Ruthenium red antagonism of the effect of capsaicin on the motility of the isolated guinea-pig ileum

Ruthenium red (1 microM), an inorganic dye which blocks transmembrane calcium (Ca) fluxes in neural tissues, selectively reduced the capsaicin (1 microM)-induced contraction of the guinea-pig ileum and protected the sensory fibers from capsaicin-induced desensitization. The ruthenium red (0.5-1 microM) antagonism of capsaicin-induced inhibition of responses to mesenteric nerve stimulation or field stimulation in the isolated guinea-pig ileum was an example of a similar antagonism of the effect of capsaicin. In view of the known action of ruthenium red on the depolarization-coupled entry of Ca into synaptosomes and the release of transmitter, our results support the proposal that ruthenium red could antagonize the action of capsaicin on the peripheral terminals of sensory nerves by a similar mechanism, thereby suppressing transmitter release and preventing the establishment of desensitization.     

The effects of ruthenium red on the response of guinea-pig ileum to capsaicin

Ruthenium red has recently been found to inhibit the effects of capsaicin on peripheral terminals of sensory neurones. Thus the effects of ruthenium red on the responses of the guinea-pig isolated ileum to capsaicin, acetylcholine (ACh), substance P (SP) and nicotine were investigated. Ruthenium red, 5 mumol/l, abolished responses to capsaicin 1.5 mumol/l and nicotine 2 mumol/l, and shifted the concentration-response lines to ACh and SP to the right. Pretreatment of ileum preparations with ruthenium red, 12.5 mumol/l for 2 min, prevented desensitization of ileum responses to capsaicin tested 30 min later. Tetrodotoxin, 1 mumol/l, abolished the response to capsaicin on control preparations and those pretreated with atropine, 5 mumol/l, ruthenium red, 12.5 mumol/l or spantide, 10 mumol/l. It is proposed that capsaicin acts via a specific receptor coupled to a receptor-operated membrane calcium channel, and that ruthenium red binds irreversibly to the calcium channel part of the complex but reversibly to some other site which prevents the action or binding of capsaicin at its specific receptor.     

Inhibitory action of capsaicin on cholinergic responses induced by GABAA agonists in the guinea-pig ileum

Pretreatment of the guinea-pig ileum with capsaicin resulted consistently in depression of the neurogenic cholinergic contractions induced by the GABAA receptor agonists 3-aminopropane sulphonic acid (3-APS) and muscimol. Since capsaicin acts mainly by releasing and depleting substance P from its stores in intestinal nerves, it is likely that substance P plays a role in the response caused by GABAA-mimetic compounds, On the whole, our results suggest that excitatory responses to 3-APS and muscimol result from both direct and indirect activation of intrinsic intestinal cholinergic neurons innervating smooth muscle cells.     

Possible mechanism of ruthenium red antagonism of capsaicin-induced action in the isolated guinea pig ileum.

Ruthenium red (3-5 microM) antagonism of the inhibitory effect of capsaicin (1 microM) on the contractile response to mesenteric nerve stimulation in the presence of hexamethonium (50 microM) and guanethidine (2 microM) was reversed significantly by sialic acid (2 mM) or neuraminidase (0.1 U/ml). These results suggested that ruthenium red at low concentrations inhibits the capsaicin-induced desensitization of activated Ca2+ influx into sensory nerves at least in part by binding to sialic acid residues.     

Inhibitory effect of enkephalins on contractions of the guinea-pig ileum elicited by PGE1

Methionine-enkephalin and leucine-enkephalin (m-enk and 1-enk) as well as D-Ala2-methionine-enkephalin amide have been shown to antagonize contractions of the isolated guinea-pig intestine elicited by PGE1, the last mentioned being the most potent of the three. The inhibitory effect of these pentapeptides is abolished by naloxone.     

The antagonism induced by ruthenium red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies

Ruthenium Red, an inorganic dye which blocks transmembrane calcium (Ca) fluxes in neural tissues, reduced the capsaicin-induced release of substance P-like immunoreactivity from muscle strips of the guinea-pig urinary bladder in a concentration-dependent (30 nM - 3 microM) manner, and protected the sensory fibers from capsaicin-induced densensitization. A similar antagonism of the actions of capsaicin was observed in functional experiments (capsaicin-induced contraction of the isolated guinea-pig bladder or inhibition of twitches of the isolated rat vas deferens). In view of its established action on the depolarization-coupled entry of Ca into synaptosomes and the secretion of transmitter, we propose that Ruthenium Red could antagonize the action of capsaicin on the peripheral terminals of sensory nerves by a similar mechanism, thereby suppressing transmitter secretion and preventing the establishment of desensitization.     

The effect of prazosin on the guinea-pig ileum.

1 The alpha-adrenoceptor blocking activity of prazosin was compared with that of phentolamine, yohimbine and phenoxybenzamine in the guinea-pig isolated ileum. 2 Phentolamine (2.56 X 10(-8) to 2.56 X 10(-6) M) and yohimbine (5.12 X 10(-8) to 5.12 X 10(-6) M) antagonized the sympathetic inhibition of twitches induced by transmural stimulation, whereas prazosin (5.28 X 10(-8) to 5.28 X 10(-6) M) and phenoxybenzamine (4.2 X 10(-9) to 4.2 X 10(-7) M) had no effect. 3 Phentolamine, yohimbine, prazosin and phenoxybenzamine antagonized the relaxant response of the ileum to periarterial nerve stimulation and to exogenous noradrenaline in the absence of transmural stimulation. 4 The pA2 value for phentolamine against noradrenaline in the transmurally-stimulated gut (presynaptic alpha-adrenoceptor site; 8.17 +/- 0.04) was significantly different from that in the non-stimulated gut (postsynaptic alpha-adrenoceptor site; 6.88 +/- 0.12). 5 Phentolamine and prazosin probably block noradrenaline responses at the same alpha-adrenoceptor site on the postsynaptic membrane.     

Effects of piperine on the motility of the isolated guinea-pig ileum: comparison with capsaicin.

Piperine (1 microM), a congener of capsaicin, produced an initial contraction blocked the capsaicin-sensitive contractile response to mesenteric nerve stimulation and inhibited the twitch response induced by field stimulation in the isolated guinea-pig ileum. These three effects of piperine (1 microM) were rapidly desensitized and significantly antagonized by ruthenium red (0.5-1 microM), an inorganic dye known to antagonize the effects of capsaicin. The contractile effect of piperine was abolished by application of tetrodotoxin plus desensitization with substance P or by extrinsic denervation. The inhibitory effect of piperine (1 microM) on the twitch response was antagonized by desensitization with calcitonin gene-related peptide (CGRP). Moreover, cross-tachyphylaxis between piperine and capsaicin was observed, suggesting that a similar mechanism may be involved in the effects of these agents. The contractile effects induced by piperine (10 microM) and the subsequent inhibitory effects on the twitch response were not desensitized and largely persisted after extrinsic denervation. The contractile effects of piperine (10 microM) were not strongly inhibited by tetrodotoxin plus desensitization with substance P. It was concluded that the lower concentration of piperine caused contraction and inhibited the twitch responses by releasing substance P and CGRP, respectively, from sensory nerves, and blocked the response to mesenteric nerve stimulation by a mechanism similar to that of capsaicin. At higher concentrations, piperine had non-specific direct actions on the smooth muscle.     

Functional antagonism by calcium of an intrinsic opioid mechanism in the guinea-pig isolated ileum

In the guinea-pig isolated ileum, elevation of the extracellular concentration of calcium attenuated the excitatory influence of the opiate antagonist, naloxone, upon peristalsis. Application of 4-aminopyridine, which enhances calcium influx into cells, had a still more pronounced effect. Further, 4-aminopyridine impaired the inhibitory action of both morphine and the presynaptic α-adrenergic agonist, clonidine, upon peristalsis.The present data are consistent with the view that the intestinal mechanism subjected to naloxone blockade may function similarly to that proposed to explain the action of narcotics or of presynaptic α-adrenergic agonists upon other preparations. This mode of action may involve a reduction in the quantity of intracellular free ionized calcium available to the stimulus-release-coupling mechanism.     

Anti-spasmogenic effect of cyproheptadine on guinea-pig ileum

Antagonistic activity of cyproheptadine against common spasmogens, like acetylcholine, histamine, serotonin, bradykinin and angiotensin, was studied on isolated guinea-pig ileum. Cyproheptadine produced a reversible antagonism of non-competitive type and was most effective against serotonin. It was less potent against histamine, bradykinin and angiotensin and least potent against acetylcholine.     

Ruthenium red as a selective capsaicin antagonist of the motor response to capsaicin in the human isolated ileum.

We have investigated the effect of ruthenium red, omega-conotoxin fraction GVIA (CTX) and tetrodotoxin (TTX) on the relaxation produced in the circular muscle of the human isolated ileum by capsaicin, electrical field stimulation (EFS) or vasoactive intestinal polypeptide (VIP). Ruthenium red (10 microM) selectively blocked the capsaicin-evoked relaxation while leaving the response to EFS or VIP unaffected. CTX had no significant effect on the various stimuli. TTX blocked the relaxation due to EFS but not that due to capsaicin or VIP. It is concluded that capsaicin excitation of primary afferents in the human ileum, leading to VIP release and muscle relaxation, occurs with mechanisms similar to those operating in animal tissues and that ruthenium red acts as a selective capsaicin antagonist in the human ileum.     

The effect of thymoxamine on guinea-pig ileum contractions

The effects of thymoxamine on the contractions induced in guinea-pig isolated ileum were examined. The drug was tested in a large range of concentrations (10(-8)-10(-4) M). The twitch response induced by acetylcholine was potentiated by low concentrations and inhibited by high concentrations of the alpha 1-blocking drug thymoxamine. The contractions in response to carbachol were always inhibited. Thymoxamine also affected the responses of the ileum to other stimulants: angiotensin, pentagastrin, cholecystokinin. The drug (8 X 10(-5), 5 X 10(-4) M) inhibited the calcium contraction of high K+ depolarized preparations.     

A quantitative study of the calcium-magnesium antagonism in isolated strips of guinea-pig ileum

The effect of increasing magnesium concentration (0.61, 1.22, 2.44 and 4.88 mM) on the acetylcholine dose— response curve of isolated strips of the longitudinal muscle of the guinea pig ileum was studied qualitatively and quantitatively in Krebs solution with 2.55 and 5.10 mM calcium. The quantitative study was based on a hypothesis assuming the competition of both ions for a common binding site in the cell “stores” from which the calcium is mobilized following cholinergic receptor activation. The theoretical equations fit the experimental data and allow the calculation of the calcium and magnesium equilibrium constants. As a cross-check two groups of experiments were done to compare the influence on the acetylcholine dose—response curve of a gradual decrease of extracellular calcium either without or with magnesium. The amounts of magnesium used were those theoretically suitable for maintaining a constant amount of calcium in the hypothetical cell “stores”. The results agreed with the predictions from the model proposed thus confirming both the hypothesis and the values obtained for the calcium and magnesium equilibrium constants.     

Functional antagonism between nitric oxide and ATP in the motor responses of guinea-pig ileum

1. The interaction of nitric oxide and ATP in the non-adrenergic, non-cholinergic (NANC) motor responses and the presence of NADPH-diaphorase and quinacrine-positive myenteric neurones were studied on guinea-pig ileum using mechanographic, histochemical and quinacrine-fluorescence techniques. In the presence of phentolamine, propranolol and atropine, the non-precontracted longitudinally oriented organ bath preparations responded to sodium nitroprusside (1-100 microM) or ATP (5-50 microM) with tetrodotoxin (0.1 microM)-resistant relaxation or contraction, respectively. The effects of ATP were suramin (50 microM)- and apamin (5 microM)-sensitive. 2. The NANC motor responses elicited by electrical stimulation (0.8 ms, 1-20 Hz, 20 s) consisted of tetrodotoxin-sensitive relaxation phase followed by a phase of twitch-like and tonic contractions. 3. NG-nitro-L-arginine (L-NNA, 0.1-0.5 mM) inhibited or abolished the relaxation phase. L-arginine (0.5 mM), but not D-arginine (0.5 mM), restored the relaxation phase in L-NNA-pretreated preparations. The relaxation phase increased after ATP-induced desensitization of purinoceptors and in the presence of suramin (50 mciroM) but was abolished by apamin (5 microM). 4. The phase of contractions was enhanced by L-NNA (0.1-0.5 mM) and restored by L-arginine (0.5 mM). The twitch-like and tonic contractions were decreased during ATP-induced purinoceptor desensitization and in the presence of suramin (50 microLM). Apamin (5 microM) inhibited the tonic contractions. 5. The desensitization of purinoceptors by ATP did not change the L-NNA-induced inhibition of the relaxation phase but decreased the L-NNA-increased phase of contractions. L-NNA reduced the relaxation phase and increased the phase of contractions during purinoceptor desensitization. 6. We conclude that in the longitudinal muscle layer of the guinea-pig ileum, nitric oxide mediates the relaxation phase while ATP contributes via smooth muscle P2 purinoceptors to the phase of contractions suggesting a postjunctional functional antagonism between nitric oxide and ATP. The presence of NADPH-diaphorase- and quinacrine-positive neuronal cells and processes running to the muscle cells confirms a physiological role of nitric oxide and ATP in the ileal neurotransmission.     

Contractile effect of (+)-glaucine in the isolated guinea-pig ileum.

The intestinal effects of (+)-glaucine [(S)-1,2,9,10-tetramethoxyaporphine] were studied using the guinea-pig ileum. (+)-Glaucine (10-300 microM) induced ileal contractions. The contraction was not affected by tetrodotoxin, atropine, hexamethonium, propranolol, naloxone, methysergide, N(G)-nitro-L-arginine methyl ester, SR141716A (a cannabinoid CB1 receptor antagonist) or SR140333 (a tackykinin NK1 receptor antagonist) plus SR48968 (a tackykinin NK2 antagonist). (+)-Glaucine-induced contraction was reduced by indomethacin, nordihydroguaiaretic acid or bisindolylmaleimide I and abolished by verapamil and nifedipine. These results suggest that (+)-glaucine-induced contraction involves activation of voltage-dependent Ca2+ channels and protein kinase C and could be mediated by the release of arachidonic acid metabolites.