Stimulation of acetylcholine release from guinea-pig ileal synaptosomes by cyclic nucleotides and forskolin

Various agents which are known to affect intracellular levels of cAMP have been assessed for their ability to induce the release of [3H]acetylcholine ([3H]ACH) from a synaptosomal preparation derived from the guinea-pig ileum myenteric plexus. 8-Bromo-cAMP increased the release of [3H]ACh above basal levels. While 8-bromo-cGMP also increased the release, this nucleotide was far less potent than 8-bromo-cAMP. Comparison of the release caused by the cyclic nucleotides to the release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP) suggested that there is some relationship, as yet undefined, between the 8-bromo-cAMP-induced and the DMPP-induced release, while no relationship was evident between the release induced by 8-bromo-cGMP and that caused by DMPP. The 8-bromo-cAMP-induced release was Ca2+-dependent. Neither adenosine, clonidine, nor oxotremorine (all of which modulate the nicotinically-induced release) affected the 8-bromo-cAMP-induced release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine stimulated the release of [3H]ACh as did the adenylate cyclase activator forskolin. The forskolin-induced release was not affected by adenosine, clonidine or oxotremorine. The ability of the modulators to block the nicotinically-induced release but not the release caused by the cyclic nucleotides indicates that the modulation of release evoked by nicotinic activity does not occur at a step involving protein phosphorylation.     

The effect of ribose and purine modified adenosine analogues on the secretion of histamine from rat mast cells induced by ionophore A23187

The purine nucleosides adenosine and 2',5'-dideoxyadenosine (2',5'ddAdo) enhance and inhibit respectively the anti-IgE-induced secretion of histamine and transient rise in cellular levels of cyclic AMP in rat mast cells. These findings have provided evidence for a role for cyclic AMP in the activation of mast cell secretion. It has been generally accepted that the nucleosides mediate their effects on mast cells by altering adenylate cyclase activity. We have investigated the effect of various purine and ribose modified analogues of adenosine on secretion of histamine from rat mast cells induced by ionophore A23187 for which there is no associated elevation in cyclic AMP and no evidence for the activation of adenylate cyclase in its mechanism of action. Adenosine and N6, phenylisopropyladenosine (0.01-1000 microM) (activators of adenylate cyclase in many tissues) enhanced the secretion of histamine induced by ionophore A23187 and anti-IgE. Two inhibitors of adenylate cyclase had differential effects on secretion. 2',5'ddAdo (100-1000 microM) inhibited both A23187-and anti-IgE-mediated secretion; whilst 9-beta-D-arabinofuranosyladenine had no effect on secretion. These results suggest that the ability of these nucleosides to modulate histamine secretion is unrelated to their effects on adenylate cyclase.     

The effect of intracerebroventricularly administered dibutyryl adenosine 3',5'-cyclic monophosphate on cerebrospinal fluid and serum dopamine-beta-hydroxylase in rabbits

Intracerebrospinal injected or infused dibutyryl adenosine 3′,5′-cyclic monophosphate provoked (a) an initial small decrease of cerebrospinal fluid dopamine-β-hydroxylase activity which lasted for 2 hours, followed by a significant rise of a 4–6 hours duration, together with a rise in total cerebrospinal fluid protein concentration, (b) a significant increase in serum dopamine-β-hydroxylase activity. Pretreatment of rabbits with two intracisternal injections of 6-hydroxydopamine completely abolished the dibutyryl adenosine 3′,5′-cyclic monophosphate induced release of dopamine-β-hydroxylase in the cerebrospinal fluid but only partially the release of dopamine-β-hydroxylase in the serum.     

The utilization of adenosine triphosphate in rat mast cells during histamine release induced by anaphylactic reaction and compound 48/80

Summary The ATP content of rat peritoneal mast cells has been studied in relation to histamine release induced by compound 48/80 and antigen-antibody (anaphylactic) reaction in vitro. When the ATP content of actively sensitized mast cells was reduced to different levels by oligomycin, a good correlation was obtained between the ATP levels and the amounts of histamine released by the anaphylactic reaction. A similar linear relation has previously been demonstrated between the ATP levels of mast cells and histamine release induced by compound 48/80. The ATP content of mast cells was also studied at different intervals after the exposure of the cells to antigen or compound 48/80. No significant change in the ATP content was observed in untreated mast cells during the short period when histamine release occurs. If, however, the mast cells were preincubated with oligomycin or 2-deoxyglucose to reduce the rate of ATP synthesis while a large part of the histamine release remained unaffected—a decrease in the ATP content could be demonstrated in close time relation to both anaphylactic and compound 48/80-induced histamine release. The observations indicate an increased utilization of ATP in mast cells during the release process.     

The role of cell-fixed calcium in histamine release by compound 48/80.

1. Histamine release by compound 48/80 was substantially reduced in a time-dependent manner (maximum at 30 min) by pre-incubating mast cells in calcium-free medium at 37 degrees C but not at 2 degrees C. This effect was optimal at pH 7.0 to 7.5. 2. The re-introduction of calcium (0.1 to 3 mmol/l) restored histamine release to the control value; this effect was independent of temperature. 3. Strontium (1 to 30 mmol/l) partially reversed the effect of calcium deprivation but the same concentrations of barium and magnesium depressed histamine release even further. Magnesium (3 to 15 mmol/l) antagonized the effect of calcium replacement. 4. Results suggest that the level of cell-fixed calcium involved in compound 48/80-induced histamine release may be controlled by the combination of rapid passive influx and slow active efflux.     

Evidence for negative cooperativity among beta-adrenergic receptors in cardiac and lung tissue of guinea pig.

Stereospecific binding sites for (?)-[³H]dihydroalprenolol, a β-adrenergic antagonist, have been identified in guinea-pig myocardial cell membranes and lung tissue preparations. A close correlation between the abilities of β-adrenergic antagonists to compete with labelled antagonist binding and to block the response of isoproterenol plus Gpp(NH)p2 stimulated adenylate cyclase has been found. Equilibrium binding of (?)-[³H]dihydroalprenolol to binding sites of cardiac and lung tissue displays characteristics consistent with negative cooperativity among the DHA binding sites: Scatchard plots are curvilinear with upward concavity. The existence of site-site interactions of the negatively cooperative type was demonstrated directly by the ability of unlabelled (?)alprenolol to accelerate the dissociation of (?)-¦³H¦dihydroalprenolol under conditions where rebinding of the radioligand does not occur.     

Calcium dependency of inhibition by arachidonic acid of compound 48/80-induced histamine release from mast cells

Compound 48/80 ( compd 48/80)-induced histamine secretion from rat mast cells was inhibited almost completely by pretreatment of the cells at 37 degrees with 25 microM arachidonic acid in the presence of 1.8 mM Ca2+. As the Ca2+ concentration was reduced below 1.8 mM, 25 microM arachidonic acid became less inhibitory and, then, progressively more stimulatory for histamine release with or without compd 48/80. No additive effect on histamine release was obtained by combining compd 48/80 and arachidonic acid. Pretreatment of mast cells with lidocaine, an inhibitor of Ca2+ binding to phospholipid, or with nordihydroguaiaretic acid, an inhibitor of Ca2+ flux and lipoxygenase, stimulated arachidonic acid-induced histamine release. Arachidonic acid also inhibited a compd 48/80-induced spike increment of intracellular 45Ca2+ uptake and a decrease of total 45Ca2+ uptake by 45Ca2+-preloaded mast cells. Arachidonic acid and Ca2+ also suppressed melittin-induced histamine release and compd 48/80-induced release of radioactivity from mast cells preloaded with [3H]arachidonic acid. These results suggest that exogenous arachidonic acid or its metabolite(s) may interact with membrane-associated Ca2+, disturbing Ca2+ availability for the trigger mechanism of compd 48/80-induced histamine release or inhibiting the subsequent metabolism of arachidonic acid via the lipoxygenase pathway to form active metabolites involved in the histamine liberating mechanism.     

Comparison of histamine release induced by synthetic polycations with that by compound 48/80 from rat mast cells.

We compared the histamine release induced by polyethylenimines and polyallylamines with that induced by compound 48/80. Lidocaine inhibited the histamine release induced by polyethylenimine with a molecular weight of 600 (PEI6), but disodium cromoglycate did not. The histamine releases induced by all polyethylenimines and polyallylamines tested were inhibited by lidocaine, but not by disodium cromoglycate. Islet activating protein inhibited the histamine release induced by PEI6. Its effects on the release by other polyethylenimines and polyallylamines were less than that on PEI6. It is likely that the inhibition of G proteins by islet activating protein resulted in a decrease of the histamine release. This possibility was supported by the finding that guanyl-5'-(beta, gamma-imino) triphosphate enhanced the histamine release. An inhibitor of polyphosphoinositide phosphodiesterase, neomycin, did not affect the histamine releases induced by these polymers. The effect of PEI6 seemed to resemble that of compound 48/80. After pretreatment of mast cells with wheat germ agglutinin and with Limax flavus agglutinin, releases of histamine induced by PEI6 and compound 48/80 decreased, suggesting that the binding sites of PEI6 and compound 48/80 had sialic acid and/or N-acetyl glucosamine residues. The binding site for PEI6 seemed to especially overlap those of compound 48/80.     

Blockade by haloperidol of the increase in tryptophan hydroxylase activity induced by incubation of slices of brain stem with dibutyryl cyclic AMP

Pretreatment of rat brain stem slices with dibutyryl cyclic AMP, caffeine, theophylline and 3-isobutyl-1-methylxanthine increased the activity of tryptophan hydroxylase in supernatant preparations of enzyme made from the slices. This effect does not appear to be mediated by a cyclic AMP sensitive mechanism since it was not reproduced by exposure of the slices to 8-bromo cyclic AMP, to papaverine, a nonxanthine phosphodiesterase inhibitor, or to other treatments known to raise tissue cyclic AMP levels. The ability of haloperidol to block this increase in enzyme activity is consistent with a role for calmodulin and calcium as mediators of the enzyme activation, particularly when this observation is considered in conjunction with the evidence that supernatant preparations of this enzyme was activated under phosphorylating conditions by a calcium-calmodulin dependent process [5]. Nevertheless, in view of the high concentrations of haloperidol employed in the present experiments, the possibility that this drug may produce its effects in the brain stem slices through some other action, unrelated to its ability to bind to calmodulin, should be kept in mind.     

Plasma kallikrein-generating activity evoked by rat peritoneal-fluid mast cells following treatment with epinephrine, 8-bromo-cyclic 3',5'-guanosine monophosphate or compound 48/80

Acting in a dose-dependent fashion, l-epinephrine caused rat peritoneal-fluid cells to rapidly deplete rat plasma kininogen in vitro; 8-bromo-cyclic 3′,5′-guanosine monophosphate (8-Br-cGMP) behaved similarly; N⁶-2¹-O-dibutyril-cyclic 3′,5′-adenosine monophosphate (di Bu-cAMP) inhibited this effect of epinephrine or 8-Br-cGMP. After fractionation of peritoneal-fluid cells by differential centrifugation, this kininogen-depleting activity was observed only in mast cells; eosinophils, lymphocytes, and monocytes were inactive. Epinephrine-treated mast cells were ablt to hydrolyze the trypsin substrates N-p-toluenesulfonyl-arginine-methyl-ester (TAME) or N-benzoyl-arginine-ethyl-ester and to generate the capacity to hydrolyze these substrates in rat plasma; because this activity accompanied kininogen depletion, it was attributed to plasma kininogenase (plasma kallikrein). Diisopropyl-fluorophosphate (DFP) inhibited the mast cell esterase activity toward TAME but did not prevent activated cells from depleting plasma kininogen. Thus, mast cell-bound argininase ester esterase may not have been necessary for the activation of plasma kininogenase. Mast cell heparin, exposed following epinephrine or 8-Br-cGMP treatment, may have been the activator of plasma kallikrein. Unlike DFP, Trasylol [polyvalent bovine proteinase inhibitor (BPTI)] inhibited both mast cell esterase and kininogen-depleting activity. This inhibitor may have acted on mast cells both as a heparin antagonist and and a non-specific esterase inhibitor. Compound $\text{48}\text{80}$, at concentrations causing 40 per cent release of mast cell histamine, failed to cause mast cells to exhibit the ability to active plasma kallikrein. At high concentrations it activated the kininogen depleting action of mast cells, but to a lesser degree than did epinephrine or 8-Br-cGMP. These compounds did not release histamine; it may be concluded, therefore, that the ability to activate plasma kininogenase was present in non-histamine releasing mast 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).     

The inhibition of mast cell damage and histamine release in anaphylaxis by pyridine and diphosphopyridine nucleotidase inhibitors. Comparison with compound 48/80

In rats and guinea-pigs both mast cell damage and histamine release in anaphylaxis were inhibited by pyridine, nicotinamide, diethylnicotinamide, nicotinic acid, isonicotinic acid and isonicotinic acid hydrazide, which are known to be inhibitors of diphosphopyridine nucleotidases. On the other hand these compounds potentiated histamine release and mast cell damage by compound 48/80 in guinea-pigs although inhibiting the same phenomena in rats. The relationship of these findings with the mechanism of histamine liberation in anaphylaxis is discussed.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

Potentiation by homocysteine of adenosine-stimulated elevation of cellular adnosine 3',5'-monophosphate.

The novel ability of l-homocysteine (Hcy) to potentiate the cellular elevation of adenosine 3′,5′-monophosphate (cAMP) caused by adenosine (Ado) is described. This effect of Hcy is highly selective in that it is not mimicked by l-cysteine, and Hcy does not potentiate the elevation of cellular cAMP caused by either 2-chloroadenosine or prostaglandin E₁. Hcy also augments the Ado-stimulated increase in 2-fluoroadenosine 3′,5′-monophosphate in cells preloaded with nucleotides of 2-fluoroadenosine. Addition of Hcy to cells during their incubation with radioactive Ado results in a decrease in the cellular content of radioactive Ado and a concomitant buildup of S-adenosylhomocysteine. The enhancive effect of Hcy on the Ado-stimulated elevation of cAMP may be due to this associated reduction in the intracellular pool of Ado (due to condensation of Ado with Hcy via S-adenosylhomocysteinase) and to a resultant reduction in inhibition of adenylate cyclase by intracellular Ado, thereby allowing greater net stimulation of the cyclase by extracellular Ado.     

维生素C对氟化钠和化合物48-80诱导大鼠肥大细胞释放组胺的影响

氟化钠(NaF)和化合物48-80均可诱发大鼠胸腔，腹腔肥大细胞释放组胺，前者依赖细胞外钙参与，后者与细胞外钙关系不大，二者的最适诱导浓度分别为10 mmol·L^-1和0.2 mg·L^-1. 维生素C可促进NaF诱导的组胺释放，但对化合物48-80所诱导的组胺释放无明显影响. 将肥大细胞置于37 ℃，2 h，NaF或化合物48-80诱导的组胺释放率均下降.预先加入维生素C 1 mmol·L^-1，此现象显著减弱.由此提示：NaF激发肥大细胞，增敏其对Ca²⁺分泌作用不是由自由基引发的，另一方面也不排除37 ℃长时间温育细胞产生自由基可能与降低细胞对NaF-Ca²⁺系统和化合物48-80的敏感性有关.     

维生素C对氟化钠和化合物48-80诱导大鼠肥大细胞释放组胺的影响

氟化钠（ＮａＦ）和化合物４８－８０均可诱发大鼠胸腔，腹腔肥大细胞释放组胺，前者依赖细胞外钙参与，后者与细胞外钙关系不大，二者的最适诱导浓度分别为１０ｍｍｏｌＬ－１和０．２ｍｇＬ－１．维生素Ｃ可促进ＮａＦ诱导的组胺释放，但对化合物４８－８０所诱导的组胺释放无明显影响．将肥大细胞置于３７℃，２ｈ，ＮａＦ或化合物４８－８０诱导的组胺释放率均下降．预先加入维生素Ｃ１ｍｍｏｌＬ－１，此现象显著减弱．由此提示：ＮａＦ激发肥大细胞，增敏其对Ｃａ２＋分泌作用不是由自由基引发的，另一方面也不排除３７℃长时间温育细胞产生自由基可能与降低细胞对ＮａＦ－Ｃａ２＋系统和化合物４８－８０的敏感性有关．     

In vitro evidence that endothelium-dependent vasodilatation induced by clozapine is mediated by an ATP-sensitive potassium channel

BackgroundThere is a definite association between antipsychotic drugs and arterial hypertension. However, endothelium functions are scarcely considered. This investigation was carried out to study the mechanisms involved in clozapine endothelium-dependent vascular reactivity.MethodsThe experimental animals were male Wistar rats with a mean age of 70–90 days (250–300 g). The endothelium-dependent vascular reactivity was studied by measuring the isometric force and then constructing clozapine concentration-response curves. The force registrations were obtained in the aorta rings with and without the endothelium precontracted with phenylephrine (PE10^?6M) treatment; this followed incubation for 30 min in “organ chambers” with different inhibitors: l- NAME (nitric oxide/cGMP); indomethacin (PGI2/cAMP); tetraethylammonium (TEA), and specific hyperpolarization blockers (paxillin, apamin, glibenclamide). The data were presented as the mean ± standard error of the mean (SEM) and were compared by one-way ANOVA or two-way ANOVA followed by the Bonferroni post-test.ResultsThe primary outcomes were: 1) Clozapine-induced endothelium-dependent relaxation was not inhibited by indomethacin, l-NAME, ODQ, and methylene blue (MB); 2) The combination of l-NAME + indomethacin partially prevented the relaxation; 3) Clozapine did not induce relaxation in vessels contracted with KCl; 4) TEA did not block the clozapine-induced relaxation in vessels precontracted with PE (10^?6 M); 5) The potassium channel blockers paxillin and apamin did not prevent relaxation but glibenclamide did.ConclusionConcerning the mechanisms involved in clozapine endothelium-dependent vascular reactivity, the present study suggests that there is synergistic participation that probably occurs through a crosstalk mechanism of the cAMP, cGMPpathways and hyperpolarization.     

Inhibitory effect of a novel anticomplementary agent, K-76COONa, on the release of histamine induced by zymosan and compound 48/80

A novel sesquiterpene anticomplementary agent, K-76COONa, if locally applied, was revealed to have an activity to prevent histamine release from rat connective tissue mast cells not only in a zymosan-induced inflammation of the air pouch type (zymosan-air-pouch inflammation) but also in compound 48/80-induced air-pouch inflammation. Moreover, K-76COONa inhibited in vitro histamine release from rat peritoneal mast cells induced by compound 48/80.     

Inhibition by calcium antagonists of histamine release and calcium influx of rat mast cells: Difference between induction of histamine release by concanavalin A and compound 48/80

The relation between calcium influx and histamine release from rat mast cells was investigated. When purified mast cells pretreated with a calcium antagonist (MnCl₂ or methoxyverapamil (D-600)) were exposed to concanavalin A or compound 48/80 in Tyrode solution (pH 7.4) at 37°C, the calcium antagonists inhibited the extracellular calcium-dependent component of concanavalin A-induced histamine release. MnCl₂ also inhibited the extracellular calcium-dependent component of compound 48/80-induced histamine release, whereas D-600 did not inhibit the release. D-600 inhibited the ⁴⁵Ca uptake induced by concanavalin A, but did not inhibit the ⁴⁵Ca uptake induced by compound 48/80. It was found that the inhibitory action of calcium antagonists depended on the uptake of extracellular calcium. These observations suggest that concanavalin A and compound 48/80 stimulate different mechanisms of calcium influx. Studies on inactivation of the mechanisms of calcium influx showed that calcium influx into cells activated by concavalin A stopped when concanavalin A was washed out, whereas the influxactivated by compound 48/80 was still operative after compound 48/80 had been washed out.