Guinea Pig Myenteric Neurons Research Articles

Selectivity of antagonists for theCys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons

The three most common Cys-loop receptors expressed by myenteric neurons are nACh, 5-HT3 and GABAA . To investigate the function of these proteins researchers have used channel inhibitors such as hexamethonium (antagonist of nACh receptors), ondansetron (antagonist of 5-HT3 receptors), picrotoxin and bicuculline (both antagonists of GABAA receptors). The aim of this study was to investigate the specificity of these inhibitors on Cys-loop receptors of primary cultured neurons obtained from the guinea-pig small intestine. The whole-cell configuration of the patch clamp techniques was used to record membrane currents induced by ACh (IACh ), 5-HT (I5-HT ) and GABA (IGABA ) in the absence and the presence of various concentrations of hexamethonium, ondansetron, picrotoxin or bicuculline. The three Cys-loop receptors present in enteric neurons are expressed independently and they do not cross-desensitized. Hexamethonium inhibited IACh without affecting I5-HT and IGABA . Ondansetron inhibited I5-HT and also IACh but did not affect IGABA . Picrotoxin and bicuculline inhibited I5-HT , IACh and IGABA with different potency, being the lowest potency on 5-HT3 receptors. All these inhibitory effects were concentration dependent and reversible. Our observations showed that except for hexamethonium, all other inhibitors used here show different degrees of selectivity, which has to be considered when these antagonists are used in experimental studies aimed to investigate the functions of these receptors. In particular, in tissues expressing nACh receptors because these are the targets of all other inhibitors used here. The low potency of picrotoxin and bicuculline to inhibit 5-HT3 receptors suggests that these receptors are heteromeric proteins.

Dibenzo[1,2,5]thiadiazepines Are Non-Competitive GABAA Receptor Antagonists

A new process for obtaining dibenzo[c,f][1,2,5]thiadiazepines (DBTDs) and their effects on GABAA receptors of guinea pig myenteric neurons are described. Synthesis of DBTD derivatives began with two commercial aromatic compounds. An azide group was obtained after two sequential reactions, and the central ring was closed via a nitrene to obtain the tricyclic sulfonamides (DBTDs). Whole-cell recordings showed that DBTDs application did not affect the holding current but inhibited the currents induced by GABA (IGABA), which are mediated by GABAA receptors. These DBTDs effects reached their maximum 3 min after application and were: (i) reversible, (ii) concentration-dependent (with a rank order of potency of 2c = 2d > 2b), (iii) mediated by a non-competitive antagonism, and (iv) only observed when applied extracellularly. Picrotoxin (which binds in the channel mouth) and DBTDs effects were not modified when both substances were simultaneous applied. Our results indicate that DBTD acted on the extracellular domain of GABAA channels but independent of the picrotoxin, benzodiazepine, and GABA binding sites. DBTDs used here could be the initial model for synthesizing new GABAA receptor inhibitors with a potential to be used as antidotes for positive modulators of these receptors or to induce experimental epilepsy.

Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

Activity of the four known protease-activated receptors (PARs) has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (APs). Application of the PAR1-AP (TFLLR) or PAR4-AP (GYPGQV) evoked spike discharge in 79 or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs) of PAR1/PAR2 in 51%, PAR1/PAR4 in 43%, and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I ATP) in guinea pig myenteric neurons. I ATP was recorded in the great majority of tested neurons. ATP concentration-response curve (0.01–10 mM) showed two phases, the first mediated by high-sensitive P2X receptors (hsP2X receptors), observed between 0.01–0.3 mM and the second mediated by low-sensitive P2X receptors (lsP2X receptors). The calculated EC 50 values of these phases were 38 and 1759 μM, respectively. 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) concentration-response curve was monophasic (0.01–1 mM), and less potent (EC 50 142 μM) than ATP to activate hsP2X receptors. A strong inward rectification was noticed when hsP2X receptors were activated with ATP (0.1 mM) and for BzATP-induced currents (0.1 mM; I BzATP) but a significant lower rectification was noticed when lsP2X receptors were activated (5 mM). Brilliant blue G (BBG) at a concentration of 0.3 μM (known to inhibit only P2X7 receptors) reduced I ATP when lsP2X receptors contributed to it but neither affect hsP2X receptors nor I BzATP. However, hsP2X receptors and I BzATP were both inhibited by concentrations ≥ 1 μM of this antagonist. BzATP inhibited hsP2X receptors and therefore, it behaves as partial agonist on these receptors. Using the single-cell RT-PCR technique P2X7 mRNA was detectable in 7 out of 13 myenteric neurons exhibiting P2X2 mRNA. Altogether, our results show that low-sensitive P2X receptors are likely P2X7, whereas, the high-sensitive P2X channels are probably constituted, at least in part, by P2X2 subunits.

Recordings from human myenteric neurons using voltage-sensitive dyes

Voltage-sensitive dye (VSD) imaging became a powerful tool to detect neural activity in the enteric nervous system, including its routine use in submucous neurons in freshly dissected human tissue. However, VSD imaging of human myenteric neurons remained a challenge because of limited visibility of the ganglia and dye accessibility. We describe a protocol to apply VSD for recordings of human myenteric neurons in freshly dissected tissue and myenteric neurons in primary cultures. VSD imaging of guinea-pig myenteric neurons was used for reference. Electrical stimulation of interganglionic fiber tracts and exogenous application of nicotine or elevated KCl solution was used to evoke action potentials. Bath application of the VSDs Annine-6Plus, Di-4-ANEPPS, Di-8-ANEPPQ, Di-4-ANEPPDHQ or Di-8-ANEPPS revealed no neural signals in human tissue although most of these VSD worked in guinea-pig tissue. Unlike methylene blue and FM1–43, 4-Di-2-ASP did not influence spike discharge and was used in human tissue to visualize myenteric ganglia as a prerequisite for targeted intraganglionic VSD application. Of all VSDs, only intraganglionic injection of Di-8-ANEPPS by a volume controlled injector revealed neuronal signals in human tissue. Signal-to-noise ratio increased by addition of dipicrylamine to Di-8-ANEPPS (0.98±0.16 vs. 2.4±0.62). Establishing VSD imaging in primary cultures of human myenteric neurons led to a further improvement of signal-to-noise ratio. This allowed us to routinely record spike discharge after nicotine application. The described protocol enabled reliable VSD recordings from human myenteric neurons but may also be relevant for the use of other fluorescent dyes in human tissues.

Two suramin binding sites are present in guinea pig but only one in murine native P2X myenteric receptors

Whole-cell patch clamp recordings were used to characterise the physiological and pharmacological properties of P2X receptors of mouse and guinea pig myenteric neurons from the small intestine. ATP application induced a rapid inward current in 95% of recorded neurons of both species when were voltage clamped at − 60 mV. Concentration–response curves for ATP (1–3000 µM) yielded EC 50 values of 114 and 115 μM for mouse and guinea pig myenteric neurons, respectively, with a Hill coefficient value of 1.02 and 0.79, respectively, which were not significantly different of unity. α,β-methylene ATP (100 µM) was virtually inactive in both species. Pyridoxalphophate-6-azophenyl-2′,4′-disulphonic acid (0.01–30 µM) inhibited the ATP-induced currents ( I ATP) with a different potency; being the IC 50 0.6 and 1.8 μM in mouse and guinea pig, respectively. In mouse myenteric neurons, I ATP were inhibited by suramin whereas in guinea pig neurons we observed two effects, potentiation and inhibition of these currents. On guinea pig, both effects of suramin had different recovering kinetics and concentration dependency, indicating that they are mediated by at least two different binding sites. Our observations indicate that myenteric P2X receptors in these two species have different pharmacological properties.

Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones

Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB(1) receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI-related drug development such as in obesity treatment. The aim of the study was to investigate how CB(1) ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea-pig myenteric neurones were loaded with different fluorescent markers: Fluo-4 to measure network activity, FM1-43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB(1) receptors was confirmed by RT-PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB(1) receptor antagonists (AM-251 and SR141716), abolished by CB(1) activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA-5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB(1) agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin-positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB(1) receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.

P4.15 Regulation of N-type Ca 2+ channels by protein kinases in the guinea-pig myenteric neurons following inflammation

P4.15 Regulation of N-type Ca 2+ channels by protein kinases in the guinea-pig myenteric neurons following inflammation

P4.11 Exploring short term plasticity in the enteric nervous system: Voltage-sensitive dye recordings of guinea pig myenteric neurons

P4.11 Exploring short term plasticity in the enteric nervous system: Voltage-sensitive dye recordings of guinea pig myenteric neurons

606 Contribution of Na-K-2Cl Cotransporter to Gamma-Amino Butyric Acid (GABAA) Receptor-Mediated Membrane Depolarization in Guinea Pig Myenteric Neurons

606 Contribution of Na-K-2Cl Cotransporter to Gamma-Amino Butyric Acid (GABAA) Receptor-Mediated Membrane Depolarization in Guinea Pig Myenteric Neurons

P2Y1 receptors mediate inhibitory neuromuscular transmission and enteric neuronal activation in small intestine

There is increasing evidence that adenosine 5'-triphosphate or a related purine plays a crucial role in smooth muscle relaxation and enteric synaptic neurotransmission. Accordingly, the aim of the present work is to investigate the role P2Y(1) receptors in purinergic inhibitory neurotransmission (pig ileum) and enteric neuronal activation in the small intestine (guinea-pig ileum). Using contractility measurements, micro-electrode recordings and Ca(2+) imaging we found that (i) adenosine 5'-Omicron-2-thiodiphosphate (ADPbetaS) (10 micromol L(-1)) caused smooth muscle relaxation and hyperpolarization that was antagonized by MRS2179 (10 micromol L(-1)) a P2Y(1) receptor antagonist and apamin (1 micromol L(-1)); (ii) electrical field stimulation (EFS) caused a non-nitrergic inhibitory junction potential (IJP) and relaxation that was antagonized by MRS2179 (10 micromol L(-1)); (iii) P2Y(1) receptors were immunolocalized in smooth muscle cells and enteric neurons; (i.v.) superfusion of ADPbetaS (1 micromol L(-1)) induced Ca(2+) transients in myenteric neurons that were inhibited by MRS2179 (1 micromol L(-1)), but not by tetrodotoxin (1 micromol L(-1)); and (v) EFS induced calcium transients were partially inhibited by MRS2179 (1 micromol L(-1)). We conclude that in the small intestine purinergic neuromuscular transmission responsible for the IJP and non-nitrergic relaxation is mediated by P2Y(1) receptors located in smooth muscle cells. Functional P2Y(1) receptors are also present in guinea-pig myenteric neurons. Therefore, P2Y(1) receptors might be an important pharmacological target to modulate gastrointestinal functions.

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.

GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways

GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca 2+ concentration of guinea-pig myenteric neurons (at 35°C) using Fura-2-AM. Neurons were identified by 75mM K + depolarization (5 s), which evoked a transient intracellular Ca 2+ concentration increase. GABA (10 s) induced a dose dependent (5nM–1μM) transient intracellular Ca 2+ concentration rise in the majority of neurons (500nM GABA: 251±17nM, n=232/289). Interestingly, the response to 5μM GABA ( n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly ( P<0.001) reduced by GABA A and GABA B receptor antagonists (10μM) bicuculline and phaclofen. The GABA A agonist isoguvacine (10μM) and GABA B agonist baclofen (10μM) induced similar responses as 50nM GABA, while the GABA C agonist cis-4-aminocrotonic acid (CACA) (10μM) only elicited small responses in a minority of neurons. Removal of extracellular Ca 2+ abolished all responses while depletion of intracellular Ca 2+ stores by thapsigargin (5μM) did not alter the responses to 500nM GABA ( n=13), but reduction of Ca 2+ influx through voltage-dependent Ca 2+ channels did. The nicotinic antagonist hexamethonium (100μM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and the 5-HT 3 receptor blocker ondansetron only had minor effects. Conclusion: GABA elicits transient intracellular Ca 2+ concentration responses in the majority of myenteric neurons through activation of GABA A and GABA B receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5μM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.

CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca(2+) imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10(-6) M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K(+)-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10(-6) M and CRF 10(-7) M during 30 s induced a Ca(2+) response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10(-7) M, and its effect at 10(-6) M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10(-6) M (n = 70) could not block the CRF-induced Ca(2+) transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca(2+) blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced Ca(2+) transients. Blocking N- or P/Q-type Ca(2+) channels did not reduce the responses significantly. Combined L- and R-type Ca(2+) channel blocking (SNX-482 10(-8) M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca(2+) transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca(2+) transients highly depend on somatic calcium influx through voltage-operated Ca(2+) channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca(2+) signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.

Glutamate stimulation of acetylcholine release from myenteric plexus is mediated by endogenous nitric oxide

Glutamate was found to be an excitatory neurotransmitter in the enteric nervous system. Although several lines of evidence indicate a role of glutamate in the regulation of gut motility and secretion the physiological significance of glutamatergic transmission is not clear yet. We studied the effect of glutamate on [ 3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining in longitudinal muscle strips with attached myenteric plexus of guinea pig ileum. l-Glutamate (100 μM) significantly enhanced both the evoked [ 3H]acetylcholine release and the optical density of nicotinamide adenine dinucleotide phosphate-diaphorase positive neurones, i.e. the intensity of staining. The non-competitive N-methyl- d-aspartate receptor antagonist MK-801 (3 μM) abolished the stimulatory effect of l-glutamate on acetylcholine efflux. Similarly, the nitric oxide synthase inhibitor N ω-nitro- l-arginine (100 μM) significantly reduced the effect of l-glutamate on [ 3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining. Our data suggest that endogenous nitric oxide seems to mediate the stimulatory effect of glutamate on acetylcholine release from guinea pig myenteric neurons.

TEA- and apamin-resistant K(Ca) channels in guinea-pig myenteric neurons: slow AHP channels.

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K(+) channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (P(o)) of TEA-resistant K(+) channels following action potentials. The P(o) increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca(2+)-activated K(+) (K(Ca)) channels were present in inside-out patches excised from AH neurons. The P(o) of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca(2+)] from < 10 nM to either 500 nM or 10 microM. P(o) was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant K(Ca) channels measured approximately 60 pS under symmetric K(+) concentrations between -60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K(+)], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant K(Ca) channels by internal Ca(2+) in excised patches was not reversed by washing out the Ca(2+)-containing solution and replacing it with nominally Ca(2+)-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant K(Ca) channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca(2+) dependence of the activation of TEA- and apamin-resistant K(Ca) channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca(2+) to about 7 ms in the presence of submicromolar cytoplasmic Ca(2+). The Ca(2+)-insensitive closed dwell time had a time constant of about 1 ms. We propose that these small-to-intermediate conductance TEA- and apamin-resistant Ca(2+)-activated K(+) channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.

Functional overlap of IP(3)- and cADP-ribose-sensitive calcium stores in guinea pig myenteric neurons.

In myenteric neurons two different receptor subtypes govern the intracellular Ca(2+) stores: the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) and the ryanodine receptor (RyR). Their degree of functional overlap was determined by examining Ca(2+) release in these cells through both superfusion techniques and intracellular microinjection. Microinjection of IP(3) (50 microM) and cADP-ribose (cADPr, 50 microM), specific ligands for the IP(3)R and RyR, respectively, demonstrated mobilization of intracellular Ca(2+) stores. Perfusion with cinnarizine (50 microM) or dantrolene (10 microM), antagonists of the IP(3)R and RyR, respectively, eliminated the Ca(2+) response to microinjected IP(3) and cADPr. Superfusion of the neurons with 100 microM ATP, an IP(3)-mediated Ca(2+)-mobilizing agonist, caused intracellular Ca(2+) increments, which were antagonized by cinnarizine, and the RyR antagonists dantrolene, procaine (5 mM), and ryanodine (1 microM). Caffeine (10 mM) was applied repetitively in Ca(2+)-free conditions to deplete RyR-sensitive stores; subsequent perfusion with ATP demonstrated a Ca(2+) response. Conversely, caffeine caused a Ca(2+) response after repetitive ATP exposures. The internal Ca(2+) stores of myenteric neurons are governed by two receptor subtypes, IP(3)R and RyR, which share partial functional overlap.

Alosetron.

Alosetron is a potent and highly selective serotonin 5-HT3 receptor antagonist which has been evaluated for the management of irritable bowel syndrome (IBS). It blocked the fast 5HT3-mediated depolarisation of guinea-pig myenteric and submucosal neurons in vitro, with half-maximal inhibition at approximately 55 nmol/L. Alosetron attenuated the visceral nociceptive effect of rectal distension in conscious or anaesthetised dogs. It increased the compliance of the colon to distension in patients with IBS and delayed colonic transit in patients with IBS or carcinoid diarrhoea and in healthy volunteers. A single dose of alosetron 4 mg increased in vivo fluid absorption in normal human small intestine. In clinical trials in patients with IBS, alosetron 1 mg twice daily was effective in relieving abdominal pain and discomfort. Alosetron was most effective in female patients and particularly in those with diarrhoea-predominant IBS. In patients with IBS and healthy volunteers who received alosetron, the most common adverse event was constipation.

Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2.

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Non‐additive interaction between nicotinic cholinergic and P2X purine receptors in guinea‐pig enteric neurons in culture

1. Acetylcholine (ACh)-activated currents and their interaction with ATP-activated currents were studied in primary cultures of myenteric neurons from guinea-pig small intestine using patch clamp techniques. Peak currents caused by co-application of ACh (1 mM) and ATP (300 microM) were 78 +/- 2 % of the sum of currents activated by each agonist alone (P < 0.05, n = 29). Reversal potentials measured during co-application of ACh and ATP did not differ from those measured during application of ACh or ATP alone. Addition of BAPTA (10 mM) to the pipette solution or replacement of extracellular Ca2+ with Na+ did not prevent occlusion. 2. Responses caused by co-application of 5-HT (300 microM), acting at 5-HT3 receptors, and ACh (3 mM) or ATP (1 mM) were additive (94 +/- 3 or 96 +/- 4 %, respectively, of the sum of currents activated by 5-HT and ACh or ATP alone; P > 0.05). Currents caused by GABA (1 mM), acting at GABAA receptors, and ACh (3 mM) or ATP (1 mM) were also additive (105 +/- 4 or 100 +/- 3 %, respectively, of the sum of currents activated by GABA and ACh or GABA and ATP applied separately; P > 0. 05). 3. Single channel currents caused by ACh and ATP in the same outside-out patches were less than additive (85 +/- 10 % of the predicted sum, P < 0.05). 4. P2X receptors and nicotinic cholinergic receptors (nAChRs) are linked in a mutually inhibitory manner in guinea-pig myenteric neurons. The functional interaction does not involve ligand binding sites, Ca2+-dependent mechanisms, a change in the driving force for Na+ or cytoplasmic signalling mechanisms.