Modulation of the delayed K+ current by histamine in guinea pig ventricular myocytes

Summary The effects of histamine on delayed K⁺ current (I_K) were investigated in patch-clamped single guinea pig ventricular myocytes. Histamine increased I_K with a maximal fractional response of 2.7 and a k_d of 9.4 × 10^–7 mol/l. At a concentration of 10^–8 mol/l, histamine did not increase I_K significantly, but increased I_Ca by 52% ± 12%. The voltage-dependence of I_K activation, the reversal potential and the time course of the I_K tail decay were not changed by histamine. Under pretreatment with 10^–4 mol/l of ranitidine, neither histamine (10^–6 mol/l) nor 2-pyridylethylamine (10^–4 mol/l) caused any sizable increase in I_K. When the cell was pretreated with a saturating dose of isoproterenol (10^–6 mol/l), histamine did not additively enhance I_K. The I_K enhancement by 3 × 10^–7 mol/l histamine was partially antagonized by concurrent exposure to 5 × 10^–6 mol/l carbachol. Whereas, use of a higher concentration of histamine (10^–6 mol/l) obscured the inhibitory effect of carbachol. It is concluded that histaminergic action of I_K is attributed exclusively to H₂ receptor-mediated reactions involving G_s protein and adenylate cyclase. Send offprint requests to Y. Habuchi at the above address     

Relations between the effects of histamine,pheniramin and metiamide on spontaneous motility and the formation of cyclic AMP in the isolated rat uterus

Summary Histamine (5×10^–6 to 10^–3M) depressed the spontaneous motility of the isolated rat uterus in a dose-dependent manner. Under these conditions uterine cyclic AMP was raised up to 92%. Both effects, uterine relaxation and cyclic AMP accumulation after 2 min could be inhibited dose dependently by the H₂-antihistaminic compound metiamide (1.7×10^–6 M to 1.7×10^–4 M). By contrast, the H₁-antagonist pheniramin (4.4×10^–5 M) was ineffective. It was concluded that the histamine-induced inhibition of rat uterine motility is mediated by cyclic AMP which is formed in response to stimulation of H₂-histaminergic receptors.     

Indirect adrenergic effect of histamine in cat cerebral arteries

Summary Histamine (10(^–4 M) induced an increase in the tritium outflow from cat cerebral arteries preloaded with ³H-noradrenaline. Pretreatment with reserpine (3 mg/kg, i.p., total dose) or removal of both superior cervical ganglia two weeks before the experiment abolished that increase. The presence of cocaine or diphenhydramine also prevented the rise in tritium efflux induced by histamine.Histamine (10(^–8 M to 10(^–3 M) elicited dose-dependent contractions in the isolated posterior communicating artery of the cat which were reduced in the presence of diphenhydramine at all doses except the highest three. The addition of phentolamine to the bath decreased the contractile responses at the doses lower than 10(^–6 M. Pretreatment with reserpine or removal of both superior cervical ganglia also diminished the responses at doses of histamine below 10(^–6 M and 10(^–5 M, respectively. When cocaine was added to the bath there was a decrease in the contraction elicited at all doses except the last one.These results suggest the existence of an indirect adrenergic mechanism in the contractile response to histamine in cat cerebral arteries.     

Characteristics of histamine tachyphylaxis in canine tracheal smooth muscle

Summary In isolated canine tracheal smooth muscle, repeated administrations of histamine result in a rapid reduction in contractile response to about 15% of the initial contraction (tachyphylaxis). Development of this tachyphylaxis is specific inasmuch as: 1) it does not develop to acetylcholine (10^–6 M or 10^–4 M), or serotonin (10^–5 M); and 2) maximally developed histamine tachyphylaxis is not associated with a parallel reduction in response to acetylcholine. Pretreatment with propranolol (10^–5 M) or phentolamine (10^–4 M) does not prevent tachyphylaxis: however, pretreatment with atropine (10^–4 M) does prevent tachyphylaxis in about 50% of the animals tested.Tachyphylaxis to histamine can be reversed in a dose- and time-dependent fashion with prostaglandin synthesis inhibiting agents. The order of potency obtained with such compounds (indomethacin > mefenamic acid > oxyphenbutazone > acetylsalicylic acid) is consistent with potencies for inhibition of prostaglandin synthesis found in the literature. Also, in indomethacin pretreated strips in which tachyphylaxis to histamine was prevented, exogenous addition of PGE₂ (1.42×10^–10 M to 2.84×10^–9 M) and PGA₂ in a high concentration (2.9×10^–9 M) are capable of selectively reducing the response to histamine without an effect on acetylcholine-induced contractions. These data suggest that the mechanism of histamine tachyphylaxis in the canine tracheal smooth muscle preparation involves prostaglandin synthesis.This report is part of a dissertation to be presented by W. H. A. to the University of South Florida College of Medicine in partial fulfillment of the requirements for a Doctor of Philosophy degree     

Characterization of the effects of histamine on the transmembrane electrical activity of guinea-pig and rabbit SA- and AV-node cells

Summary The effects of histamine on the transmembrane electrical activity of cells of small preparations (0.5 × 0.5 mm) of guinea-pig and rabbit sinoatrial- and atrioventricular-nodes were studied. Histamine at concentrations above 10^–7 mol/l increased the firing rate, the rate of diastolic depolarization, the maximum diastolic potential, the amplitude and the maximum rate of depolarization of the action potential of pacemaker cells of rabbit and guineapig sinoatrial cells and rabbit atrioventricular cells. These effects were antagonized by the HZ-receptor blocker cimetidine (2.5 × 10^–6 mol/1) but they were not modified by the H₁-receptor blocker chlorphenamine (2.5 and 5×10^–6 mol/1). Small preparations of guinea-pig atrioventricular node did not exhibit spontaneous activity, but it was induced by histamine and blocked by cimetidine. Histamine increased the maximum upstroke velocity of propagated action potential of cells of the central part of complete atrioventricular node in both species studied. These effects were blocked by cimetidine, but not by chlorphenamine. It is concluded that the increase in automaticity induced by histamine in guinea-pig and rabbit sinoatrial and atrioventricular nodes was due to stimulation of H₂receptors. Histamine did not depress electrical activity of atrioventricular node cells, but rather increased it. This effect was due to H₂-receptor stimulation. Send offprint requests to: J. Sanchez-Chapula at the above address     

Human gastric mucosal adenylate cyclase: Activation by histamine-H2-receptor stimulation and inhibition by cimetidine in vitro and in peptic ulcer patients

Summary Biopsy specimens of human fundic mucosa from 96 subjects were assayed for adenylate cyclase activity to characterize specificity of the histamine receptor with selective agonists and antagonists in vitro, and to study the effect of cimetidine therapy. Histamine and the selective histamine H₂-receptor agonist dimaprit were almost equally potent throughout the concentration-response curve (10^–7–10^–3 mol/l); maximal stimulation was obtained with concentrations of 10^–4–10^–3 mol/l, and half maximal stimulation with about 10^–5 mol/l. The selective H₁-receptor agonist PEA 10^–5 and 10^–3 mol/l failed to stimulate adenylate cyclase. Mepyramine 10^–6 mol/l, a selective H₁-receptor antagonist, and cimetidine 10^–6 mol/l, a selective H₂-receptor antagonist, did not affect basal adenylate cyclase activity. Histamine-stimulated adenylate cyclase was inhibited in a concentration dependent manner by cimetidine 10^–7 and 10^–5 mol/l, but not by the same concentrations of mepyramine. Almost identical basal adenylate cyclase activities of about 150 pmol cAMP/mg protein/20 min were found in gastric mucosal biopsies from controls and from peptic ulcer patients, whether or not treated with cimetidine. Histamine 10^–5 mol/l doubled adenylate cyclase activity in the controls and the untreated ulcer group, but was completely ineffective in specimens from the cimetidine-treated peptic ulcer patients. The data underline the concept that the effects of histamine on acid secretion and on adenylate cyclase activity are linked together and that the therapeutic effect of cimetidine in ulcer treatment is related to histamine H₂-receptor blockade followed by inhibition of adenylate cyclase.     

Effect of triphenyltin chloride on the release of histamine from mast cells

The effects of triphenyltin chloride (TPTC) on the release of histamine from rat and mouse mast cells in vitro and in vivo were investigated. The results obtained are summarized as follows: (a) At doses between 1 and 3 mg/kg, TPTC inhibited the dye leakage due to passive cutaneous anaphylaxis mediated by IgE antibody in mouse ear. At the same doses, TPTC inhibited the swelling due to reversed cutaneous anaphylaxis mediated by IgG antibody in rat. However, TPTC did not affect dye leakage due to histamine, serotonin, and LTC₄ in rat skin; (b) Histamine release by antigen and IgE antibody in rat peritoneal cavity was inhibited by the administration of TPTC at doses between 0.3 and 3 mg/kg; (c) Histamine release by calcium ionophore A 23187 from purified rat peritoneal mast cells in vitro was inhibited by TPTC at concentrations between 10^–7 and 10^–6 M. At the same concentration, TPTC, itself, caused neither the release of histamine nor any change in cell viability which was supported by the activity of lactate dehydrogenase (LDH) from purified rat peritoneal mast cells. All this evidence suggests that TPTC has an inhibitory effect on the release of histamine from mast cells without direct cytotoxicity.     

Adenylate cyclase of the dog gastric mucosa: Stimulation by histamine and inhibition by metiamide

Summary The activity of the non-stimulated, basal adenylate cyclase of the dog gastric mucosa is reduced by the histamine H₂-receptor antagonist metiamide but not by the histamine H₁-receptor antagonist mepyramine. Histamine activates the adenylate cyclase only slightly. In the presence of 10^–5 M metiamide a concentration-dependent stimulation of the enzyme by histamine was found. These data indicate that endogenous histamine in dog gastric mucosal homogenate is contributing at least in part to what is measured as basal adenylate cyclase activity. This effect is mediated by H₂-receptor excitation and in earlier studies has prevented the demonstration of a stimulatory effect of exogenous histamine on this enzyme.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Histamine inhibits activation of human neutrophils and HL-60 leukemic cells via H2-receptors

Summary The effects of prostaglandin E₁ (PGE₁) and histamine on activation of superoxide (O_22/su–) formation, exocytosis of -glucuronidase and aggregation in human neutrophils and HL-60 leukemic cells were studied. PGE₁, histamine and impromidine, a potent H₂-agonist, inhibited O₂ ^– formation in neutrophils induced by the chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe) with IC₅₀ values of 0.5 µM, 8 µM and 2 µM, respectively. The full H₁-agonist and weak partial H₂-agonist, betahistine, was much less potent and effective than histamine. Dibutyryl cyclic AMP and forskolin mimicked the effects of histamine and PGE₁, on O₂ ^– formation. The H2-antagonist, famotidine, competitively reversed histamine-induced inhibition of O₂ ^– formation with a pA₂ value of 7.5. Histamine inhibited O₂ ^– formation when added prior to or after fMet-Leu-Phe. fMet-Leu-Phe-induced aggregation and release of -glucuronidase in neutrophils were less sensitive to inhibition by PGE₁, histamine, dibutyryl cyclic AMP and forskolin than_OZ formation. The inhibitor of cyclic AMP-specific phosphodiesterase, rac-4-(3-butoxy4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), additively enhanced the inhibitory effects of histamine and PGE₁, on the above cell functions. In HL-60 cells differentiated by dimethyl sulfoxide or dibutyryl cyclic AMP, histamine, impromidine and PGE₁, but not betahistine inhibited fMet-Leu-Phe-induced O₂ ^– formation as well. Our data suggest that histamine inhibits activation of neutrophils and HL-60 cells via H2-receptors through activation of adenylyl cyclase and increased formation of cyclic AMP. As stimulated basophils and mast cells release high quantities of histamine, this intercellular signal molecule may play an inhibitory role in the activation of cytotoxic functions of myeloid cells. Send offprint requests to R. Seifert at the above address     

Effect of histamine on human bronchial arteries in vitro

Summary Histamine caused a concentration-dependent relaxation at lower concentrations (1 pmol/1–1 mol/l) and contraction at higher concentrations (0.01–1 mmol/l) of isolated precontracted human bronchial arteries. In the vessels at resting tension only concentration-dependent contraction was evoked by histamine (0.01–1 mmol/l). Both the contractile and relaxant responses were significantly antagonised by mepyramine (1 gmol/l), with an estimated pK_B value of 8.4, but not by cimetidine (100 mol/l). Our results indicate that histamine induces biphasic effects on human bronchial arteries via H₁-re-ceptors. Send offprint requests to P. J. Barnes at the above address     

Effect of some new histamine H2-receptor antagonists on the guinea-pig papillary muscle

Summary Several histamine H₂-receptor antagonists (cimetidine, ranitidine, oxmetidine and tiotidine) were tested for their activity on the papillary muscle of the guinea pig stimulated by histamine. All of the compounds exerted a competitive antagonism against histamine the order of potency being tiotidine > oxmetidine > ranitidine > cimetidine. Oxmetidine was the only drug which at high concentrations (10^–6 M) decreased the maximum response of histamine probably because of non specific effects of the molecule already described in the literature. As it was expected, the H₁-receptor antagonist, mepyramine, exerted a non-competitive antagonism.     

Characterization of histamine H2-receptors in human neutrophils with a series of guanidine analogues of impromidine

Summary Human neutrophils possess an NADPH oxidase which catalyzes superoxide (O _inf2 ^sup– ) formation and is activated by chemotactic peptides. Histamine inhibits O _inf2 ^sup–1 formation via H₂-receptors (Burde et al. 1989). We characterized the neutrophil H₂-receptor with a series of new guanidine-type H₂-agonists structurally derived from impromidine. Histamine inhibited O _inf2 ^sup– formation with an IC₅₀ value of 6.7 ± 1.2 M. Five aryloxy- and arylthioalkylguanidines were less potent and effective than histamine. Several arpromidine-like phenyl(pyridylalkyl)guanidines were either full or partial H₂-agonists. Some guanidines possess a three-membered carbon chain connecting the aromatic rings and the guanidine group; they were similarly potent and effective as histamine. Shortening or elongation of the carbon chain substantially decreased the potency and intrinsic activity of the guanidines. Halogenation of the phenyl ring did not substantially affect the potency and intrinsic activity of the compounds in comparison to the non-substituted parent compound. The H₂-antagonist, famotidine, competitively antagonized inhibition of O _inf2 ^sup– formation caused by the guanidine, arpromidine, with a pA₂ value of 6.84. The H₂-antagonist, cimetidine, differentially counteracted inhibition caused by partial and full H₂-agonists. Partial H₂-agonists antagonized the effects of histamine. The inhibitor of phosphodiesterases, 3-isobutyl-lmethylxanthine, additively enhanced the inhibitory effects of histamine and guanidines. The properties of the neutrophil H₂-receptor were compared with literature data concerning properties of the H₂-receptor of the guinea pig atrium. In the latter system, guanidines are full H₂-agonists with potencies of up to 125-fold of that of histamine. Our data indicate that guanidines inhibit O _inf2 ^sup– formation in human neutrophils via H₂-receptors. The structure/activity relationship for the neutrophil H₂-receptor substantially differs from the one for the H₂-receptor in the guinea pig atrium, suggesting that the neutrophil H₂-receptor has cell type-specific properties. Other possibilities to explain the differences between H₂-receptors in these systems are discussed. Send offprint requests to R. Seifert at the above address     

Separate receptors mediating the positive inotropic and chronotropic effect of histamine in guinea-pig atria

The direct positive inotropic effect of histamine was studied on paced left atrial preparations from guinea pigs. Histamine (10^?8 to 10^?4 M) increased the maximum tension developed in left atria incubated at 35°C and driven at 2 Hz. The maximum increase in tension was 60% of that observed with norepinephrine. Metiamide (3 × 10^?5 M; a specific H₂-receptor antagonist) did not alter the inotropic response of left atria to histamine. However, tripelennamine (a typical H₁-receptor antagonist) competitively shifted the histamine inotropic dose—response curve to the right at concentrations from 10^?8 to 10^?7 M. Higher concentrations (3 × 10^?7 and 10^?6 M) caused little further additional shift to the right. The positive chronotropic effect of histamine on spontaneously beating atria was competitively antagonized by metiamide (10^?6 and 3 × 10^?6 M). These results demonstrate that in guinea-pig atria histamine increases myocardial contractility by an interaction with receptors closely related to classical H₁-receptors while its chronotropic effects is mediated by interaction with H₂-receptors.     

Mechanism underlying histamine-induced intracellular Ca movement in PC3 human prostate cancer cells

The effect of histamine on intracellular free Ca ²⁺levels ([Ca ²⁺] _i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca ²⁺dye. Histamine at concentrations between 0.1 and 50 μM increased [Ca ²⁺] _iin a concentration-dependent manner with an EC ₅₀value of 1 μM. The [Ca ²⁺] _iresponse comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca ²⁺removal inhibited 50% of the [Ca ²⁺] _isignal. In the absence of extracellular Ca ²⁺, after cells were treated with 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺pump inhibitor), 10 μM histamine did not increase [Ca ²⁺] _i. After pretreatment with 10 μM histamine in a Ca ²⁺-free medium for several minutes, addition of 3 mM Ca ²⁺induced [Ca ²⁺] _iincreases. Histamine (10 μM)-induced intracellular Ca ²⁺release was abolished by inhibiting phospholipase C with 2 μM 1-(6-((17 β-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 μM pyrilamine but was not altered by 50 μM cimetidine. Collectively, the present study shows that histamine induced [Ca ²⁺] _itransients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca ²⁺release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca ²⁺entry.     

Evidence for two types of 5-hydroxytryptamine receptor on secretomotor neurons of the guinea-pig ileum

Summary Flat sheet preparations of the mucosa plus submucosa from the guinea-pig ileum were placed in Ussing chambers so that short circuit currrent (I _sc), an index of electrolyte movement across the mucosa, could be measured. In these preparations, 5-hydroxytryptamine (5-HT) increasesI _sc indirectly by stimulating both cholinergic and non-cholinergic secretomotor neurons. The 5-HT₃ receptor antagonist, ICS 205–930 (10^–13–10^–5 M), substantially depressed the secretory response due to 5-HT (10^–6 M), but not that produced by direct activation of muscarinic receptors on the mucosal epithelium with carbachol (10^–6 M), or by stimulation of secretomotor neurons with substance P (10^–8 M) or 1,1-dimethyl-4-phenylpiperazinium (10^–5 M). The residual response to 5-HT, after the addition of a maximally effective concentration of ICS 205–930 (10^–6 M), was further reduced by hyoscine (10^–7M). When that part of the 5-HT response attributable to the release of acetylcholine was blocked by hyoscine (10^–7M), ICS 205–930 did not further modify the response to 5-HT. The hyoscine-resistant component was, however, sustantially depressed by tetrodotoxin (3.5 × 10^–7 M). The response remaining after ICS 205–930 and hyoscine was not affected by methysergide (2 × 10^{– 5} M) or cyproheptadine (10^–7 M). We conclude that there are ICS 205–930 sensitive 5-HT receptors on cholinergic secretomotor neurons, and ICS 205–930, methysergide, and cyproheptadine insensitive 5-HT receptors on non-cholinergic secretomotor neurons.     

Long-term increase of hippocampal excitability by histamine and cyclic AMP

The action of histamine (HA) on rat hippocampal CA1 pyramidal cells in vitro was investigated in slices perfused with solution containing 0.2 mM Ca²⁺/4.0 mM Mg²⁺. Extracellular recordings of the spontaneous discharges occurring under these conditions revealed that HA caused a long-lasting increase in cell firing. The HA-effects were dose-dependent, in that low concentrations of HA (0.1–0.5 μM) exhibited an initial transient depression of cell firing and practically no long-lasting action, whereas higher concentrations of HA (1–10 μM) exerted strong, non-declining increases. The H₁-receptor antagonist mepyramine (1 μM) blocked the initial depression of firing and attenuated the long-lasting HA-mediated excitation. Pure H₁-receptor activation, tested with the H₁-receptor agonist 2-(3-fluorphenyl)histamine (1–10 μM) depressed cell firing, similar to the low dose effects of HA. HA-induced excitations were prevented by the H₂-receptor antagonist cimetidine (10–50 μM), and mimicked by the very potent H₂-receptor agonist impromidine (1 or 3 μM) which was, however, less effective compared to equal concentrations of HA. H₃-receptor activation by R-α-methylhistamine had no significant effect on cell firing. Thus, histamine H₁ and H₂ receptors seem to cooperate in producing this long-lasting augmentation of excitability. 8-Bromo-cyclic AMP monophosphate (8-Br-cAMP, 50–100 μM) mimicked the long-term excitation, whereas the adenylyl-cyclase inhibitor 9-tetrahydro-2-furyladenine (THFA, 100–500 μM) or the PKA-inhibitor Rp-adenosine-3′5′-cyclic monophosphate (Rp-cAMPS, 10 μM) blocked it, indicating that the HA-mediated increase of excitability in the hippocampus is dependent on the adenylate cyclase/PKA-signal transduction cascade. -2-Amino-5-phosphonopentanoic acid (APV, 50 μM) significantly attenuated the magnitude of the HA-induced enhancement, indicating an NMDA receptor-dependent component. Other biogenic amines, acting through receptors positively coupled to adenylyl cyclase, elicited similar responses as HA, indicating common mechanisms by which these substances modulate excitability in CA1 pyramidal cells.     

Effects of compound 48/80 on mast cells,histamine and cyclic AMP in isolated superior cervical ganglia

Summary Superior cervical ganglia of the rat contain mast cells which are sensitive to degranulation by compound 48/80. The granulation process is shown to the independent of the ATP content of the ganglion. Compound 48/80 released histamine into the incubation medium, thereby decreasing the histamine content of the ganglia. Moreover, the release of ³H-noradrenaline was accelerated by the compound. Histamine and adrenaline induced a rapid accumulation of cyclic AMP in the ganglia. This effect of the amines was specifically blocked by diphenhydramine or propranolol with an ID₅₀ of 1.5×10^–9 M and 2.2×10^–7 M, respectively.In contrast to other findings with isolated mast cell preparations, compound 48/80 induced a rapid and marked accumulation of cyclic AMP in intact ganglia and an enhanced release of cyclic AMP into the incubation fluid. Diphenhydramine prevented the accumulation in the tissue but only partly inhibited the enhanced appearance of cyclic AMP in the medium. The accumulation of the cyclic nucleotide in the tissue was partly blocked by propranolol, suggesting an additional action of compound 48/80 on cyclic AMP through catecholamines.The cyclic nucleotide phosphodiesterase activity in homogenates of superior cervical ganglia was completely inhibited by compound 48/80 at 7 g/ml when low cyclic AMP concentrations were used.In addition to cyclic AMP release, rapid and marked efflux of ATP into the medium was observed during incubations with compound 48/80. The lactate dehydrogenase activity in the incubation medium was significantly enhanced with incubation periods of 40 to 60 min indicating rather slowly occurring toxic damage to cell membranes by compound 48/80.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70).     

Brain histamine N-methyltransferase purification, mechanism of action, and inhibition by drugs

Histamine N-methyltransferase (E.C., 2.1.1.8; HMT) has been purified approximately 200-fold with about 20 per cent yield from guinea pig brain. The purification steps involved centrifugation at 225,000g for 12hr, calcium phosphate gel adsorption, DEAE-cellulose chromatography and final chromatography on hydroxylapatite. The enzyme obtained is the most highly purified brain HMT so far prepared. It is specific for histamine with a K_m of (4.30 ± 0.14) × 10^?5 M; it fails to methylate norepinephrine, serotonin and betazole. a histamine analogue. The kinetics and mechanism of action of histamine N-methyltransferase were investigated. Initial velocity studies at low substrate concentrations suggested that this methyltransferase action proceeded through two half-reactions in a ping pong mechanism with the intermediate formation of a methylated enzyme. This type of mechanism was also supported by results of product inhibition studies, in which methylhistamine was found to inhibit the enzyme competitively with respect to S-adenosylmethionine and noncompetitively with respect to histamine.Several drugs and chemicals were found to inhibit the enzyme; they included mercurial diuretics, antimalarials, antihistaminics, local anesthetics and ethylamine derivatives. The inhibition by each of these compounds, usually at a concentration of 10^?4 M or lower, was competitive with respect to histamine and appeared to be mixed with respect to S-adenosylmethionine. These patterns of inhibition also have a strong support for the ping pong mechanism.     

Voltage clamp analysis of the kinetics of piperidine-induced chloride current in isolated Aplysia neurons

Summary The effect of piperidine (Pip) on isolated Aplysia neurons was investigated using the voltage clamp and concentration clamp techniques in which neurons were perfused internally and externally with Na⁺,K^s+ free solution.Pip induced a Cl-current (I_Cl) in a dose-dependent manner for doses ranging from 10^–4 M to 10^–2 M. The dose-response curve gave an apparent dissociation constant of 8.4 × 10^–4 M and a Hill coefficient of 1.7. The current-voltage relationship was linear in the voltage range examined (t-70 to +30 mV). The equilibrium potential for Pip induced current was close to the calculated equilibrium potential for chloride ions (E_Cl), (–10.7 mV). The activation phase of the I_Cl, was characterized by a single exponential at all concentrations. The time constant of this phase decreased with increasing concentrations of Pip but did not depend on the membrane potential. The deactivation phase of the I_Cl proceeded on a single exponential curve at concentrations of Pip less than 5 × 10^–4 M, but on a double exponential at concentrations of 5 × 10^–4 M and higher. The deactivation time constant also decreased with increasing concentrations of Pip, but showed no potential dependence. Pip- and ACh-induced Ic,s were not blocked by 10^–4 M atropine. However, Pip-induced Ic, was abolished with 10^–4 M d-tubocurarine (dTC), and the ACh-induced I_Cl, was depressed by the same dose of dTC. These results suggest that Pip acts on at least two components of the nicotinic receptor-Cl channel complex in Aplysia neurons to elicit the I_Cl.Send offprint requests to M. R. Klee at the above address     

Investigations of nerve populations influencing ion transport that can be stimulated electrically,by serotonin and by a nicotinic agonist

Summary It is known that the majority of the mucosal nerve fibres in the guinea-pig small intestine arise from submucous ganglia. There are a number of neurochemically distinct populations of nerve cells in these ganglia, approximately half of them being cholinergic. In these studies we have stimulated isolated preparations of mucosa and submucosa with electrical field stimulation (EFS), 5-hydroxytryptamine (5-HT) and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) and monitored changes in ion transport.Segments of intestine were dissected free of external muscle and myenteric plexus and mounted in Ussing chambers. Short-circuit current (I _sc) was measured as an indication of net ion transport across the tissue. EFS consisted of passing bipolar rectangular stimulus pulses through two platinum wires, one placed on each of the mucosal and submucosal sides of the tissue.EFS, 5-HT and DMPP each caused a transient increase inI _sc. Tetrodotoxin (TTX) abolished all of the EFS response and the majority of the response observed with 5-HT or DMPP, suggesting that the action of these stimuli on the mucosa is primarily nerve-mediated. The TTX-sensitive responses to 5-HT (>5×10^–7 M) and DMPP consisted of two components, appearing with different latencies. The response to EFS also consisted of two components. Hyoscine abolished the first component of each of these responses and significantly reduced the amplitude of the second, by 40% (for EFS and 5-HT) and 84% (for DMPP). At lower 5-HT concentrations, only the later component was seen, and this was unaffected by hyoscine. These results suggest that the early component of each response is due to the release of acetylcholine from cholinergic nerves. The hyoscine-resistant responses to EFS and DMPP were reduced by a substance P antagonist (d-Arg¹,d-Pro²,d-Trp^7,9, Leu¹¹), suggesting that these responses involve activation of substance P receptors in the mucosa.The studies suggest that EFS and 5-HT (>5×10^–7 M) stimulate both cholinergic and non-cholinergic nerves effectively, that 5-HT (10^–8–5×10^–7 M) preferentially stimulates non-cholinergic nerves and that DMPP preferentially stimulates cholinergic nerves.