, (2) IPSPs are revealed by ADO, and (3) ongoing release of endogenous ADO inhibits synaptic transmission.

Effects of PACAP on morphologically identified myenteric neurons in guinea pig small bowel.

Intracellular microelectrodes were used to examine the actions of pituitary adenylate cyclase-activating peptide (PACAP) on morphologically identified myenteric neurons and glial cells of the guinea pig small bowel. PACAP-27 and PACAP-38 evoked excitatory responses in 96% of after hyperpolarizing (AH)/type 2 neurons. The half-maximal concentration for PACAP-27 was 1.5 nM. The responses consisted of membrane depolarization in association with increased input resistance, suppression of hyperpolarizing afterpotentials, and repetitive spike discharge. Forskolin mimicked the action of PACAP in all AH/type 2 neurons. PACAP excited 36% of S/type 1 neurons. Most of the AH/type 2 neurons had Dogiel II morphology, whereas the S/type 1 neurons were uniaxonal with morphology characteristics of Dogiel I or filamentous neurons. No glial cells responded to PACAP. A selective A1 adenosine receptor agonist blocked the excitatory action of PACAP, and this was reversed by a selective A1 antagonist. The results suggest that excitatory PACAP receptors and inhibitory adenosine A1 receptors are linked to adenylate cyclase in AH/type 2 myenteric neurons.

Suppression of nicotinic synaptic transmission by adenosine in myenteric ganglia of the guinea-pig gastric antrum.

Conventional intracellular recording techniques were used to investigate actions of adenosine on nicotinic cholinergic transmission in myenteric neurons of the gastric antrum. Adenosine or the more potent derivatives, 5'-N-ethylcarboxamidoadenosine (NECA), 5'-N-cyclopropylcarboxamidoadenosine, 1-deaza-2-chloro-N6-cyclopentyladenosine or N6-cyclopentyladenosine reversibly and dose dependently inhibited the fast excitatory postsynaptic potentials (fast EPSPs) in 60% of the gastric neurons. Neither adenosine nor NECA affected excitatory responses to the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodine. The EC50 concentration for inhibition of the fast excitatory postsynaptic potential (EPSP) by adenosine was 55 microM NECA was a more potent inhibitor than adenosine. The specific adenosine receptor antagonists 1,3-dipropyl-8-p-sulfophenyl xanthine or 1,3-dipropyl-8-(cyclopentyl) xanthine blocked the inhibitory effects of adenosine or NECA. Fast EPSPs were enhanced by superfusion of the antagonists alone, suggestive of ongoing inhibition of nicotinic transmission by endogenous adenosine. The antagonists had no effect on resting membrane properties, excitability or antidromic action potentials. In neurons with suppression of fast EPSPs, adenosine did not suppress all cholinergic inputs to the same neuron. The results suggest that adenosine inhibits nicotinic transmission by interacting with presynaptic P1 adenosine receptors located at cholinergic release sites.

Neuropharmacology of the muscarinic antagonist telenzepine in myenteric ganglia of the guinea-pig small intestine.

Intracellular recording methods were used to investigate the actions of the putative M1 muscarinic receptor antagonist telenzepine on the electrical and synaptic behavior of myenteric neurons. Telenzepine had no effect on resting membrane potential, input resistance, excitability and antidromic potentials in both AH/type 2 and S/type 1 neurons, when applied in concentrations of 0.1-2000 nM, although higher concentrations (10-100 microM) did have a significant non-specific effect on the postsynaptic membrane. Micromolar concentrations of telenpzepine (1-2 microM) had no effect on excitatory responses to substance P, vasoactive intestinal peptide, the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium or the nicotinic action of acetylcholine. Nicotinic fast excitatory postsynaptic potentials were also unaffected by 2 microM telenzepine. In contrast, at submicromolar concentrations (100 nM), telenzepine abolished responses to either muscarine or the muscarinic component of the acetylcholine response. The excitatory effect of muscarine at postsynaptic M1 receptors was dose dependently inhibited by telenzepine (0.1-1000 nM) at concentrations which had no effect on the electrical properties of the cells. This effect was slowly reversible, usually requiring more than 60 min for significant recovery. The threshold dose of telenzepine as an antagonist of the muscarinic depolarization in AH/type 2 neurons was in the range of 0.1-1 nM. The IC50 concentration of telenzepine needed to abolish the response was 8.5 nM. A small proportion of stimulus-evoked slow excitatory postsynaptic potentials in both AH/type 2 and S/type 1 cells were abolished by 1 microM telenzepine, while the majority of them remained unaffected, indicating that some slow excitatory postsynaptic potentials are mediated by the muscarinic action of released acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide release from isolated myenteric nerve endings derived from the guinea pig myenteric plexus.

Isolated myenteric nerve varicosities prepared from the myenteric plexus of the guinea pig ileum were investigated as a suitable model system with which to study the release of several neuropeptide-like immunoreactivities (-LI). Basal release of substance P-LI, neurokinin A-LI, Leu-enkephalin-LI and Met-enkephalin-LI was determined, and clear depolarization-induced release of the enkephalin-LI's and neurokinin A-LI was obtained using this preparation, providing further support for their roles as putative mediators in the enteric nervous system. Evoked-release of these peptides was dependent on the presence in the incubation mixture of certain antagonists to known endogenous neuronal mediators. In the absence of such antagonists, no unequivocal evidence of release was seen. Clear evoked release of Leu-enkephalin-LI occurred only in the presence of the adenosine receptor antagonist 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX), atropine and naloxone. Release of Met-enkephalin-LI occurred in the presence of either atropine or naloxone. The release of neurokinin A-LI was evident in the presence of DPSPX. These findings suggest the existence of either distinct subpopulations of nerve varicosities or distinct neuronal pools containing each peptide and that these peptides may be under differential regulation by endogenous inhibitory mediators. It is concluded that, under suitable conditions, isolated myenteric nerve varicosities provide a useful model system for the study of release, and the modulation of release, of endogenous neuropeptides.