In vitro pharmacology of inosine, with special reference to possible interactions with capsaicin-sensitive mechanisms and inflammatory mediators.

The in vitro pharmacology of inosine (Ino), a putative anti-inflammatory compound, has been investigated in smooth muscle preparations, with emphasis on its possible interaction with known inflammatory mediators, as well as capsaicin, an inducer of "neurogenic inflammation". The highest concentration of Ino routinely studied was 1 mM, since 10 mM nonspecifically inhibited many types of smooth muscle motor responses. In the guinea pig isolated ileum or trachea, Ino (1 mM) failed to influence the excitatory effect of capsaicin. The nitric oxide (NO)-mediated relaxant effect of capsaicin in the human colonic circular muscle was not influenced by Ino. Ino only weakly reduced the contractile effect of histamine on the guinea pig ileum. Substance P-mediated nonadrenergic, noncholinergic (NANC) contractions evoked by electrical stimulation in the guinea pig ileum were inhibited by half by Ino (1 mM). Ino showed no or only a weak inhibitory effect on NANC relaxation of the rat ileum. Arachidonic acid- or leukotriene D(4)-induced contractions of the guinea pig ileum were only moderately inhibited by Ino. Collectively, these results indicate that Ino (up to 1 mM) shows no major antagonist activity at histamine H(1) receptors, leukotriene CysLT(1) receptors, the transient receptor potential channel TRPV1 or tachykinin NK(1) or NK(2) receptors, or cyclooxygenase-inhibitory activity. Therefore, its anti-inflammatory activity is probably not associated with these mechanisms. The in vitro methods used in this study are capable of detecting a wide range of biological effects and hence may be recommended as a screening procedure for potential drugs or natural products.

Role of extrinsic afferent neurons in gastrointestinal motility.

Capsaicin-sensitive extrinsic afferent nerves have been demonstrated to release biologically active substances in the gastrointestinal (GI) tract. This fact may be useful for identifying sensory transmitter substances in isolated organ experiments. In the GI tract of animals neuropeptides like tachykinins and calcitonin gene-related peptide (CGRP) mediate specific excitatory and inhibitory effects of capsaicin; some evidence indicates a participation of purinergic mechanisms as well. The human gut (especially the circular musculature) is powerfully relaxed by capsaicin, and this effect seems to have a completely different transmitter background (nitric oxide (NO) and maybe VIP, neither of them of intrinsic neuronal origin). We propose that NO may be a sensory neurotransmitter. The "local efferent" (mediator-releasing) effect of extrinsic afferent neurons can also be demonstrated in vivo, both in animals and man. Yet, nearly normal motility of the small and large intestines (i.e., the most "autonomous" part of the GI tract) is maintained in animals with functionally inhibited capsaicin-sensitive nerves. The importance of this system in regulating GI movements may be exaggerated under pathopysiological conditions, first of all inflammation. The afferent function of capsaicin-sensitive nerves plays a role in sympathetic reflexes, such as the inhibition of GI motility after laparotomy or by peritoneal irritation.

P2 purinoceptor antagonists inhibit the non-adrenergic, non-cholinergic relaxation of the human colon in vitro.

Neurotransmitters released by myenteric neurons regulate movements of intestinal smooth muscles. There has been little pharmacological evidence for a role of purinergic mechanisms in the non-adrenergic, non-cholinergic (NANC) relaxation of the human large intestine. We used P(2) purinoceptor antagonists to assess whether such receptors are involved in the NANC relaxation of the circular muscle of the human sigmoid colon. It was also investigated whether the guanylate cyclase enzyme mediates the NANC response. Human colonic circular strips were tested in organ bath experiments with isotonic recording. NANC, non-nitrergic relaxations induced by electrical field stimulation (1 and 10 Hz, in the presence of atropine, guanethidine, and 100 microM N(G)-nitro-L-arginine [L-NOARG]) were strongly inhibited by a combination of the P(2) purinoceptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-sulfonic acid (PPADS) (50 microM) and suramin (100 microM). PPADS plus suramin was ineffective in the absence of L-NOARG. L-NOARG alone significantly reduced the NANC relaxation to electrical stimulation. PPADS plus suramin strongly inhibited the relaxation in response to exogenous alpha,beta-methylene ATP. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (3 microM) inhibited the NANC relaxation, but did not add to its reduction by L-NOARG. L-NOARG was still slightly effective in the presence of ODQ. Vasoactive intestinal polypeptide tachyphylaxis failed to influence the non-nitrergic NANC relaxation. It is concluded that nitric oxide (NO) and ATP co-mediate, in a non-additive manner, the NANC relaxation. NO probably acts through the guanylate cyclase, though a small fraction of its effect might be mediated by other mechanisms. Activators of the guanylate cyclase other than NO do not seem to participate in the NANC relaxation.

Pharmacological evidence for the presence of cholecystokinin-containing neurones in the mesenteric nerves supplying the guinea-pig ileum.

In guanethidine-treated, capsaicin-desensitized segments of guinea-pig isolated ileum electrical stimulation of mesenteric nerves resulted in contractions reaching 10 to 30% of the maximal longitudinal spasm. These responses were abolished by tetrodotoxin or atropine, and were reduced by half by the selective cholecystokinin antagonist lorglumide. It is concluded that neuronal processes whose endings release cholecystokinin-like material within the gut wall run in the mesenteric nerves and contribute to the capsaicin-resistant contractile response by releasing endogenous acetylcholine.

Is ganglionic transmission through nicotinic receptors essential for the peristaltic reflex in the guinea-pig ileum?

Peristaltic reflex activity in the guinea-pig isolated ileum was elicited by slow intraluminal infusion of Tyrode solution. The reflex was abolished by the ganglionic blocking drug hexamethonium. However, in more than half of the preparations, the peristaltic reflex was restored by the opioid antagonist naloxone. Hexamethonium-resistant peristaltic waves were blocked by tetrodotoxin or atropine. These data suggest that ganglionic transmission through nicotinic acetylcholine receptors may not be essential for the peristaltic reflex provided that the inhibitory action of endogenous opioids has been eliminated.

Tachykinin NK1 and NK2 receptor-mediated control of peristaltic propulsion in the guinea-pig small intestine in vitro.

The tachykinins substance P and neurokinin A are excitatory cotransmitters of cholinergic enteric neurons, their actions being mediated by NK1, NK2 and NK3 receptors. This study examined which of these receptors are part of the neural circuitry of peristalsis. Peristaltic propulsion in luminally perfused segments of the guinea-pig isolated ileum was elicited by a rise of the intraluminal pressure. The pressure threshold at which peristaltic contractions were triggered was used to quantify drug effects on peristalsis, inhibition of peristalsis being reflected by an increase in the pressure threshold. The NK1, NK2 and NK3 receptor antagonists SR-140333, SR-48968 and SR-142 801 (each at 0.1 microM), respectively, had little effect on peristaltic activity as long as cholinergic transmission was left intact. However, both the NK1 and NK2 receptor antagonist (each at 0.1 microM) abolished peristalsis after cholinergic transmission via muscarinic receptors had been blocked by atropine (1 microM) and peristalsis rescued by naloxone (0.5 microM). When cholinergic transmission via nicotinic receptors was suppressed by hexamethonium (100 microM) and peristalsis restored by naloxone (0.5 microM), only the NK2 receptor antagonist (0.1 microM) was able to attenuate peristaltic performance as deduced from a rise of the peristaltic pressure threshold by 106%. The NK3 receptor antagonist (0.1 microM) lacked a major influence on peristalsis under any experimental condition. It is concluded that tachykinins acting via NK1 and NK2 receptors sustain intestinal peristalsis when cholinergic neuroneuronal and neuromuscular transmission via muscarinic receptors has been suppressed. NK2 receptors help maintaining peristalsis once cholinergic neuroneuronal transmission via nicotinic receptors has been blocked, whereas NK3 receptors play little role in the neural pathways of peristalsis.

Evidence for an involvement of substance P, but not cholecystokinin-like peptides, in hexamethonium-resistant intestinal peristalsis.

It has previously been found that, in the presence of naloxone, the ganglionic blocking drug hexamethonium fails to completely block peristaltic motility in the isolated ileum of the guinea-pig. This hexamethonium-resistant peristaltic activity is coordinated by enteric nerves since it is abolished by tetrodotoxin. In the present study the neurotransmitter circuitry of this type of peristalsis was studied by means of specific antagonists. Atropine totally suppressed hexamethonium-resistant peristalsis. This type of peristalsis was also strongly inhibited by the tachykinin antagonist, spantide, if a concentration sufficient to antagonize neuronally located substance P receptors was employed. In contrast, the cholecystokinin antagonist, lorglumide, caused only a slight inhibition of hexamethonium-resistant peristalsis. Both substance P and the cholecystokinin-related peptide, ceruletide, potently stimulated the hexamethonium-resistant type of peristaltic activity. These data indicate that, after blockade of nicotinic acetylcholine receptors, tachykinins mediate neuroneuronal coordination of peristalsis whereas acetylcholine acting via muscarinic receptors may be primarily responsible for neuromuscular transmission. Cholecystokinin-like peptides appear to play a modulator rather than a mediator role in hexamethonium-resistant peristalsis.

Intestinal peristalsis associated with release of immunoreactive substance P.

The release of immunoreactive substance P into the vascular bed of the isolated small intestine of the guinea-pig was investigated. Raising the intraluminal pressure to 5 mbar for 5 min initiated peristalsis and stimulated the release of substance P. The substance P releasing effect of pressure stimulation was reduced by 46% when hexamethonium (240 microM) was added to the perfusion solution. The ganglion stimulant drug dimethylphenylpiperazinium (32 microM) also stimulated the release of substance P; its effect was completely prevented by hexamethonium (240 microM). Intraarterial infusion of capsaicin (22 microM), a neurotoxin known to act on sensory substance P-containing neurones, stimulated the release of substance P and caused intestinal contractions. The motor effect of capsaicin in the gut can thus be explained by release of substance P from sensory nerve endings in the gut. Systemic pretreatment of the guinea-pigs with capsaicin abolished the release of substance P due to capsaicin, whereas that evoked by elevated intraluminal pressure or dimethylphenylpiperazinium was not reduced. This means that substance P released in the course of peristalsis or by dimethylphenylpiperazinium originates from neurones intrinsic to the intestine. These findings indicate that intestinal peristalsis is associated with the release of substance P from enteric neurones. Substance P is likely to be a neurotransmitter involved in the coordination of the peristaltic reflex.

Tachykinin receptors are involved in the "local efferent" motor response to capsaicin in the guinea-pig small intestine and oesophagus.

The sensory neuron stimulant drug capsaicin stimulates primary afferent nerve endings in the guinea-pig small intestine, which in turn activate myenteric cholinergic neurons by an unknown mechanism. The tachykinins substance P and neurokinin A are present in primary afferent neurons. This study was performed to assess the possible involvement of endogenous tachykinins acting via neurokinin-1, neurokinin-2 and neurokinin-3 receptors in the contractile effect of capsaicin in the isolated guinea-pig ileum and oesophagus by using the receptor-specific antagonists GR 82334 (3 microM) for neurokinin-1 receptors, MEN 10627 (3 microM; ileum) or MEN 11420 (1 microM; oesophagus) for neurokinin-2 receptors and SR 142801 (0.1 microM) for neurokinin-3 receptors. In the ileum, the peak contraction evoked by capsaicin (2 microM) was not reduced when tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors were blocked separately, whereas an inhibition of neurokinin-3 receptors diminished the area under the curve of the capsaicin response. A combined blockade of neurokinin-1 and neurokinin-3 receptors significantly depressed the effect of capsaicin; the amplitude of the contractile response was 53.3+/-3.7% of the maximal longitudinal spasm in control preparations, whereas in the presence of GR 82334 plus SR 142801 it reached only 27.6+/-5% (P<0.001, Kruskal-Wallis test; n=9 and 10, respectively). Also, the area under the curve of the contractile response to capsaicin was more than 85% lower in the group of preparations treated with GR 82334 plus SR 142801 than in the control group (P<0.001). Including a neurokinin-2 blocker in the combination did not produce any further inhibition. A concomitant tachyphylaxis to substance P (natural neurokinin-1 receptor stimulant) and the neurokinin-3 receptor agonist senktide (5 and 1 microM, respectively) also reduced the contractile effect of capsaicin. In the oesophagus, capsaicin (1 microM) induced biphasic contractions which were strongly inhibited by atropine (1 microM) or capsaicin pretreatment (1 microM for 10 min). Here again, a blockade of tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors separately failed to inhibit the response to capsaicin, whereas a combined blockade of any two tachykinin receptors caused a partial inhibition. The reduction of the contractile effect of capsaicin was strongest when all three tachykinin receptors were blocked. In seven control preparations, peaks for the first and second phases of contraction reached 35.3+/-3.7% and 20+/-3.2% of maximal longitudinal spasm; the corresponding values in the presence of a combination of GR 82334, MEN 11420 and SR 142801 were 7.5+/-0.8% and 9.1+/-2.2%, respectively (n=6, P<0.001 and 0.05, respectively). Tetrodotoxin (0.5 microM) practically abolished the contractile effect of capsaicin in both tissues studied. It is concluded that an interplay of neuronal tachykinin neurokinin-1 and neurokinin-3 receptors (ileum) and neurokinin-1, neurokinin-2 and neurokinin-3 receptors (oesophagus) is involved in the contractile action of capsaicin, probably in mediating excitation of myenteric neurons by tachykinins released from primary afferents. In both tissues, there also seems to be a non-tachykininergic component of the capsaicin-induced contraction.

The mechanism of the motor response to periarterial nerve stimulation in the small intestine of the rabbit.

In the presence of guanethidine, periarterial nerve stimulation caused a frequency-dependent motor response in the rabbit isolated ileum. Tachyphylaxis to capsaicin greatly diminished the effect of periarterial stimulation; the remaining portion of the contractile response was susceptible to hexamethonium. The motor effect of periarterial stimulation is mediated by two different mechanisms: one via parasympathetic preganglionic nerves and the other sensitive to capsaicin.

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine.

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.

Theophylline-sensitive modulation of non-cholinergic excitatory neurotransmission in the guinea-pig ileum.

Atropine-resistant longitudinal contractions of the guinea-pig ileum due to field stimulation were greatly augmented by theophylline (150 microM). Adenosine exerted an opposite effect. Theophylline did not potentiate contractions evoked by exogenous substance P. It is suggested that a theophylline-sensitive inhibitory mechanism (possibly mediated by adenosine or a related substance) controls transmitter release from enteric non-cholinergic excitatory neurones.

Different receptors mediating the inhibitory action of exogenous ATP and endogenously released purines on guinea-pig intestinal peristalsis.

1 Adenosine 5'-triphosphate (ATP) is an enteric neurotransmitter which acts at purine receptors on intestinal nerve and muscle. This study set out to shed light on the receptor mechanisms by which exogenous and endogenous ATP influences intestinal peristalsis. 2 Peristalsis in isolated segments of the guinea-pig small intestine was triggered by a perfusion-induced rise of the intraluminal pressure. Motor changes were quantified by alterations of the peristaltic pressure threshold (PPT) at which propulsive muscle contractions were elicited. 3 ATP (>/= 3 microM) increased PPT and abolished peristalsis at concentrations of 100-300 microM. Adenosine 5'-O-2-thiodiphosphate (ADPbetaS, 3-100 microM) was more potent, whereas alpha,beta-methylene ATP (alpha,beta-meATP, 3-100 microM) was less potent, than ATP in depressing peristalsis. 4 8-Phenyltheophylline (10 microM) attenuated the anti-peristaltic effect of 10 and 30 microM ATP but not that of higher ATP concentrations. Apamin (0.5 microM) counteracted the ability of ATP, ADPbetaS and alpha,beta-meATP to enhance PPT. Suramin (300 microM) and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 150 microM) antagonized the inhibitory effect of alpha,beta-meATP on peristalsis but did not alter the effect of ATP and ADPbetaS. 5 PPADS (50-150 microM) reduced PPT by as much as 50%. This stimulant effect on peristalsis was prevented by suramin (300 microM) but left unaltered by apamin (0.5 microM) and NG-nitro-L-arginine methyl ester (300 microM). 6 These data show that exogenous and endogenous ATP inhibits intestinal peristalsis via different apamin-sensitive purinoceptor mechanisms. Exogenous ATP depresses peristalsis mostly via suramin- and PPADS-insensitive P2 receptors, whereas endogenous purines act via P2 receptors sensitive to both suramin and PPADS.

Evidence for the involvement of P2-purinoceptors in the cholinergic contraction of the guinea-pig ileum.

In the isolated ileum of the guinea-pig the P2-purinoceptor antagonists PPADS and suramin: (a) strongly inhibited the cholinergic contractile effect of alpha,beta-methylene ATP, (b) did not influence contractions evoked by exogenous acetylcholine (ACh) but, (c) moderately (by about 30%) inhibited cholinergic contractions due to electrical field stimulation (EFS), in a non-additive manner. These results suggest that an endogenous ligand that stimulates P2-purinoceptors (possibly ATP) is involved in the contractile effect of EFS, as a positive modulator of ACh release.

Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct.

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK1, NK2 and NK3 receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle. 2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK1 and NK3 receptor-selective agonists. 3. Among the natural tachykinins neurokinin B (EC50 = 3.2 nM; 95% c.l. = 2.0-5.1; n = 4) was the most potent, being about 40 and 25 fold more potent than substance P (EC50 = 121.6 nM; 95% c.l. = 94-157; P < 0.01; n = 4) and neurokinin A (EC50 = 83.4 nM; 95% c.l. = 62-112; P < 0.01; n = 4), respectively. Among the synthetic analogues the NK3 receptor-selective agonist senktide (EC50 = 1.1 nM; 95% c.l. = 0.7-1.8; n = 8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar9]substance P sulfone (NK1 receptor-selective) (EC50 = 130.4 nM; 95% c.l. = 99-172; P < 0.01; n = 8), [beta Ala8]NKA (4-10) (NK2 receptor-selective) (EC50 = 120.1 nM; 95% c.l. = 95-151; P < 0.01; n = 8) and septide (NK1 receptor-selective) (EC50 = 22.6 nM; 95% c.l. = 18-28; P < 0.01; n = 8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar Emax, averaging about 50% of that produced by KCl (80 mM). 4. Atropine (1 microM) did not affect the responses to either NK1 or NK2 receptor-selective agonists, whereas it reduced the Emax of senktide by about 50%, without affecting its potency (EC50). Tetrodotoxin (1 microM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK1 and NK2 receptor-selective antagonists GR 82334 and MEN 11420 (1 microM each), respectively. 5. GR 82334 (1 microM) blocked with apparent competitive kinetics septide- (apparent pKB = 7.46 +/- 0.10; n = 5) and [Sar9]substance P sulfone- (apparent pKB = 6.80 +/- 0.04; n = 4) induced contractions. MEN 11420 (30-300 nM), a novel potent NK2 receptor antagonist, potently antagonized [beta Ala8]NKA (4-10), with competitive kinetics (pKB = 8.25 +/- 0.08; n = 12: Schild plot slope = -0.90; 95% c.l. = -1.4; -0.35). The NK3 receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (Emax inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration-response curve to methacholine (0.1-300 microM). 6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK1, NK2 and NK3 receptors. The responses obtained by activating NK1 and NK2 receptors are atropine-resistant. The contraction obtained by stimulating NK3 receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK1/NK2 receptors. The role of the NK3 receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

Tachykinin NK1 and NK2 receptor antagonists and atropine-resistant ascending excitatory reflex to the circular muscle of the guinea-pig ileum.

1. The aim of this study was to investigate the effect of various antagonists, selective for the tachykinin NK1 or NK2 receptor, on the atropine-resistant ascending excitatory reflex (AER) to the circular muscle of the guinea-pig ileum elicited by radial stretch (balloon distension) or electrical field stimulation. 2. Submaximal and maximal atropine- (1 microM) resistant AER elicited by balloon distension averaged about 40-50% and 70-90% of maximal circular spasm to 80 mM KCl, respectively. The NK1 receptor antagonist, (+/)-CP 96,345 (1 microM) inhibited both maximal and submaximal AER. FK 888 (1-3 microM) inhibited submaximal AER only. RP 67,580 (1 microM) was ineffective. The NK2 receptor antagonist, GR 94,800, inhibited both maximal and submaximal AER at all concentrations tested (0.1-3.0 microM), while SR 48,968 was effective only at 1.0 microM. The NK2 receptor antagonists, MEN 10,376 and MEN 10,573 inhibited both submaximal and maximal AER at 10 and 1.0 microM, respectively. 3. In other experiments, an NK1 receptor antagonist, (+/-)-CP 96,345 or FK 888 (1.0 microM in each case) was administered first and the effect of GR 94,800 (1.0 microM) on the residual AER response was determined; or GR 94,800 was administered first and the effect of (+/-)-CP 96,345 or FK 888 was determined. The results of these experiments indicated an additive effect produced by the combined treatment with NK1 and NK2 receptor antagonists. 4. Electrical field stimulation (10 Hz for 0.5 s, 10-20 V, 0.15-0.3 ms pulse width) with electrodes placed at 1.4-1.8 cm anal to the recording site, produced ascending contractions which were almost abolished by 10 MicroM hexamethonium (electrically-evoked AER). In the presence of apamin (0.1 MicroM) and N0-nitro-L-arginine (30 MicroM) these contractions were reproducible over 10 consecutive stimulation cycles.GR 94,800 (1 MicroM) and FK 888 (1 MicroM) both produced a partial inhibition of the electrically-evoked AER and their combined administration produced an inhibitory effect which was larger than that induced by each antagonist alone.5. FK 888 (1-3 MicroM), GR 94,800 (1-3 MicroM), MEN 10,573 (1 MicroM) and MEN 10,376 (10 MicroM) did not significantly affect the atropine-sensitive twitch contractions produced by electrical field stimulation of the guinea-pig ileum longitudinal muscle-myenteric plexus preparation, which were abolished by 10-30 MicroM procaine, 1 MicroM tetrodotoxin or 1 MicroM atropine. (+/-)-CP 96,345 (1 MicroM) and SR 48,968 (1 ILM)produced 12% and 27% inhibition of cholinergic twitches in the longitudinal muscle of the ileum,respectively.6. We conclude that both NK1 and NK2 receptors mediate the atropine-resistant AER to the circular muscle of the ileum. NK2 receptor activation plays a more important role than NK1 receptor activation in the AER evoked by radial stretch. Since a consistent fraction of the distension- and electrically evoked atropine-resistant AER persists in the presence of combined NK1 and NK2 receptor blockade,the existence of a third excitatory transmitter to the circular muscle of the ileum, in addition to acetylcholine and tachykinins, is suggested.

Evaluation of a new and potent cholecystokinin antagonist on motor responses of the guinea-pig intestine.

The potency and selectivity of D,L-4-(3,4-dichloro-benzoyl-amino)-5-(dipentyl-amino)-5-oxo-pen tan oic acid (CR 1409) as a cholecystokinin (CCK) antagonist was investigated on motor responses of the longitudinal and circular muscles of the guinea-pig isolated ileum. CR 1409 was further used to examine whether nerve-mediated motor responses to electrical field stimulation or distension of the gut wall may involve the release of CCK-like peptides. CR 1409 (0.06-2.1 microM) antagonized longitudinal muscle responses to ceruletide (caerulein, a CCK-related decapeptide) in a concentration dependent and competitive manner (pA2 7.77); responses to CCK-octapeptide (CCK-8) were antagonized with a similar potency. Contractions of the circular muscle evoked by ceruletide were also blocked by CR 1409 (0.2-0.4 microM). Longitudinal muscle contractions in response to dimethylphenylpiperazinium, bethanechol, histamine, substance P, or 5-hydroxytryptamine (5-HT), and circular muscle contractions evoked by acetylcholine, 5-HT, substance P, or substance K were not altered by CR 1409 (0.4 microM). Longitudinal muscle contractions induced by electrical field stimulation (with pulses delivered at 0.05 and 1 Hz in the absence, and at 5 Hz in the presence of atropine) were not or only slightly reduced by CR 1409 (0.4 microM). Longitudinal contractions due to activation of extrinsic nerves by capsaicin remained unaltered in the presence of CR 1409 (0.4 microM). Reflex contractions of the circular muscle, induced by balloon distension and recorded orally to the site of distension, and peristaltic activity elicited by intraluminal infusion of Tyrode solution remained unaffected by CR 1409 (0.4 microM). 5 These findings indicate that CR 1409 is a potent and selective antagonist of CCK-like peptides in the guinea-pig ileum. The results do not provide any evidence that CCK-like peptides, released from extrinsic or intrinsic neurones, are involved in nerve-mediated contractions of intestinal muscle and in the peristaltic reflex.

Tachykininergic transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of NK2 receptors.

1. The effect of newly developed, receptor-selective tachykinin antagonists (GR 71,251 for NK1 receptors, MEN 10,376 and L 659,877 for NK2 receptors) on noncholinergic transmission to the circular muscle of the guinea-pig ileum has been investigated. 2. In circular muscle strips of the ileum, electrical field stimulation in the presence of atropine (2 microM) and apamin (0.1 microM) evoked a complex motor response. The tonic primary contraction in this response was reduced by GR 71,251 (10 microM) and MEN 10,376 (3-10 microM) but not by L 659,877 (up to 10 microM). The presence of apamin was necessary in this experimental arrangement to unmask an atropine-resistant primary contraction, sensitive to tachykinin antagonists. The motor response was abolished by tetrodotoxin. 3. In circular strips of the ileum GR 71,251 (10 microM) inhibited the tonic contraction produced by [Sar9] substance P sulphone, a selective NK1 receptor agonist but not that produced by [beta Ala8] neurokinin A (4-10), a selective NK2 receptor agonist. By contrast, MEN 10,376 antagonized the effect of the NK2 agonist while leaving the response to the NK1 agonist unaffected. 4. In whole segments of the ileum, distension of the gut wall by an intraluminal balloon placed at about 1 cm from the point of recording of mechanical activity of the circular muscle produced atropine-sensitive phasic contractions (ascending enteric reflex). In the presence of atropine (2 microM), a noncholinergic response was elicited, which required larger volumes of distension that the cholinergic one. The atropine-resistant ascending enteric reflex was enhanced by apamin (0.1 microM) and abolished by tetrodotoxin, either in the presence or absence of apamin.5. MEN 10,376 (3-lOmicroM) inhibited the atropine-resistant ascending enteric reflex in the presence of apamin while GR 71,251 or L 659,877 (10 microM each) were ineffective. MEN 10,376 inhibited the atropine-resistant ascending enteric reflex to a larger extent in the absence than in the presence of apamin and also slightly inhibited the ascending enteric reflex in the absence of atropine.6. These findings provide evidence for an involvement of NK2 tachykinin receptors in excitatory transmission to the circular muscle of the guinea-pig ileum. NK2 receptors are also involved in the physiological-like circular muscle activation produced by stimulation of intramural neuronal pathways which subserve the atropine-resistant ascending enteric reflex.

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct.

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.

Capsaicin-sensitive afferents and their role in gastroprotection: an update.

The pivotal role of capsaicin-sensitive peptidergic sensory fibers in the maintenance of gastric mucosal integrity against injurious interventions was suggested by the authors 20 years ago. Since then substantial evidence has accumulated for the local sensory-efferent function of the released CGRP, tachykinins and NO in this gastroprotective mechanism. This overview outlines some recent achievements which shed light on new aspects and further horizons in this field. (1) Cloning the capsaicin VR-1 receptor (an ion channel-coupled receptor) and raising the VR-1 knockout mice provided a definite molecular background for the existence of capsaicin-sensitive afferents with both sensory and mediator releasing functions in the stomach. This cation channel is also sensitive to hydrogen ions. (2) VR-1 agonists (capsaicin, resiniferatoxin, piperine) protect against gastric ulcer of the rat parallel with their sensory stimulating potencies. (3) Antidromic stimulation of capsaicin-sensitive vagal and somatic afferents results in the release of CGRP, tachykinins, NO and somatostatin. Somatostatin with gastroprotective effect is released from D cells and sensory nerve endings. (4) The recent theory for the existence of spinal afferents without sensory function [P. Holzer, C.A. Maggi, Dissociation of dorsal root ganglion neurons into afferent and efferent-like neurons, Neuroscience 86 (1998) 389-398] is discussed. Data proposed to support this theory are interpreted here on the basis of a dual sensory-efferent function of VR-1 positive afferents, characterized by a frequency optimum of discharges for release vasodilatory neuropeptides below the nociceptive threshold. (5) Recent data on the effect of capsaicin in healthy human stomach are summarized. These results indicate that the gastroprotective effect of capsaicin in the human stomach involves additional mechanisms to those already revealed in the rat.