Rank-order inhibition by omega-conotoxins in human and animal autonomic nerve preparations.

The inhibitory effects of the omega-conotoxins GVIA, MVIIA and MVIIC on electrically-evoked, tetrodotoxin (10(-7) M)-sensitive, autonomic nerve activity were studied using human, rat or guinea-pig vas deferens and intestinal tissues. In each preparation from each species, nM concentrations of omega-conotoxins GVIA and MVIIA prevented the neuronally-mediated contractions, whereas omega-conotoxin MVIIC was either markedly less potent (IC(50)'s 1.4 or 2.9 log units more than for omega-conotoxin GVIA in guinea-pig ileum and rat vas deferens, respectively) or was without significant activity (human vas deferens, human Taenia coli) when tested at similar concentrations. In contrast the differences in potency between omega-conotoxins GVIA and MVIIC were considerably less when assayed directly on Ca(2+) channel currents evoked from rat superior cervical ganglion neurons in culture (approximately 0.1 log unit difference) and from a stable cell line expressing rat alpha(1B), alpha(2)delta, beta(1b) Ca(2+) channel subunits (approximately 0.9 log unit). These different rank-orders of inhibitory activity of the conotoxins support the suggestion that there are pharmacologically distinct N-type Ca(2+) channels in the peripheral nervous system, and that this tissue-dependent difference is seen in man.

SB-207266: 5-HT4 receptor antagonism in human isolated gut and prevention of 5-HT-evoked sensitization of peristalsis and increased defaecation in animal models.

SB-207266 is a new 5-HT4 receptor antagonist which in a pilot study reduced the symptoms of irritable bowel syndrome. To help validate this and further studies, we examined the ability of SB-207266 to antagonize at the human 5-HT4 receptor (human isolated intestine) and to affect the mechanisms of peristalsis (guinea-pig isolated ileum) and defaecation (conscious, fed mice). In the human intestine, the potency of 5-HT4 receptor antagonism (pKB 9.98) was similar to that previously demonstrated using a guinea-pig model of the receptor, validating the use of SB-207266 in clinical trials. In each of the animal models, SB-207266 did not affect normal patterns of intestinal motility measured in the absence of exogenous 5-HT. However, SB-207266 10-1000 pM concentration-dependently antagonized the ability of 5-HT (0.1 microM) to sensitize the peristaltic reflex and lower the distension threshold at which peristalsis was evoked. In mice, oral or subcutaneous (s.c.) doses of SB-207266 dose-dependently prevented the ability of the 5-HT precursor, 5-hydroxytryptophan (5-HTP, 10 mg kg-1 s.c.) to increase both the rate of defaecation of formed faecal pellets and their fluid content. SB-207266 was maximally active at 10 micrograms kg-1 s.c. and 1000 micrograms kg-1 p.o. SB-207266 may therefore represent a new class of therapeutic agent, capable of preventing the actions of an important sensitizer of gut function.

Pharmacological characterization of the vanilloid receptor in the rat dorsal spinal cord.

1. In the present study a novel 96-well plate assay system was used to characterize pharmacologically the vanilloid receptor in the dorsal spinal cord of the rat. When activated, this receptor stimulates release of calcitonin gene-related peptide (CGRP) from the central terminals of the afferent nerves. 2. Capsaicin, resiniferatoxin (RTX) and olvanil each evoked a concentration-dependent increase in CGRP release with pEC50 values of 6.55 +/- 0.07, 7.90 +/- 0.24 and 6.19 +/- 0.15 respectively. RTX and olvanil were partial agonists with respect to capsaicin. All concentration-effect curves were bell-shaped. 3. The vanilloid receptor antagonist, capsazepine (10 microM) had no effect on basal peptide release but inhibited the CGRP release evoked by all 3 agonists to a similar extent. These results suggest that the antagonistic effects of capsazepine were agonist-independent. 4. The capsaicin-sensitive cation channel blocker, ruthenium red (10 microM) had no effect on basal CGRP release, but antagonized the peptide release evoked by capsaicin, olvanil and RTX. 5. The pharmacology of the vanilloid receptor in the rat dorsal spinal cord is not identical to that previously found in other systems. The reason for these differences is unclear, but the possibility of multiple classes of receptor cannot at this stage be ruled out.

Selective and functional 5-hydroxytryptamine4 receptor antagonism by SB 207266.

1. The pharmacology of a novel 5-HT4 receptor antagonist, SB 207266 has been evaluated in vitro in the guinea-pig distal colon longitudinal muscle myenteric plexus (LMMP) and in vivo in the dog Heidenhain pouch. 2. SB 207266 is a highly potent antagonist of 5-HT-evoked, cholinergically-mediated contractions in the guinea-pig distal colon. Low concentrations (0.1-10 nM) produced a parallel shift to the right of the concentration-effect curve (apparent pA2 10.6 +/- 0.1) with no significant effect on the maximum response. With higher concentrations of SB 207266 (30 nM and above) the maximum response to 5-HT was reduced. 3. The antagonism seen with SB 207266 cannot be attributed to a non-selective effect since high concentrations (1 microM) had no effect on cholinergically-mediated contractions evoked by the nicotinic receptor agonist DMPP in the same preparation. 4. SB 207266 is not an irreversible antagonist since the effects of the compound were reversible upon washing of the tissue. 5. In the dog Heidenhain pouch, oral (0.1-100 micrograms kg-1) and intravenous (0.1-100 micrograms kg-1) administration of SB 207266 produced a dose-dependent antagonism of the contractions evoked by a bolus intravenous injection of 5-HT. An ID50 for SB 207266 of 1.3 micrograms kg-1 was obtained following i.v. administration and 9.6 micrograms kg-1 following oral administration. 6. The antagonistic effects of SB 207266 (0.1-100 micrograms kg-1) in the dog Heidenhain pouch were long lasting since, following oral administration, the response to 5-HT was reduced for at least 135 min. 7. SB 207266 is a highly potent, highly selective and orally active 5-HT4 receptor antagonist. This compound is the first orally active amide to be identified in this class of antagonists and as such is an important new tool in the evaluation of 5-HT4 receptor function both in vitro and in vivo.

Pharmacological characterization of the vanilloid receptor in the rat isolated vas deferens.

The present study set out to further characterize the vanilloid receptor in the rat isolated vas deferens. In this preparation, both capsaicin and resiniferatoxin (RTX) evoked a concentration-dependent inhibition in the amplitude of electrically-evoked contractions with pEC50 values of 7.62 +/- 0.03 and 12.2 +/- 0.21 respectively. Responses to capsaicin were fast in onset and faded rapidly over a 30-min exposure period, whereas those to RTX were slow in onset and well maintained, an observation believed to reflect pharmacokinetic differences in the rate of penetration to the vanilloid receptor. Responses to both agonists showed mutual cross-desensitization and were antagonized by both the vanilloid-receptor antagonist capsazepine and the ion-channel blocker ruthenium red. The capsaicin analogue, olvanil failed to either mimic or antagonize capsaicin-evoked responses in the rat isolated vas deferens, an effect at variance with previous observations in other tissues. The reason for these differences is unclear, but the possibility of multiple classes of receptor cannot at this stage be ruled out.

Azabicyclic indole esters as potent 5-HT4 receptor antagonists.

The synthesis of a series of azabicyclic indole esters is described and their potency reported as 5-HT4 receptor antagonists. Optimization of the most potent compound (19) by preparing the corresponding oxazino[3,2-a]indole ester afforded 34, which had a pIC50 of 9.5 in the guinea pig distal colon longitudinal muscle myenteric plexus preparation.

Constipation evoked by 5-HT3-receptor antagonism: evidence for heterogeneous efficacy among different antagonists in guinea-pigs.

The abilities of selective 5-HT3-receptor antagonists to evoke constipation were examined in conscious guinea-pigs and in preparations of guinea-pig isolated colon. Compared with vehicle-treated guinea-pigs, acute doses of granisetron (0.1, 1 and 10 mg kg-1, i.p.) and tropisetron (10 mg kg-1, i.p., but not 1 and 0.1 mg kg-1, i.p.) significantly (P < 0.05) reduced the total number of faecal pellets excreted during a 12-h observation period. By contrast, BRL 46470 (0.1-10 mg kg-1, i.p.) had no significant effect on the incidence of defecation. Mid-to-distal lengths of guinea-pig isolated colon spontaneously expelled faecal pellets. Granisetron (0.1 and 1 microM) and tropisetron (1 microM) reduced or prevented the rate at which they were spontaneously expelled. Morphine (0.1 microM) and clonidine (10 nM) also showed faecal pellet transit time. Naloxone (0.1 microM) had no effects alone, but reversed the actions of granisetron, morphine and clonidine. BRL 46470 (1 microM) had no significant effect on the transit of faecal pellets in guinea-pig isolated colon. In segments of guinea-pig isolated colon which did not contain faecal pellets, granisetron, tropisetron and BRL 46470 antagonized the ability of 5-HT to evoke cholinergically-mediated contractions of the longitudinal muscle. The respective pA2 values and slopes of the Schild plots were 8.5 +/- 0.05, slope 1.06 +/- 0.03; 8.5 +/- 0.1, slope 0.91 +/- 0.04; and 7.9 +/- 0.1, slope 0.93 +/- 0.05. Our experiments suggest that not all 5-HT3-receptor antagonists are the same.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of SB 204070, a highly potent and selective 5-HT4 receptor antagonist, on guinea-pig distal colon.

1. The pharmacology of a novel 5-HT4 receptor antagonist, SB 204070 has been evaluated in the guinea-pig isolated distal colon longitudinal muscle-myenteric plexus (LMMP). 2. SB 204070 is a highly potent antagonist of 5-HT-evoked cholinergically-mediated contractions in the guinea-pig distal colon. Low concentrations (10-100 pM) produced a shift to the right of the curve (apparent pA2 10.8 +/- 0.1) with no significant effect on the maximum response. With higher concentrations of SB 204070 (300 pM and above), the maximum response to 5-HT was reduced. 3. When tested against the partial 5-HT4 receptor agonist, BIMU 1, SB 204070 was active at similar low concentrations (10 pM and above) but produced a reduction in maximum, with no prior shift to the right of the curve, at all concentrations tested (10-300 pM). 4. The antagonism seen with SB 204070 is unlikely to be due to a non-selective effect since high concentrations (10 nM and 1 microM) of the compound had no effect on cholinergically-mediated contractions evoked by the nicotinic receptor agonist, DMPP, in the same preparation. SB 204070 is unlikely to be an irreversible antagonist since the effects of the compound could be reversed upon washing of the tissue. 5. Radioligand binding studies show that SB 204070 has a greater that 5000 fold selectivity for the 5-HT4 receptor over 5-HT1A, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2C, 5-HT3, GABAA, BDZ, TBPS, A1 adenosine receptors, alpha 1, alpha 2, beta 1, beta 2 adrenoceptors and D1, D2 and D3 dopamine receptors. 6. SB 204070 is a highly potent, highly selective 5-HT4 receptor antagonist and as such is an important new tool in evaluating the functional role of the 5-HT4 receptor.

The guinea-pig distal colon--a sensitive preparation for the investigation of 5-HT4 receptor-mediated contractions.

1. Experiments were designed to characterize pharmacologically the contractile responses to 5-hydroxytryptamine (5-HT) in the guinea-pig isolated distal colon longitudinal muscle-myenteric plexus preparation (LMMP). 2. In the presence of methiothepin (100 nM) and granisetron (1 microM), 5-HT (10 pM-10 nM) produced concentration-dependent contractile responses of the guinea-pig distal colon LMMP, with a pEC50 of 9.2 +/- 0.08. 3. Responses to 5-HT were mimicked by a series of tryptamine analogues, with the following rank order of potency; 5-HT > 5-MeOT >> 5-CT > tryptamine > 2-Me-5-HT. All were found to be full agonists. 4. Responses to 5-HT were also mimicked by a series of substituted benzamide analogues. Their rank order of potency was 5-HT > renzapride > cisapride > (S)-zacopride > (R)-zacopride > metoclopramide. All were full agonists relative to 5-HT. 5. The benzimidazolone derivatives, BIMU 1 and BIMU 8 were approximately equipotent partial agonists (intrinsic activities of 0.8 +/- 0.07 and 0.5 +/- 0.08 respectively) in the guinea-pig distal colon. 6. Tropisetron produced a rightward displacement of the 5-HT concentration-effect curve, yielding an apparent pA2 of 6.4 +/- 0.1. The slope of the Schild plot (1.3 +/- 0.1) was significantly greater than unity. 7. SDZ 205,557 produced a concentration-dependent shift to the right of the 5-HT concentration-response curve, yielding an estimated pA2 of 7.8 +/- 0.1 and a slope which did not significantly deviate from unity. SDZ 205 557 produced similar pKB estimates (7.3-7.9) when tested against 5-MeOT,renzapride and 5-CT, indicating a common site of action.8. The pharmacological profile of the 5-HT-evoked contractions of the guinea-pig distal colon LMMPare consistent with activity at the 5-HT4 receptor. Furthermore, of the models of this receptor described in the literature, the guinea-pig distal colon appears to be the most sensitive model to date, making it a useful tool in the investigation of 5-HT4 receptor-mediated events.

Vascular smooth muscle responses in normo- and hypertensive rats to sympathetic nerve stimulation and putative transmitters.

1. Using changes in perfusion pressure as a measure of end organ response, the effects of field stimulation (0.5 ms supramaximal voltage), noradrenaline (NA, 10(-5)-10(-3) M), adrenaline (ADR, 10(-6)-10(-4) M) and adenosine triphosphate (ATP, 10(-4)-10(-2) M) on the tail artery and mesenteric bed preparations in both normotensive (WKY) and spontaneously hypertensive (SHR) rats were examined. 2. The pressor responses in both preparations from SHR rats to field stimulation, NA and ADR were significantly (P less than 0.05) greater than those from age-matched WKY controls. Responses of both preparations to ATP in normo- and hypertensive rats did not differ significantly. 3. In both preparations from either WKY or SHR rats, pressor responses to ATP (10(-4)-10(-2) M) were inhibited by alpha,beta-methylene ATP (alpha,beta MeATP, 1 x 10(-6) M) while those to field stimulation were not. Phentolamine (2 x 10(-6) M) and prazosin (1 x 10(-7) M) each inhibited the pressor responses to both field stimulation, NA and ADR. 4. [3H] was released by field stimulation from tail arteries pre-incubated with either [3H]-NA or [3H]-adenosine in both normotensive and hypertensive rats. Release in each case was abolished by tetrodotoxin (1 x 10(-6) M). 5. There was no significant difference in the stimulation-evoked [3H]-NA overflow between SHR & WKY rats, alpha,beta MeATP had no significant inhibitory effect on the overflow of [3H] following incubation with [3H]-NA from either group of animals. 6. In the presence of diltiazem (2 x 10(-6) M) and prazosin (5 x 10(-7) M) to abolish any squeezing effect of muscle contractions on ATP release, there was no significant difference in the [3H] overflow between tail arteries from SHR and WKY rats following incubation with [3H]-adenosine. 7. The results confirm the increased response to nerve stimulation in hypertensive animals, an effect probably mediated postsynaptically via alpha-adrenoreceptors. There was no evidence for the involvement of ATP in the hypertensive state.

The electrical and mechanical basis of co-transmission in some vascular and non-vascular smooth muscles.

1. The intracellularly-recorded electrical and mechanical responses to field stimulation of intramural nerves in three sympathetically-innervated smooth muscles--the mouse vas deferens, the rabbit ear artery and the rabbit mesenteric bed preparation were investigated. 2. In each tissue there was evidence for co-transmission involving noradrenaline (NA) and adenosine 5'-triphosphate (ATP) or a closely related nucleotide. 3. The electrical response in each tissue consisted of excitatory junction potentials (ejps) which were abolished by alpha, beta-methylene ATP (alpha, beta MeATP, 1-10 X 10(-6) M), suggesting that they were mediated by ATP. Only in the rabbit ear artery was there an additional electrical event mediated by NA. This took the form of a small slow membrane depolarization which followed the ejps and which was antagonized by either of the alpha-adrenoreceptor blocking agents phentolamine (1 X 10(-6) M) or prazosin (1 X 10(-7) M). 4. In the mouse vas deferens and rabbit mesenteric artery, both transmitter substances (NA and ATP) played a role in the contractile response to field stimulation. In the rabbit ear artery, NA alone appeared to mediate the contractile event. 5. Contractile responses to nerve-released ATP were accompanied by a membrane potential change, whereas those to NA appeared to be mediated largely by a voltage-independent mechanism. 6. In the mouse vas deferens, the ejps and action potentials evoked by field stimulation appeared to be mediated by a discrete increase in permeability to Na+ and K+. 7. In the mouse vas deferens, local application of bradykinin (1-100 X 10(-7) M) produced a small, slow membrane hyperpolarization. VIP (1-100 X 10(-7) M), neuropeptide Y (1-100 X 10(-7) M), substance P (1-100 X 10(-7) M), somatostatin (1-100 X 10(-7) M), leu-enkephalin (1-100 X 10(-7) M), metenkephalin (1-100 X 10(-7) M) and bombesin (1-100 X 10(-7) M), similarly applied, each produced no significant change in membrane potential. None of these peptides appear to be the transmitter mediating the ejp in this tissue.

Is co-transmission involved in the excitatory responses of the rat anococcygeus muscle?

1 Electrical and mechanical responses to field (transmural) and extrinsic nerve stimulation were recorded simultaneously in the rat anococcygeus muscle. Membrane potential changes recorded intracellularly following either method of stimulation were indistinguishable. Single stimuli usually produced a slow depolarization; trains of pulses produced a fast excitatory junction potential (e.j.p.) initially, followed by a slow depolarization similar to that produced by single pulses. The fast e.j.ps, the slow depolarizations and the accompanying contractions were abolished by the alpha-adrenoceptor antagonists, phentolamine (1 X 10(-6)M) or prazosin (1 X 10(-7)M) and by tetrodotoxin (TTX, 1 X 10(-6)M) but unaffected by alpha, beta-methylene adenosine triphosphate (alpha, beta-MeATP, 1(-10) X 10(-6)M), an agent known to desensitize purinoceptors. 2 Application of noradrenaline (NA, 1 X 10(-8)-1 X 10(-6)M), by pressure ejection from a micropipette, depolarized the membrane and produced a localized contraction, both of which were abolished by phentolamine (1 X 10(-6)M) or prazosin (1 X 10(-7)M). 3 Application of adenosine-5'-triphosphate (ATP, 1 X 10(-4)-1 X 10(-3)M), by pressure ejection from a micropipette, produced a small membrane depolarization and localized contraction which were unaffected by phentolamine (1 X 10(-6)M) or prazosin (1 X 10(-7)M) but abolished by alpha, beta-MeATP (1 X 10(-6)M). 4 The results show that, in the rat annococcygeus muscle, (1) field or extrinsic nerve stimulation released only one excitatory transmitter, namely NA, although receptors for both NA and ATP were present on the muscle, (2) alpha, beta-MeATP was selective for purinoceptors and (3) there was no evidence for excitatory co-transmission in this tissue.