The interaction of stimulants on the function of isolated canine parietal cells

With isolation, the parietal cell is removed from the effects of the many endogenous substances that may modulate its function in intact mucosa, even in the basal state. The isolated canine parietal cell responds to the major endogenous regulators of secretion: histamine, acetylcholine and gastrin. These agents act on specific receptors as evidenced by (1) the specificity of antagonist (H2 antagonists, atropine, and dibutyryl cyclic GMP respectively), (2) the binding of radiolabelled ligands, and (3) the existence of separate second messenger systems (cyclic AMP for histamine, calcium influx for acetylcholine, and an unidentified mechanism for gastrin). Potentiating interactions, which occur between histamine and acetylcholine or histamine and gastrin, do not involve extra production of second messenger. When histamine and acetylcholine are given together, the amounts of cyclic AMP generated and of calcium entering the cell are not greater than when each is acting alone. The apparent non-specific effects of inhibitors acting in vivo, such as the inhibition of all forms of stimulation by H2 antagonists, could reflect withdrawal of the potentiating action of the background histamine always present in the mucosa.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Decreased synthesis of acetylcholine accompanying impaired oxidation of pyruvic acid in rat brain minces.

The relation between pyruvate utilization and acetylcholine synthesis was investigated in minces of adult rat brain. The flux of pyruvate to acetylcholine was less than 1% of that to CO2; nevertheless, a number of agents which inhibited conversion of [1-14C]-pyruvate or [2-14C]pyruvate into 14CO2 were associated with corresponding decreases in the conversion of [2-14C]pyruvate into acetylcholine. The amount of acetylcholine produced by minces of whole rat brain, measured by g.l.c.-mass spectrometry, decreased similarly. Among the inhibitory compounds tested were 3-bromopyruvate, an irreversible inhibitor of pyruvate dehydrogenase; 2-oxobutyrate, a competitive inhibitor of pyruvate dehydrogenase; other 2-oxo acids; and amobarbital and pentobarbital. Linear-regression equations relating CO2 production to acetylcholine synthesis gave correlation coefficients of 0.89-0.93 for the combined observations. The inhibition of acetylcholine synthesis could not be attributed to inhibition of choline acetyltransferase. Incorporation of [2-14C]pyruvate into lipids, proteins and nucleic acids was effected less than that into acetylcholine. Under these experimental conditions, it was shown that pyruvate utilization can limit acetylcholine synthesis.     

The characteristics of the effect of mediator substances on the dynamic oxygen tension in the gastric mucosa]

The studies on rats were carried out to determine dynamics of pO2 in the mucous membrane of the stomach under the effect of acetylcholine, norepinephrine, serotonin, histamine, prostaglandin E2 and ATP. As to the changes in pO2 the mediator substances were arranged as follows (from more intensive effect to less pronounced one): serotonin, acetylcholine, prostaglandin E2, norepinephrine, histamine and ATP. As to the duration of the action--PGE2 acetylcholine, serotonin, norepinephrine, ATP and histamine. Under the joint action of the mediator substances (serotonin, norepinephrine, histamine) with acetylcholine the effects of domination and modulation of acetylcholine effect are found.     

Respiratory epithelium inhibits bronchial smooth muscle tone

The aim of the present study was to determine whether or not the respiratory epithelium can modulate the responsiveness of bronchial smooth muscle. Paired rings of canine bronchi (4-6 mm OD), in some of which the epithelium had been removed mechanically (by rubbing the luminal surface), were mounted in physiological saline solution, gassed with 95% O2-5% CO2, and maintained at 37 degrees C. The presence or absence of the epithelium was confirmed by histological examination. Removal of the epithelium increased the contractile responses evoked by acetylcholine, histamine, and 5-hydroxytryptamine. Transmural nerve stimulation evoked similar peak responses in the presence and absence of epithelium. In unrubbed preparations, the peak response was followed by a gradual decrease when the stimulation was continued. This decrease, which persisted in the presence of propranolol, was not observed in epithelium-denuded preparations. In bronchial rings contracted with acetylcholine, isoproterenol produced concentration-dependent relaxations which were significantly greater in rings with epithelium compared with denuded rings. These results suggest that respiratory epithelial cells may generate an inhibitory signal to decrease the responsiveness of bronchial smooth muscle to contractile agonists and augment the effectiveness of inhibitory stimuli.     

Interaction of contractile responses in canine tracheal smooth muscle

Concentration-response curves for norepinephrine, acetylcholine, and 5-hydroxytryptamine were obtained in vitro alone and after precontraction with histamine, 5-hydroxytryptamine, or acetylcholine. Responses obtained to each agonist after precontraction were greater than responses to the agonist alone after subtraction of the force due to the precontracting stimulus. Augmentation of responses after precontraction was the greatest for norepinephrine, less for 5-hydroxytryptamine, and least for acetylcholine. Verapamil had no significant effect on the augmentation of responses to either 5-hydroxytryptamine or acetylcholine caused by precontraction. When the efficacy of acetylcholine was decreased by receptor alkylation with phenoxybenzamine, the augmentation of responses to acetylcholine caused by precontraction with histamine was significantly enhanced. Differences in the magnitude of the effect of precontraction on responses to different agonists may reflect differences in their efficiency of stimulus-response coupling in canine tracheal smooth muscle, or they may result from an increased expression of distinct receptors or receptor-mediated effects uncovered by the facilitory stimuli.     

Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes

The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10-150 microM) produced a dose-dependent inhibition of high-affinity [3H]choline uptake. Low-affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high-affinity [3H]choline uptake with the following order of potency: arachidonic greater than palmitoleic greater than oleic greater than lauric; stearic acid (up to 150 microM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]choline, and this inhibition was equal in magnitude to the inhibition of high-affinity [3H]choline uptake produced by the fatty acid. A K+-stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium-3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high-affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.     

The effects in vitro of hypoglycaemia and recovery from anoxia on synaptosomal metabolism.

Synaptosomes from several regions of the rat brain were found to exhibit half-maximal rates of 14CO2 output and [14C]acetylcholine synthesis from D-[U-14C]glucose at glucose concentrations approx. 50-fold lower than those required by the brain in situ. However, synaptosomal acetylcholine synthesis was found not to be directly proportional to substrate oxidation as measured by 14CO2 output. When synaptosomes had been exposed to anoxia in vitro, their metabolic indices (14CO2 and [14C]acetylcholine synthesis, and adenine nucleotide levels) were found not to be significantly different from control aerobic values, unless they had been subjected to veratridine depolarization. This is in accord with previous findings that neither the absolute metabolic rates nor the vulnerability to hypoxic damage exhibited by brain in situ is reflected by brain slices in vitro, unless these are stimulated by depolarization. The use of synaptosomes as a model for synaptic damage in vivo is discussed.     

Airway epithelium modulates the responsiveness of porcine bronchial smooth muscle

The effect of epithelium removal on the responses of porcine airways to exogenously applied agonists and nerve stimulation was examined. Paired rings of third- (segmental), fourth- and fifth-order (subsegmental) bronchi, with and without epithelium, were placed in organ chambers in physiological salt solution (95% O2-5% CO2, 37 degrees C). Removal of the epithelium caused a leftward shift in the concentration-effect curve for acetylcholine (3rd and 4th order). A similar shift occurred for histamine (3rd and 5th order). The relaxation to isoproterenol was reduced by epithelium removal in a similar fashion in the three orders. Removal of the epithelium reduced the maximal response to KCl (3rd and 4th order) and acetylcholine (5th order). The peak response to nerve stimulation showed a significant rightward shift in the absence of epithelium. In fifth-order bronchi, tissues with epithelium showed a significantly greater degree of fade of the response to sustained electrical stimulation. Thus both epithelium-derived relaxing and contracting factors may be released in porcine airways.     

The effect of euthanasia technique on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility

This study was designed to determine the effects that specific euthanasia methods have on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility. Rats were euthanatized by decapitation (DC), pentobarbital overdose (PB), or anesthesia with CO2, methoxyflurane or ether followed by DC (CO2-DC, Met-DC, Ether-DC, respectively). Rabbits were killed by a similar protocol, but CO2 overexposure replaced Ether-DC. The rat and rabbit aortas produced mainly 6-keto PGF1 alpha, the prostacyclin metabolite, and lesser amounts of PGE2. No qualitative differences were seen in arachidonate metabolites. However, aortic tissue from rabbits and rats killed by Met-DC produced more prostacyclin. In contrast, aorta from rabbits euthanatized by CO2-DC produced less prostacyclin than controls, whereas aorta from rats killed in the same way yielded greater amounts of prostacyclin. Aortic tissue from rabbits killed by Met-DC and CO2-OD was less responsive to acetylcholine (ACH). Intestinal contractility to ACH was increased in rabbits when Met-DC was used as the method of euthanasia, while colon from rats sacrificed by Met-DC showed decreased responsiveness to ACH. Colon from rats killed by intraperitoneal PB exhibited altered contractility to ACH and norepinephrine. The results of this study show that methoxyflurane, carbon dioxide (rabbit) and pentobarbital (rat) alter the vascular synthesis of prostacyclin and smooth muscle contractility. We conclude that the method of euthanasia affects certain physiologic parameters and careful consideration should be given to the selection of a particular euthanasia technique.     

Effects of (S)-alpha -fluoromethylhistidine and metoprine on locomotor activity and brain histamine content in mice.

We examined the effects of (S)-alpha -fluoromethylhistidine (FMH), an inhibitor of histidine decarboxylase, and metoprine, an inhibitor of histamine N-methyltransferase, on the locomotor activity and the brain histamine content of ICR mice. The brain histamine content was decreased by FMH (12.5 or 50 mg/kg, i.p.) and increased by metoprine (4 mg/kg, i.p.). Under these conditions, the locomotor activity and the number of rearing were significantly decreased and increased by FMH and metoprine, respectively. The higher the brain histamine content, the greater the locomotor activity and vice versa. In a previous paper [Sakai et al., Life Sciences, 48, 2397-2404 (1991)], we showed that thioperamide, a histamine H3 antagonist, which enhances the release of histamine from histaminergic neurons, in doses of 12.5 and 25 mg/kg, i.p. increases the locomotor activity, whereas it decreases the brain histamine content. Taken together, these results support the hypothesis that central histaminergic neurons may be involved in the control of state of locomotion and rearing.     

Highly sensitive muscarinic receptors in the cerebellum are coupled to prostaglandin formation

There have been relatively few studies on the effects of neurotransmitters on the synthesis of prostaglandins in the brain. We report here that acetylcholine is very effective in stimulating prostaglandin synthesis in cerebellar cortex slices incubated in vitro, i.e., 5 microM acetylcholine increased prostaglandin levels up to 3-fold over control levels. The response was saturable and dose-dependent over the range of 0.1-5 microM acetylcholine. Atropine, at a concentration of 50 nM, abolished the response. The results indicate that high-affinity muscarinic receptors in the cerebellum are coupled to prostaglandin formation. Potassium-induced depolarization or incubation with 0.1 mM histamine also significantly increased prostaglandin formation. These findings provide support for the notion that certain neurotransmitters can modulate prostaglandin levels in the mammalian brain.     

Heterogeneity in the effects of epithelium removal in the canine bronchial tree

The effect of removing the airway epithelium on the responses of canine airways of decreasing diameter to contractile and relaxing agonists was explored. Three orders of canine bronchus were studied: second order (lobar bronchus), third order (segmental bronchus), and fourth order (subsegmental bronchus). Paired rings of tissue, with and without epithelium, were placed in organ chambers in physiological salt solution gassed with 95% O2-5% CO2 and maintained at 37 degrees C. For second- and third-order bronchi, epithelium removal caused significant left-ward shifts of the concentration-effect curves for 5-hydroxytryptamine, histamine, and acetylcholine. In fourth-order bronchi, there was no significant shift for any of the contractile agonists. Isoproterenol (during contractions evoked by acetylcholine) induced concentration-dependent relaxations that were significantly greater in bronchi with than in those without epithelium. This effect was most prominent in fourth-order bronchi. These results suggest that 1) the canine airway epithelium releases a relaxing factor, 2) in larger airways the major effect is reduction of contractile responses, and 3) in smaller airways the major effect is enhancement of relaxing responses.     

止嗽散药理作用研究

为比较止嗽散及其改进后中药复方的药理作用,用小鼠氨水引咳法进行镇咳实验;祛痰作用采用小鼠肺酚红排泌法;实验性支气管痉挛性哮喘采用豚鼠组胺和乙酰胆碱混合喷雾引喘法。实验表明,止嗽散可明显延长小鼠引咳潜代期,减少咳嗽次数,增加酚红排泌量,但平喘作用不明显;改进方具有明显的镇咳、化痰作用外,还可延长豚鼠引喘潜伏期。     

Strict additivity of the antispasmodic effects of papaverine and tiemonium: an example of sequential blockage]

The interaction of papaverine and tiemonium alone or combined, with BaCL2 and histamine on guinea pig ileum and with acetylcholine on rat jejunum have been studied with the help of molecular pharmacology techniques. The competitive antagonist effects of tiemonium and the non competitive antagonist effects of papaverine are evidenced and shown to be strictly additive when the two drugs are combined. This reflects a sequential blockage of the effects of acetylcholine, histamine and barium ions at the smooth muscle level. No such antagonism has been previously described in the case of the interaction with barium chloride with any other combination of two spasmolytic drugs.     

Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea

Contractions of guinea pig trachea in the absence and presence of indomethacin to LTD4 greater than LTC4 greater than K+ greater than histamine greater than acetylcholine were reduced following a 45 minute exposure of the tissues to calcium-free Krebs' solution (Ca2+-free Krebs' solution), were further reduced by a transient exposure to EGTA (1.25 mM) in Ca2+-free Krebs' solution and were virtually abolished when tested in the presence of EGTA (0.125 mM) in Ca2+-free Krebs' solution. In normal Krebs' solution (2.5 mM Ca2+) the Ca2+ entry blockers nifedipine (N) much greater than D-600 greater than verapamil (V) greater than diltiazem (D) almost completely abolished the contractions to K+ but blocked only a component of the maximum response to the other agonists. After exposure to Ca2+-free Krebs' solution for 45 minutes, any residual contractions to LTC4 & LTD4, were reversed by low concentrations of N (0.3 microM) or D-600 (2.1 microM). Leukotrienes appear to mobilize a superficial and a bound store of Ca2+ which gains entry through at least two types of Ca2+ channels (or mechanisms), one of which is blocked by N and D600. K+-induced contractions appear to be dependent on superficial and tightly bound Ca2+ but entry is solely through channels which are blocked by the Ca2+ entry blockers studied. Contraction to histamine and acetylcholine persisted following exposure of the tissues to Ca2+ free Krebs' solution but contractile activity was virtually abolished in Ca2+ free Krebs' solution containing EGTA. Residual contractions to histamine and part of the residual contractions to acetylcholine in Ca2+-free Krebs' solution were blocked by low dose N (0.3 microM) or D600 (2.1 microM). These findings suggest a major role for extracellular Ca2+ during spasmogen-induced contraction in this tissue.     

Antagonistic effect of KC-404, a new anti-asthmatic agent, on leukotriene D4-induced contractile responses in isolated guinea pig smooth muscles

The inhibitory effects of KC-404, a novel clinically available anti-asthmatic drug, on leukotriene(LT) D4-, LTC4-, histamine- and acetylcholine(ACh)-induced contractile responses in isolated guinea pig lung parenchymal, tracheal and ileal longitudinal strips were compared using an organ bath system. In lung parenchyma, KC-404 antagonized LTD4 in a competitive fashion, whereas it antagonized histamine noncompetitively. The pA2 value against LTD4 was 7.39. KC-404 hardly antagonized LTC4 and ACh. A ranked order of potency estimated from its minimum effective concentrations (MEC) was LTD4 greater than histamine greater than LTC4 greater than ACh. In trachea, KC-404 antagonized LTC4 and LTD4 in a competitive fashion, while it antagonized histamine noncompetitively. The pA2 values against LTC4 and LTD4 were 5.99 and 6.51, respectively. KC-404 hardly antagonized ACh. A ranked order of the potency estimated from MEC was LTD4 greater than LTC4 greater than histamine greater than ACh. The pA2 values of KC-404 against LTD4 in lung parenchyma and trachea were little or not altered, while its inhibitory effect on histamine-induced contraction in trachea was markedly diminished by the pretreatment of tissues with indomethacin. In ileum, KC-404 noncompetitively antagonized all of the agonists used. A ranked order of the potency estimated from pD2 values was LTD4 divided by LTC4 greater than histamine greater than ACh. These results suggest that KC-404 is a selective antagonist of LTD4 and that it might interact with LTD4 receptor in airway smooth muscles but not in ileum. Another possibility that the drug might interact with LTD4 specific excitation-contraction coupling mechanism was also discussed.     

Effects of acetylcholine and other vasodilatory substances in the vascular bed of skeletal muscles during and after muscular contractions.

In dogs and cats perfusion pressure or femoral venous outflow and changes in the volume of the hind limb were measured. The influence of muscular contractions on the effects of acetylcholine, histamine, isoprenaline and papaverine in the vascular bed of the limb was investigated. It was found that after muscular excerise the effects of i. a. injection of acetylcholine into the isolated limb were markedly potentiated, whereas the effects of histamine and isoprenaline were diminished.     

Differential effects of prostaglandin E2 on contractions of airway smooth muscle

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3',5'-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.     

Possible role of histamine in brain function: neurochemical, physiological, and pharmacological indications

Recently accumulated neurochemical, physiological, and pharmacological evidence strongly supports a role for histamine as a central neurotransmitter. Neurochemical methods, which became available within the last years, allow determination of small amounts of histamine and its metabolites in the brain and make possible future studies of central histamine regulation. The demonstration of histamine H1 and H2 receptors in the brain of several species suggests a possible role for histamine in brain function. Microelectrophysiological studies on single central neurones suggest both excitatory and depressant effects of histamine which are receptor mediated. In addition, brain histamine has been demonstrated to be subject to cyclic variations, to play a role in hormonal regulation, and to be altered by stressful conditions. Several psychotropic drugs significantly affect brain histamine regulation and elicit inhibitory effects on central histamine receptors. These findings bring new approaches and stimulus to further research on the significance of brain histamine.