Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation.

Desensitization of the neurokinin-1 receptor (NK1-R) in neurons: effects of substance P on the distribution of NK1-R, Galphaq/11, G-protein receptor kinase-2/3, and beta-arrestin-1/2.

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and beta-arrestins mediate desensitization and endocytosis of G-protein-coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, Galphaq/11, GRK-2 and -3, and beta-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive Galphaq/11, GRK-2 and -3, and beta-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R-mediated increase in [Ca2+]i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of beta-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of beta-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons Galphaq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and beta-arrestin-1 and -2. This regulation will determine whether NK1-R-expressing neurons participate in functionally important reflexes.

Nitric oxide targets in the guinea-pig intestine identified by induction of cyclic GMP immunoreactivity.

The immunohistochemical localization of cyclic GMP was used to determine potential physiological sites of action of nitric oxide in the guinea-pig small intestine and colon. In control tissue, cyclic GMP-immunoreactivity was observed only in macrophages, whose identity was confirmed by double-label experiments using either F4/80, a macrophage-specific antibody, or fluorescein isothiocyanate-labelled dextran injected intravenously. Following exposure to the nitric oxide donor, sodium nitroprusside, cyclic GMP-immunoreactivity was induced in subpopulations of neurons in the myenteric and submucosal plexuses of the ileum and colon. In the colon, cyclic GMP-immunoreactivity was induced in 5-10% of myenteric neurons. The cyclic GMP-immunoreactive neurons did not contain nitric oxide synthase. In the ileum, cyclic GMP-immunoreactive neurons comprised about 2% of myenteric neurons and 40% of submucosal neurons; these cyclic GMP-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide, but they did not contain nitric oxide synthase. Interstitial cells between the mesothelium and the longitudinal muscle layer, vascular smooth muscle and vascular pericytes also showed sodium nitroprusside-induced cyclic GMP-immunoreactivity. The interstitial cells of Cajal at the inner surface of the circular muscle layer and the smooth muscle cells of the circular and longitudinal muscle layers showed increases in cyclic GMP-immunoreactivity that varied in extent from animal to animal. The results suggest that nitric oxide could act at several sites in the intestine through the stimulation of guanylyl cyclase.

Activation and internalization of the mu-opioid receptor by the newly discovered endogenous agonists, endomorphin-1 and endomorphin-2.

The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal miro-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for micro-opioid receptors. Since the miro-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the micro-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged micro-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the micro-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced micro-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 approximately 8.4) is consistent with antagonism at the micro-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the micro-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced micro-opioid receptor activation can be visualized by receptor endocytosis.

Electrophysiological analysis of the actions of pituitary adenylyl cyclase activating peptide in the taenia of the guinea-pig caecum.

The actions of pituitary adenylyl cyclase activating peptide (PACAP) on membrane potential and conductance were investigated in the taenia of the guinea-pig caecum. The possible role of PACAP in inhibitory transmission was also investigated. Membrane potentials of smooth muscle cells were measured by intracellular microelectrodes, in the presence of hyoscine and nifidepine (both 10(-6)M. To determine conductance changes, current was passed from external plate electrodes using the technique of Abe and Tomita (1968). PACAP-27 caused a concentration dependent hyperpolarization of the muscle with a maximum of 12-15 mV at 10(-6)M. The hyperpolarization caused by PACAP was associated with a substantial increase in membrane conductance. The hyperpolarization was abolished by apamin (10(-6)M), a blocker of small conductance, calcium-dependent, potassium channels, and was reduced to about 50% by suramin (10(-4)M), which is an antagonist of P2 receptors for purines. The hyperpolarization was not reduced by tetrodotoxin (2 x 10(-6)M), suggesting PACAP acts directly on the muscle. With continued exposure to PACAP, the hyperpolarization decayed back to resting membrane potential after several minutes, possibly due to receptor desensitization. Inhibitory junction potentials (IJPs) were markedly reduced in amplitude in the period of presumed receptor desensitization to PACAP, were abolished by tetrodotoxin, but were not affected by suramin. Apamin abolished the IJP and revealed a small excitatory junction potential. This study implies that PACAP released from nerve fibres in the taenia caeci hyperpolarizes the muscle via an opening of apamin-sensitive potassium channels. The action is probably through type I PACAP receptors.

Projections of nitric oxide synthesizing neurons in the guinea-pig colon.

The neuronal form of the enzyme nitric oxide synthase, which is an obligatory constituent of neurons that utilise nitric oxide as a transmitter, was revealed histochemically in this study by its ability to transfer a proton from reduced nicotinamide adenine dinucleotide phosphate to nitro-blue tetrazolium. In the guinea-pig colon, nitric oxide synthase was located in numerous irregularly-shaped myenteric neurons with single axons. In the submucosa, a small number of neurons had strong enzyme activity, whereas many were weakly stained. Nerve fibres were found in the longitudinal muscle, circular muscle, muscularis mucosae and ganglia of the two plexuses. No nerve fibres were found in the lamina propria of the mucosa. The same distribution of nerve cells and fibres was revealed using immunohistochemistry for nitric oxide synthase. Lesion studies showed that the axons of myenteric neurons all projected anally. Myenteric cells were the source of nerve fibres in the circular muscle and in more anally located myenteric ganglia. The sparse innervation of submucous ganglia was intrinsic to the submucous plexus. It is suggested that nitric oxide synthase is one of the transmitters of inhibitory neurons to the muscle and is also utilized by descending interneurons of the myenteric plexus.

Gastrointestinal neurotransmitters.

The enteric nervous system contains neurones that are intrinsic to the gastrointestinal tract and the axons of extrinsic neurones. More than 30 functional types of neurone are present and about 25 different possible neurotransmitters have been identified in enteric neurones. Most neurones utilize several transmitters; amongst the transmitters of an individual neurone, one is usually a primary transmitter and other substances are subsidiary transmitters or neuromodulators. The primary transmitter is the substance that has the major role in acutely changing the excitability of the innervated cell. Current evidence indicates that primary transmitters are strongly conserved; that is, the same substance will be the neurotransmitter in functionally equivalent neurones in different regions of the gastrointestinal tract and in different species. In contrast, subsidiary transmitters and neuromodulators of equivalent neurones in different regions are not necessarily the same. Only about seven of the approximately 25 enteric neurotransmitters are known to be primary transmitters. Acetylcholine is the primary transmitter of vagal and pelvic preganglionic neurones, of enteric interneurones, of one class of secretomotor neurone in the intestine and of motor neurones controlling gastric acid secretion. Acetylcholine and tachykinins are co-primary transmitters of muscle motor neurones, with acetylcholine appearing to have the greater role. Tachykinins are probably primary transmitters of enteric sensory neurones at neuroneuronal synapses. Serotonin may also be a transmitter to neurones in the enteric ganglia. Nitric oxide appears to be the usual primary transmitter of enteric inhibitory motor neurones to the muscle. ATP and vasoactive intestinal peptide are subsidiary transmitters of these neurones, although in some regions they may have a primary transmitter role. Vasoactive intestinal peptide is the primary transmitter of non-cholinergic secretomotor neurones. Gastrin releasing peptide is the primary transmitter of motor neurones to gastrin cells. Noradrenaline is the primary transmitter of sympathetic neurones that supply the intestine.

Calretinin immunoreactivity of motor neurons in the guinea-pig distal colon and taenia coli.

Calretinin is a calcium-binding protein which occurs in neurons and endocrine cells, including neurons throughout the gastrointestinal tract. Calretinin-immunoreactive (IR) neurons innervate the circular muscle in the guinea-pig distal colon and have descending as well as ascending projections. This suggests that calretinin-IR is in motor neurons, but whether it might be in excitatory or inhibitory motor neurons or both was previously undetermined. The presence of calretinin-IR in neurons innervating the taenia coli has not been previously reported. Numerous fibres in the circular muscle of the distal colon and in the taenia coli displayed immunoreactivity for calretinin. Tachykinin (TK), vasoactive intestinal peptide (VIP), calretinin, and gamma-aminobutyric acid (GABA) immunoreactivity was also in fibres innervating these targets. The abundances of these fibres was estimated to be TK > VIP > calretinin > GABA. Double label immunohistochemistry revealed the presence in both tissues of populations of calretinin-IR fibres which were also TK-IR, and fibres with calretinin and GABA-IR in the colon, but calretinin-IR fibres were never VIP-IR. TK- and VIP-IR were in separate populations of nerve fibres as were GABA- and TK-IR. It is concluded that calretinin-IR does not provide a definitive labelling of a physiologically known subgroup of motor neurons, either in the distal colon or in the taenia coli, but that calretinin is most likely to be in excitatory motor neurons.

G protein-coupled receptors in gastrointestinal physiology. II. Regulation of neuropeptide receptors in enteric neurons.

Neuropeptides exert their diverse biological effects by interacting with G protein-coupled receptors (GPCRs). In this review we address the question, What regulates the ability of a target cell, in particular a neuron, to respond to a neuropeptide? Available evidence from studies of many GPCRs in reconstituted systems and transfected cell lines indicates that much of this regulation occurs at the level of the receptor and serves to alter the capacity of the receptor to bind ligands with high affinity and to couple to heterotrimeric G proteins. Although some of the knowledge gained from these studies is applicable to the regulation of neuropeptide receptors on neurons, at present there are far more questions than answers.

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon.

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.

Direct measurement of the release of ATP and its major metabolites from the nerve fibres of the guinea-pig taenia coli.

1. The release of adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate and adenosine from guinea-pig taenia coli in response to electrical stimulation of intramural nerves was measured directly using high performance liquid chromatography separation and fluorometric detection. 2. Purines were released in a frequency-dependent manner by trains of transmural electrical pulses at 1-30 Hz. 3. Electrically evoked release of ATP was abolished by tetrodotoxin (10(-6)mol/L) but was not affected by nicardipine (10(-6)mol/L). 4. The release of purines was reduced in the presence of atropine. 5. Pituitary adenylyl cyclase-activating peptide did not evoke the release of any purines and did not modify the electrically evoked release of purines. 6. The results suggest that ATP and its major metabolites are released from a neuronal source, possibly the enteric inhibitory nerves, in the guinea-pig taenia coli.

Mechanisms attenuating cellular responses to neuropeptides: extracellular degradation of ligands and desensitization of receptors.

Neuropeptides make up one of the largest and functionally most diverse groups of signaling molecules. They exert their effects by interacting with members of the large family of G-protein-coupled receptors, which transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins. Cellular responses to neuropeptides are usually rapidly attenuated. Mechanisms of signal attenuation include removal of peptides from the extracellular fluid and receptor desensitization. Peptides are removed from the extracellular fluid principally by enzymatic degradation by cell surface enzymes, exemplified by neutral endopeptidase. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second messenger kinases, interaction of receptors with arrestins, and consequent receptor uncoupling from G-proteins. Peptides also induce endocytosis of their receptors, which may contribute to desensitization by depleting the cell surface of high-affinity receptors. Recycling and processing of internalized receptors, which include dissociation of receptors from their ligands and receptor dephosphorylation, contribute to resensitization of cellular responses. These regulatory mechanisms are important for they determine the ability of cells to respond to agonists, and defects may result in uncontrolled stimulation of cells, which could cause disease. A greater understanding of the processes that modulate signaling by neuropeptides may lead to the development of novel receptor antagonists and agonists and help to explain the mechanism of drug tolerance.

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon.

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.

Histochemical, pharmacological, biochemical and chromatographic evidence that pituitary adenylyl cyclase activating peptide is involved in inhibitory neurotransmission in the taenia of the guinea-pig caecum.

The possibility that pituitary adenylyl cyclase-activating peptide (PACAP) is an inhibitory neurotransmitter has been investigated in the taenia of the guinea-pig caecum. The action of PACAP on muscle contractility and its ability to alter levels of adenosine-3':5'-cyclic monophosphate (cyclic AMP) and guanosine-3':5'-cyclic monophosphate (cyclic GMP) were investigated. PACAP-1-27 was an effective agonist, giving relaxations comparable in magnitude to isoproterenol; its EC50 was 3.4 x 10(-7) M. PACAP (10(-6) M) caused an almost two-fold increase in cyclic AMP levels; but the level of cyclic GMP was not affected. The relaxation caused by PACAP was slow in onset, with a latency of 5.8 +/- 0.8 s and reached a maximum at 9.1 +/- 1.1 s after onset. The relaxation was significantly reduced by apamin (10(-6) M) and suramin (10(-4) M) but was not reduced by tetrodotoxin (10(-7) M). Relaxation of the taenia coli caused by electrical stimulation of the inhibitory nerves was greatly reduced by apamin but only slightly reduced by suramin. PACAP-like immunoreactivity (-IR) was localised immunohistochemically in varicose nerve fibres within the taenia coli and in the underlying myenteric plexus and circular muscle. Approx. 50% of vasoactive intestinal peptide (VIP)-IR nerve fibres in the taenia also had immunoreactivity for PACAP; conversely, almost all PACAP-IR fibres were immunoreactive for VIP. PACAP-IR and substance P (SP)-IR were generally in separate fibres; only about 5% of SP-IR fibres were PACAP-IR. Radioimmunoassay revealed tissue concentrations of PACAP-1-27 and PACAP-1-38 of 1.0 +/- 0.1 and 2.1 +/- 0.3 (SEM) pmol/g wet weight of tissue, respectively. Material with PACAP-1-27 immunoreactivity co-eluted with authentic PACAP-1-27 on gel filtration chromatography, and PACAP-1-38 immunoreactivity also co-eluted with the authentic peptide. This study provides structural, chemical and pharmacological evidence that PACAP could be involved in inhibitory neurotransmission to the taenia coli of the guinea-pig caecum.

Plurichemical transmission and chemical coding of neurons in the digestive tract.

The enteric nervous system contains neurons with well-defined functions. However, when neurons of the same function are examined in different regions or species, they are found to show subtle differences in their pharmacologies of transmission and different chemical coding. Individual enteric neurons use more than one transmitter, i.e., transmission is plurichemical. For example, enteric inhibitory neurons have three or more primary transmitters, including nitric oxide, vasoactive intestinal peptide, and possibly adenosine triphosphate and pituitary adenylyl cyclase activating peptide. Primary transmitters are highly conserved, although their relative roles vary considerably between gut regions. Multiple substances, including transmitters and their synthesizing enzymes and nontransmitters (such as neurofilament proteins), provide neurons with a chemical coding through which their functions and projections can be identified. Although equivalent neurons in different regions have the same primary transmitters, other chemical markers differ substantially. Caution must be taken in extrapolating pharmacological and neurochemical observations between species or even between regions in the one species. On the other hand, careful interregion and interspecies comparisons lead to an understanding of the features of enteric neurons that are highly conserved and can be used in valid extrapolation.

Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2.

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1alpha from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

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.