Inhibition of histamine N-methyltransferase (HNMT) in vitro by neuromuscular relaxants

There have been reports of hypotension and flushing following vecuronium administration. The etiology of these symptoms, which are similar to those of histamine release, is not clear. The steroidal neuromuscular relaxants (NMRs), unlike muscle relaxants structurally similar to curare, have been shown not to cause histamine release after the administration of typical clinical doses. Histamine levels in plasma reflect a balance between release and catabolism. In humans, histamine N-methyl-transferase (HNMT) is the enzyme primarily degrading for histamine. Therefore, we performed in vitro kinetic studies of purified HNMT to determine the effects of the steroidal and curare-like NMRs and also of gallamine on histamine catabolism. We demonstrated that all NMRs tested were inhibitors of HNMT in vitro. The inhibition was competitive with respect to the cosubstrate S-adenosyl-L-[3H-methyl] methionine, and noncompetitive with respect to histamine. The rank order of inhibition was vecuronium greater than pancuronium greater than gallamine greater than d-tubocurarine greater than metocurine greater than atracurium greater than pipecuronium, with Ki values ranging from 1.2 to 44.8 microM. Our data suggest that HNMT-based radioenzymatic assays for histamine should be susceptible to inhibition by concurrent use of NMRs, particularly vecuronium.     

Disturbance of the prejunctional modulation of cholinergic neurotransmission during chronic granulomatous inflammation of the mouse ileum.

The effect of chronic granulomatous inflammation of the intestine was studied on the prejunctional modulation of cholinergic nerve activity in the mouse ileum. Contractions to carbachol (0.01 - 0.3 microM) and to electrical field stimulation (EFS, 0.25 - 8 Hz) of enteric neurons were higher in inflamed ileum as compared to control ileum. However, when the neurally-mediated contractions to EFS were expressed as percentage of the direct smooth muscle contraction to carbachol, the responses to EFS were similar in control and inflamed ileum. Atropine (1 microM) abolished all contractions to EFS and carbachol in control and inflamed ileum. DMPP (3 - 30 microM), a nicotinic receptor agonist, induced concentration-dependent contractions that were more pronounced in inflamed ileum as compared to control ileum. Hexamethonium (100 microM), a nicotinic receptor blocker, significantly inhibited the contractions to EFS in inflamed ileum but not in control ileum. In control ileum, histamine (10 - 100 microM) and the histamine H(1) receptor agonist HTMT (3 - 10 microM) inhibited the contractions to EFS concentration-dependently without affecting the contractions to carbachol. The inhibitory effect of histamine and HTMT was prevented by the histamine H(1) antagonist mepyramine (5 - 10 microM) but not by the H(2)- and H(3)-receptor antagonists cimetidine and thioperamide (both 10 microM). In chronically inflamed ileum however, histamine (10 - 100 microM) and HTMT (3 - 10 microM) failed to inhibit the contractions to EFS. The histamine H(2) and H(3) receptor agonists dimaprit and R(-)-alpha-methylhistamine did not affect the contractions to EFS in control and inflamed ileum. The alpha(2)-receptor agonist UK 14.304 (0.01 - 0.1 microM) inhibited the contractions to EFS in control and inflamed ileum without affecting the contractions to carbachol. The effect of UK 14.304 was reversed by the alpha(2)-receptor antagonist yohimbine (1 microM). The inhibitory effect of UK 14.304 on contractions to EFS was of similar potency in control and inflamed ileum. Our results suggest that the prejunctional modulation of cholinergic nerve activity by nicotinic and histaminic H(1) receptors is disturbed during chronic intestinal inflammation whereas the modulation by alpha(2)-receptors is preserved. Such a disturbance of cholinergic nerve activity may contribute to the motility disturbances that are often observed during chronic intestinal diseases in humans.     

Verapamil intensifies neuromuscular blockade produced by gallamine and pancuronium at the chick neuromuscular junction

Experiments were performed to study the effect of verapamil on neuromuscular transmission and muscle contraction at a chick skeletal muscle-nerve preparation. In addition, the effects and interactions of verapamil with some muscle relaxants were studied in the same preparation. These effects were explored by studying the effects of verapamil on: directly-and indirectly-elicited twitch contractions, and neuromuscular blockade produced by gallamine and pancuronium. The results showed that verapamil (2-200 microM) had a differential effect on the twitch responses; more reductions occurred in the indirectly-elicited twitch tension, whereas the directly-elicited twitch response was reduced only by 20-30% of maximum indirectly-elicited twitch tension. Furthermore, in low concentrations (1-20 microM), verapamil significantly increased the neuromuscular blockade produced by gallamine (28-1280 nM) and pancuronium (18-573 nM). In high concentrations (greater than 200 microM), verapamil completely blocked the indirectly-elicited twitch response and produced a marked contracture in the chick skeletal muscle (1.0 +/- 0.1 g, n = 6). It was concluded that by reducing twitch tension and inhibiting neuromuscular transmission, verapamil increases (intensifies) neuromuscular blockade produced by muscle relaxants, e.g. gallamine and pancuronium.     

Pharmacological evaluation of the histamine H1 and 5-HT blocking properties of 2-N-(carboxamidinonormianserin) (FCC5): in-vitro studies.

Some in-vitro pharmacological effects of a novel analogue of mianserin, 2-carboxamidino-1,2,3,4,10,14b-hexahydrodibenzo (c,f) pyrazino (1,2-alpha) azepine hydrochloride (FCC5) have been studied. FCC5 was a non-competitive antagonist of both histamine-induced contractions of the guinea-pig ileum and 5-HT-induced contractions of rat fundal strips with pD'2 values of 6.13 and 5.57, respectively. The insurmountable antihistaminic effect of FCC5, 100 nM, in the guinea-pig isolated ileum was not removed by washing. FCC5, 10-100 nM, had no effect on responses to acetylcholine or barium chloride of the guinea-pig isolated ileum. In guinea-pig isolated right atria, FCC5, 1-30 microM, had no effect on H2-receptor-mediated chronotropic responses to histamine. FCC5, 10-1000 nM, had no alpha 2-adrenoceptor antagonist activity, as assessed by lack of effect on the inhibitory responses to B-HT 920 in the electrically stimulated rat isolated vas deferens. FCC5 resembles mianserin by being a potent, non-competitive antagonist at histamine H1 and 5-HT receptors, but differs from mianserin in a number of respects including having much less effect at alpha 2-adrenoceptors.     

Characterization of histamine-induced contraction in rat isolated aorta.

High concentrations of histamine (greater than 10 microM) contract rat aortic rings and the effect is greatly enhanced when the endothelium is removed. The present study was aimed at characterizing the histamine-induced contractions of de-endothelialized rat aortic rings. These contractions were poorly inhibited by the histamine H1-receptor antagonist, mepyramine (1 and 10 microM) and insensitive to the histamine H2-receptor antagonist, cimetidine (10 microM), and to the cyclooxygenase inhibitor, indomethacin (5 microM). In contrast, the alpha-adrenoceptor antagonists, prasozin and pentholamine, antagonized these contractions in a concentration-dependent manner (respective apparent pKB values 9.7 and 7.9) and nifedipine (3 microM) reduced them by about 75%. Pretreatment of de-endothelialized rings with 8-bromo-cyclic GMP and of intact rings with methylene blue resulted in respective inhibition and enhancement of histamine-induced contractions, quite similarly to the effects in the presence and in the absence of endothelium, respectively. Histamine elicited endothelium-dependent relaxation of aortic rings precontracted by prostaglandin F2 alpha. This relaxation was abolished in the presence of mepyramine (1 microM). However, mepyramine failed to mimic the enhancing effect of endothelium removal on histamine-induced contractions of resting aortic rings. It is concluded that, in rat aorta, (1) contractions induced by high concentrations of histamine (greater than 10 microM) are probably mediated by alpha 1-adrenoceptors; and (2) spontaneous, but not histamine-stimulated, release of endothelium-derived relaxing factor is mainly involved in the modulation of histamine-induced contractions.     

A comparative study of functional 5-HT4 receptors in human colon, rat oesophagus and rat ileum.

1. The pharmacological properties of 5-hydroxytryptamine (5-HT), the 5-HT4 receptor agonists, DAU 6236 and SC 53116 and the 5-HT4 receptor antagonist, GR 1130808, were studied in the rat oesophagus, rat ileum and human colon. 2. 5-HT relaxed the longitudinal muscle of the rat oesophagus and rat ileum and the circular muscle of the human colon. Absolute values of relaxation were measured and showed the order of the maximum responses, rat oesophagus >> human colon > rat ileum with EC50 values of 189 +/- 15 nM, 157 +/- 4 nM, 306 +/- 72 nM, respectively. 5-HT also inhibited the spontaneous contractions of the human colon with an EC50 value of 119 +/- 1 nM. The effect of 5-HT on the human colon was not affected by methysergide (10 microM) or ondansetron (1 microM). 3. The use of the uptake and metabolism inhibitors, cocaine (30 microM) and pargyline (100 microM), did not increase the potency of 5-HT in the rat oesophagus or human colon. In the rat oesophagus, cocaine (30 microM) produced a reduction in carbachol-induced tone of 22.2 +/- 0.6% and reduced the 5-HT maximum effect by 52.0 +/- 0.4%. 4. The compounds, DAU 6236 and SC 53116, showed a different pattern of potencies and efficacies in the rat oesophagus, rat ileum and human colon compared to 5-HT. DAU 6236 relaxed the human colonic circular muscle with an EC50 value of 129 +/- 16 nM but its efficacy was less than that of 5-HT. DAU 6236 (1 microM) also antagonized the 5-HT-induced relaxation of the human colon with a dose-ratio of 9.9. In the rat oesophagus and rat ileum, DAU 6236 was inactive in the majority of tissues. In the minority of oesophagus tissues that did respond the EC50 value was 1.2 +/- 0.7 microM. DAU 6236 also antagonized the effect of 5-HT in the rat oesophagus in a non-surmountable fashion. SC 53116 relaxed the rat oesophagus with an EC50 value of 91 +/- 4 nM, with an efficacy less than that observed to 5-HT; however, at 200 nM it did not antagonize the 5-HT-induced relaxation of the rat oesophagus. SC 53116 showed no agonist activity in the rat ileum and human colon, but at 1 microM it did antagonize the effect of 5-HT in the human colon with a dose-ratio of 11.3 +/- 0.3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histamine-induced inositol phospholipid breakdown in the longitudinal smooth muscle of guinea-pig ileum.

The characteristics of histamine-stimulated inositol phospholipid breakdown in slices of guinea-pig ileal smooth muscle and cerebellum have been investigated. In cerebellar slices the inhibition of the inositol phospholipid response to histamine by mepyramine was consistent with competitive antagonism of histamine H1-receptors. In slices of the longitudinal smooth muscle of guinea-pig ileum, mepyramine produced only a weak inhibition of the response to histamine, at concentrations up to 1 microM. This was in striking contrast to the potent competitive antagonism of the H1-mediated contractile responses obtained with mepyramine in this tissue. The H1-receptor antagonists (+)-chlorpheniramine and promethazine similarly had no effect on the EC50 value for histamine in guinea-pig ileum, while promethazine competitively antagonized the muscarinic receptor-mediated inositol phospholipid response in this tissue (Ka 3.6 X 10(7)M-1). Cimetidine, on its own, did not significantly inhibit the inositol phosphate accumulation elicited by histamine in ileum. In the presence of 0.2 microM mepyramine, cimetidine (0.1 mM) produced a small parallel shift of the histamine concentration-response curve (Ka 3 X 10(4) M-1). This inhibition, however, was not consistent with antagonism of an H2-receptor-mediated response. The effect of a range of histamine analogues on inositol phospholipid breakdown was determined. Dose-response curves were constructed and characterized in terms of the EC50, slope and maximal response attainable relative to histamine. The H1-agonists, N alpha,N alpha-dimethylhistamine, N alpha-methylhistamine, 2-pyridylethylamine and 2-thiazolyethylamine produced the largest accumulations of [3H]-inositol-1-phosphate. A very weak response was produced by the H2-selective agonist impromidine, while dimaprit (also H2-selective) was without significant effect. Mepyramine appeared to antagonize competitively the response to the H1-selective agonist 2-pyridylethylamine. This was in contrast to the data obtained with other H1-agonists, where mepyramine produced only a small dextral shift of the agonist curves at low agonist concentrations and an increase in the Hill coefficient. This was particularly striking in the case of 2-methylhistamine. The results suggest that an H1-receptor component in guinea-pig ileum, may coexist with a larger inositol phospholipid response to histamine which is independent of the activation of H1- or H2-receptors.     

Differing actions of beta-(2-thienyl)-gamma-aminobutyric acid in central and peripheral preparations.

In the guinea-pig isolated ileum, beta-(2-thienyl)-gamma-aminobutyric acid (BTG; 100-500 microM) reversibly and competitively (pA2 = 4.3 +/- 0.1) antagonised the baclofen-induced (5-100 microM) depression of cholinergic twitch contractions, but not that to adenosine or morphine. By contrast, in rat neocortical slice preparations, BTG (100-500 microM) acted as an agonist, abolishing the frequency and amplitude of spontaneous discharges, sensitive to 2-hydroxysaclofen (100-500 microM). BTG exhibits differential actions at GABAB receptors in brain and periphery.     

Activation of 5-HT3 receptors in the rat and mouse intestinal tract: a comparative study

This study provides a comprehensive evaluation of 5-HT(3) receptor functional distribution in both the rat and mouse intestinal tract. 5-HT(3A-S) receptor splice variant mRNA was expressed throughout the intestine of the rat and mouse; the 5-HT(3A-L) variant being more common in the rat.5-HT, m-CPB, 1-PBG and 2-methyl-5-hydroxytryptamine (2m5-HT) induced contraction in the jejunum, ileum, proximal colon and distal colon of the rat (pEC(50) range: 2m5-HT, 5.86+/-0.40 to m-CPB, 7.47+/-0.27) and mouse (pEC(50) range: 1-PBG, 5.34+/-0.06 to m-CPB, 6.49+/-0.14) in the presence of nontarget 5-HT receptor antagonists, methysergide (1 muM) and GR125487 (0.1 microM). The rank orders of potency in the four regions of the rat and mouse intestine were concordant with the accepted order and the responses to 5-HT were inhibited by ondansetron (0.1 microM).5-HT(3)-induced contractions to 5-HT were reduced by tetrodotoxin (1 microM). Pargyline (10 muM) and fluoxetine (1 microM) potentiated responses in the rat jejunum. Atropine (0.1 microM) potentiated 5-HT(3)-induced responses in the rat jejunum (E(max) 49-65%), but attenuated responses in most other regions of the rat and mouse (e.g. mouse ileum: E(max) 57-26%). In the rat jejunum, L-NAME (100 microM) mimicked the effect of atropine, hexamethonium (100 microM) suppressed 5-HT(3)-induced responses, but tachykinin receptor antagonists were without effect. It is concluded that functional 5-HT(3) receptors are present in nerves along the length of the rat and mouse intestinal tract. The mouse proximal colon was found to discriminate 5-HT(3) receptor agonist profiles better than any other region in the rat or mouse. The rat jejunum shows evidence of 5-HT uptake and inactivation processes as well as inhibitory nitrergic and nontachykinin excitatory pathways associated with the 5-HT(3)-induced response.     

Characterization of histamine receptors mediating the stimulation of cyclic AMP accumulation in rabbit cerebral cortical slices.

The characteristics of histamine-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in slices of rabbit cerebral cortex have been investigated. The selective H2-receptor antagonists, cimetidine, tiotidine, metiamide and ranitidine appeared to antagonize the stimulation of cyclic AMP accumulation elicited by histamine in a competitive manner consistent with an interaction with histamine H2-receptors. The H1-receptor antagonist mepyramine (0.8 microM) produced only a weak inhibition of the response to histamine. The inhibition appeared to be non-competitive producing a decrease in the maximal response with little effect on the EC50 value. The specific H2-receptor agonist, impromidine, produced a maximum response of only 31 +/- 2% of that obtained with histamine. Studies with histamine and impromidine in combination indicated that impromidine was not acting as a partial agonist. 2-Thiazolylethylamine, a selective H1-agonist, produced only a weak response (EC50 approximately 1mM) yielding a relative potency with respect to histamine (= 100) of 2.5. In the presence of a supramaximal concentration of impromidine, histamine and 2-thiazolylethylamine further elevated the response to impromidine. In these conditions the relative potency of 2-thiazolylethylamine was increased to 59 (histamine = 100), a value which was comparable with that reported for H1-receptor-mediated contractions of guinea-pig ileum. The H1-receptor antagonists mepyramine, promethazine, triprolidine and chlorpheniramine competitively antagonized the potentiation of impromidine-stimulated cyclic AMP accumulation elicited by histamine and 2-thiazolylethylamine in rabbit cerebral cortex without affecting the response to impromidine alone. (+)-Chlorpheniramine was some 150 fold more potent than the (-)-isomer in this respect. Histamine and adenosine in combination had a much greater than additive effect on the accumulation of cyclic AMP in rabbit cerebral cortical slices. The potentiation of the adenosine response could be partially but not completely antagonized by either cimetidine or mepyramine. In the presence of H2-receptor blockade with 0.02 mM tiotidine, histamine elicited a significant potentiation (EC50 44 microM) of the response to adenosine. This response was antagonized competitively by mepyramine yielding a KB value of 0.05 microM similar to that obtained from inhibition of the potentiation of impromidine-stimulated accumulation of cyclic AMP (0.02 microM). These results suggest that there are two components in the response to histamine in rabbit cerebral cortical slices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for histamine and 5-hydroxytryptamine like effects of MK-212 on guinea pig ileum, taenia coli and rat fundus strip

MK-212 (1 x 10(-7)M -- 1 x 10(-5)M) produced dose-dependent contractions of guinea pig ileum, taenia coil and rat fundus strip. The responses to MK-212 in all three preparations were blocked competitively by cyproheptadine (1 x 10(-8)M) a 5-HT receptor antagonist. Mepyramine (1 x 10(-8)M)-H1 receptor antagonist also inhibited competitively the responses of guinea pig ileum and taenia coli to MK-212. However, it failed to block significantly the responses of rat fundus strip to MK-212. Metiamide (1 x 10(-6)M), propranolol (1 x 10(-6)M) or atropine (1 x 10(-6)M) did not produce any significant effects on MK-212 induced contractile responses of guinea pig ileum, taenia coli and rat fundus strip. Our findings suggest that MK-212 produces both 5-HT as well as histamine like effects on the guinea-pig ileum, taenia coli and rat fundus strip.     

Selective inhibition of Kir currents by antihistamines

In the present study, the effects of antihistamines on inwardly rectifying potassium (Kir) channels expressed in Xenopus oocyte were investigated using two-electrode voltage clamp technique. Firstly, effects of antihistamines on two members of Kir2.0 sub-family, Kir2.1 and Kir2.3 were compared. For antihistamines that selectively block histamine H(1) receptor, the first-generation antihistamines mepyramine and diphenhydramine inhibited Kir2.3 current by 25.0+/-2.9% and 17.3+/-0.7% at concentrations of 100 microM, respectively. In contrast, the second- and third-generation antihistamines astemizole and desloratadine were completely devoid of any inhibitory effect on Kir2.3 current. Histamine H(2) receptor antagonist cimetidine, at 100 microM, failed to inhibit Kir2.3 current. On the other hand, Kir2.1 current was not sensitive to any of these drugs. The mepyramine-induced inhibition of Kir2.3 current was significantly reduced by a single point mutation in Kir2.3 (Kir2.3(I213L)), which enhances Kir2.3-PIP(2) interaction. Secondly, the effect of mepyramine was also tested on Kir3.4*, another member of Kir family. 100 microM mepyramine produced a 30.3+/-4.6% inhibition on Kir3.4* current. These results suggest that the first-generation histamine H(1) receptor antagonists selectively inhibit Kir currents. The inhibitory effect of antihistamines on Kir currents may be involved in their neuronal and cardiac toxic effects caused by drug overdosing.     

The pharmacology of anaphylaxis in the chicken intestine.

1 The Schultz-Dale phenomenon has been demonstrated in several circular smooth muscle strips of oesophagus, crop, duodenum, jejunum and ileum taken from young and adult domestic fowl sensitized actively to crystalline bovine albumin or horse plasma. 2 The ileal strips contract to acetylcholine, histamine, 5-hydroxytryptamine (5-HT), prostaglandins E1, E2, F2alpha, bradykinin and bovine slow reacting substance of anaphylaxis (SRS-A). Marked seasonal and individual variations in the responsiveness of gut tissues to these exogenous agonists were noted. 3 Antagonism of contractions to histamine by mepyramine suggests the existence of H1-histamine receptors in chicken ileum. Blockade of 5-HT-induced contractions by methysergide shows the preponderance of 'D'-musculotropic tryptamine receptors. 4 Failure of selective receptor antagonists of acetylcholine, histamine and 5-HT to modify the Schultz-Dale reaction suggests the nonparticipation of aminergic mechanisms in this reaction. 5 Partial to complete blockade of the Schultz-Dale reaction by a prostaglandin receptor antagonist (polyphloretin phosphate, PPP); prostaglandin synthetase inhibitors (sodium meclofenamate and phenylbutazone); inhibitors of synthesis and release of histamine and SRS-A (PR-D-92-EA, M & B 22948, diethylcarbamazine citrate, and PPP) and an inhibitor of proteinases (aprotinin) strongly suggests the involvement of vasoactive lipids and polypeptides in the anaphylactic response of chicken ileum to specific antigen.     

Sex differences in the pharmacological activity of venom from the white-tailed spider (Lampona cylindrata).

This study compared the pharmacological activity of venom from male and female white-tailed spiders (L. cylindrata). In guinea-pig ileum, male L. cylindrata venom (1-10 microg/ml) caused dose-dependent contractions. The response to venom (5 microg/ml) was significantly inhibited by mepyramine (0.5 microM). Venom (5-50 microg/ml) from female L. cylindrata had no contractile activity in this tissue. However, female L. cylindrata venom (50 microg/ml) inhibited electrically-evoked twitches of guinea-pig ileum. This inhibitory effect was attenuated by 8-phenyltheophylline (10 microM) or by prior exposure of venom to adenosine deaminase. In the rat vas deferens, male (5 microg/ml) and female (50 microg/ml) L. cylindrata venom inhibited electrically-evoked twitches. 8-Phenyltheophylline (20 microM) significantly attenuated the response to female L. cylindrata venom, while the histamine H(2)- and H(3)-receptor antagonists ranitidine (10 microM) and thioperamide (0.2 microM) significantly attenuated the response to male L. cylindrata venom. Male L. cylindrata venom (5-20 microg/ml) caused dose-dependent contractions in the epididymal segment of the rat vas deferens. The response to male L. cylindrata venom (10 microg/ml) was significantly inhibited by prazosin (0.3 microM) but was unaffected by depleting monoamine stores with reserpine. Male L. cylindrata venom (5-15 microg/ml) caused dose-dependent increases in rate and force of rat atria which were significantly inhibited by propranolol (5 microM) but not by reserpine. Female L. cylindrata venom (50 microg/ml) had no effect in atria. In the anaesthetised (pentobarbitone, 100 mg/kg, i.p.) rat, male L. cylindrata venom (10-300 microg/kg, i.v.) caused dose-dependent depressor responses while venom (up to 1 mg/kg, i.v.) from female L. cylindrata had no effect on arterial pressure. A histamine content of 5 and 0.01% (dry weight) was detected in venom from male and female L. cylindrata, respectively. Venom from male L. cylindrata was found to contain 56 pg noradrenaline/microg whereas venom from the female contained negligible noradrenaline. The results of this study show the presence of histamine and noradrenaline in venom from male L. cylindrata. Although devoid of significant quantities of these amines, female L. cylindrata venom has activity at adenosine receptors.     

Pharmacological characterization of the 5-hydroxytryptamine receptor mediating relaxation in the rat isolated ileum.

1 The aim of the present study was to investigate a 5-HT4 receptor involvement in the mediation of a 5-HT-induced relaxation response in the rat isolated ileum in vitro. 2 Ileal segments were taken at regular intervals from the ileo-caecal junction to duodenum. 5-HT (1 microM) induced a relaxation or contraction response in segments taken from the terminal ileum: the relaxation decreased and finally disappeared as contractions dominated in the proximal tissues. The 5-HT-induced relaxations were enhanced in the terminal segments and the contractions attenuated in both terminal and proximal segments, in the presence of methysergide (1 microM) and atropine (0.1 microM). 3 In the presence of methysergide (1 microM) and atropine (0.1 microM), a cumulative addition of 5-HT (0.01-1 microM) induced a concentration-dependent relaxation in the terminal (1-20 cm from the ileo-ceacal junction) ileal segments which at higher concentrations of 5-HT (3-30 microM) reverted to contraction. 4 The rank order of potency of indole agonists in inducing a concentration-related relaxation response in tissues of the terminal ileum (pretreated with pargyline (100 microM) and in the presence of methysergide (1 or 100 microM) and atropine (0.1 microM) was 5-hydroxytryptamine (6.97 +/- 0.06), 5-methoxytryptamine (6.50 +/- 0.07), alpha-methyl-5-hydroxytryptamine (5.53 +/- 0.17), 5-carboxamidotryptamine (5.51 +/- 0.12) and 2-methyl-5-hydroxytryptamine (< 5), the pEC50 values (mean +/- s.e.mean) being shown in parentheses. 5 Pretreatment of tissues with pargyline (100 microM) selectively enhanced the potency of 5-methoxytryptamine by a factor of 19 but failed to modify the potency of the other indole agonists. 6 The 5-HT4 receptor antagonists, tropisetron, SDZ 205-557 and GR 113808 antagonized the relaxation response to 5-HT (in the presence of methysergide (1 or 10 microM) and atropine (0.1 microM)) with pKB values (95% CL) of 6.09 (5.94-6.24), 7.0 (6.9-7.09) and 8.95 (8.81-9.1) respectively. Apparent pKB values estimations for tropisetron (1 microM) and GR 113808 (10 nM) using the agonists 5-methoxytryptamine and 5-carboxamidotryptamine were 6.37 +/- 0.31, 5.91 +/- 0.38 and 8.83 +/- 0.11, 8.82 +/- 0.22 respectively. 7 Tropisetron (10 microM), SDZ 205-557 (3 microM) and GR 113808 (10-100 nM) caused an increase in basal tone of the rat terminal ileum when administered in the presence of methysergide and atropine. 8 The relaxation response to 5-HT in the rat terminal ileum was not antagonized by ritanserin (1 microM), ondansetron (1 microM) or N omega-nitro-L-arginine methyl ester (100 microM) and with only a twofold dextral shift of the concentration-response curve by tetrodotoxin (1 microM). 9 It is concluded that the relaxant response to 5-HT in the terminal region of the ileum is mediated directly at the smooth muscle; a ranked indole agonist potency and selective antagonism by 5-HT4 receptor antagonists tropisetron, SDZ 205-557 and GR 113808 indicate a 5-HT4 receptor involvement in the relaxation response.     

The lack of compound 48/80-induced contraction in isolated trachea of mast cell-deficient Ws/Ws rats in vitro: the role of connective tissue mast cells

In the rat trachea, two types of mast cells have been identified, connective tissue mast cells and mucosal mast cells. Their different characteristics may account for their different biological functions. The role of connective tissue mast cells in tracheal contraction as one feature of the immediate reaction of asthma was studied in vitro in isolated trachea, using tissue derived from mast cell-deficient (Ws/Ws) rats, heterozygous (Ws/+) rats and control (+/+) rats, and compound 48/80 as a potent inducer of mast cell degranulation. The contractile response of tracheas from the three types of rats was also studied upon exposure to the following spasmogens: histamine, 5-hydroxytryptamine (5-HT), and carbachol. Histamine content in tissues reflected the differing mast cell numbers in strips from the three rat types. It was found that carbachol and 5-HT elicited tracheal contraction in a similar manner in strips from the three types of rats. Histamine had no contractile effect. Compound 48/80, at a dose of 25 microg/ml, elicited contraction in tracheas from both control (+/+) and heterozygous (Ws/+), but not in trachea from Ws/Ws rats. Compound 48/80-induced contractions in tracheas from +/+ rats were inhibited by 0.1 microM ketanserin and 0.1 microM nedocromil, but not by 0.1 microM mepyramine. Enzyme histochemistry confirmed that the degranulation occurred in connective tissue mast cells, but not in mucosal mast cells. We concluded that connective tissue mast cells play an important role in rat tracheal contraction via 5-HT release induced by compound 48/80. In addition, the specific mast cell-deficient (Ws/Ws) rats provide a good tool for studying the roles of mast cells in airway system.     

Activation of a myenteric 5-hydroxytryptamine-like receptor by metoclopramide

Cholinergically mediated contractions were evoked by electrical field stimulation (EFS) of guinea-pig distal ileum longitudinal muscle-myenteric plexus strips. Metoclopramide, 0.1-100 microM, dose-dependently increased the contractions, probably by increasing acetylcholine (ACh) release; contractions evoked by exogenous ACh in the presence of tetrodotoxin were not increased by metoclopramide. 5-Hydroxytryptamine (5-HT) 0.3-30 nM similarly increased the height of the EFS-evoked contractions, although the maximum increase was less than for metoclopramide; higher concentrations of 5-HT (3 and 30 microM) had no effects or caused inhibition. The compound, ICS 205-930, 0.001-1 microM, had no effect on the EFS-evoked contractions and caused inhibition at higher concentrations. Preincubation of the tissues with 5-HT, 0.3-30 nM, did not affect the increase in EFS-evoked contractions caused by metoclopramide, 1 or 100 microM, whereas 5-HT, 3 and 30 microM, prevented the response caused by metoclopramide 1 microM, but not 100 microM. ICS 205-930, 0.1 microM, had no effect on the increase in EFS-evoked contractions caused by metoclopramide 1 or 100 microM. The drug, at least in low concentrations, may therefore increase cholinergically mediated contractions of guinea-pig ileum by stimulating 5-HT-like receptors within the myenteric plexus, which differ from those antagonized by ICS 205-930.     

Characterization of histamine H3-receptors in guinea-pig ileum with H3-selective ligands.

1. The effect of the selective histamine H3-receptor agonist R-(alpha)-methylhistamine has been investigated on the contractile responses of the longitudinal smooth muscle of guinea-pig ileum elicited by electrical field stimulation of acetylcholine release from myenteric nerve endings. 2. R-(alpha)-methylhistamine produced a concentration-dependent (EC50 = 1.4 +/- 0.2 x 10(-8) M) inhibition of the response to electrical field stimulation which was insensitive to inhibition by mepyramine (1 microM) and tiotidine (2.4 microM). 3. This response to R-(alpha)-methylhistamine could be inhibited in a competitive fashion by a range of H3-receptor antagonists including thioperamide (KB = 1.1 nM), impromidine (KB = 65 nM), norburimamide (KB = 380 nM) and SKF 91486 (KB = 34 nM). Burimamide was also a potent inhibitor of this response but the Schild slope obtained (1.3) was significantly greater than unity. 4. The estimated KB values were all within a factor of three of those values reported for the histamine H3-receptor mediating inhibition of histamine release in rat cerebral cortex. 5. These data suggest that the histamine receptor mediating inhibition of cholinergic neurotransmission by R-(alpha)-methylhistamine in guinea-pig ileum is the same as the H3-receptor present in rat cerebral cortex.     

Pharmacological studies of jumper ant (Myrmecia pilosula) venom: evidence for the presence of histamine, and haemolytic and eicosanoid-releasing factors.

Jumper ant venom was prepared by extraction of venom sacs in distilled water and centrifugation to remove insoluble material. Jumper ant venom (2 micrograms/ml) produced a biphasic response on isolated guinea-pig ileum, i.e. an initial rapid contraction followed by a slower prolonged contraction. The histamine antagonist mepyramine (0.1 microM) inhibited the first phase of this response by greater than 90%. In the isolated rat stomach fundus strip (which is insensitive to histamine), jumper ant venom (6 micrograms/ml) produced only a single contraction. No tachyphylaxis was observed to repeated doses of jumper ant venom in guinea-pig ileum or rat fundus strip. Responses to jumper ant venom of the egg-albumin-sensitised guinea-pig ileum were not significantly different before and after an in vitro anaphylactic response induced by egg albumin (0.5 mg/ml). Fluorometric assay revealed a mean value of 0.9 +/- 0.2% of the dry weight as histamine in jumper ant venom. Both the lipoxygenase/cyclo-oxygenase inhibitor BW755C and the cyclooxygenase inhibitor indomethacin significantly inhibited the second phase response to jumper ant venom of the guinea-pig ileum, and the response of the rat fundus strip. The muscarinic receptor antagonist atropine (0.1 microM), the bradykinin antagonist [Thi5,8,D-Phe7]-bradykinin (10 microM) and the angiotensin converting enzyme inhibitor captopril (20 microM) did not affect either phase of the venom response in guinea-pig ileum. Jumper ant venom caused haemolysis of guinea-pig blood. The degree of haemolysis was significantly reduced when boiled venom was used. These results suggest that jumper ant venom contains histamine and may cause the release of cyclo-oxygenase products. It also contains a heat-sensitive haemolytic factor.     

Investigation into the 5-hydroxytryptamine-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon.

1. The aim of this study was to characterize the receptors mediating the atropine-resistant neurogenic contraction to 5-hydroxytryptamine (5-HT) in the longitudinal muscle of the guinea-pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5-HT by the use of selective antagonists. 2. In the presence of atropine (0.3 microM), guanethidine (5 microM), hexamethonium (100 microM), ketanserin (0.1 microM) and indomethacin (3 microM), 5-HT (0.01-3 microM) produced concentration-dependent neurogenic contractions of colonic strips and at 0.3 microM produced a maximal effect (pEC50 = 7.39 +/- 0.09, n = 18). The 5-HT4 receptor stimulant, 5-methoxytryptamine (5-MeOT, 0.03-10 microM) also produced neurogenic contractions with similar maximum effect to those of 5-HT (pEC50 = 6.89 +/- 0.16). 3. The 5-HT4 receptor antagonist, DAU 6285 (3 microM) shifted the concentration-response curves to both 5-HT and 5-MeOT to the right without significant depression of the maximum, but the 5-HT1/5-HT2 receptor antagonist, metitepine (0.1 microM) and the 5-HT3 receptor antagonist, ondansetron (0.3 microM) had no effect on the control curves to 5-HT and 5-MeOT. 4. The selective NK1 receptor antagonist, FK 888 (1 microM) markedly attenuated the contractions to 5-HT and 5-MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5-HT and 5-MeOT. 5. These results indicate that the 5-HT-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon is due to activation of 5-HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5-HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5-HT4 receptor-mediated contraction is evoked indirectly via tachykinin release from tachykinin-releasing excitatory neurones.