Role of cyclic AMP in the prejunctional α2-adrenoceptor modulation of noradrenaline release from the rat tail artery

Summary Experiments were designed to evaluate the effect of cyclic AMP on the electrically-induced release of noradrenaline from vascular sympathetic nerve terminals. The possible implication of the inhibition of adenylate cyclase in the negative feed-back control by prejunctional α₂-adrenoceptors of neurotransmitter release was also investigated. Rat isolated tail arteries were preincubated with [³H]-noradrenaline; the preparations were subsequently perfused/superfused with [³H]-noradrenaline-free medium and their perivascular nerves were field stimulated with 24 pulses at 0.4 Hz (0.3 ms, 200 mA). 2 compounds known to enhance the intracellular concentration of cyclic AMP, namely the membrane permeant analogue 8-Br-cAMP (10–300 μmol/l) and forskolin (0.3–10 μmol/l), an activator of adenylate cyclase, concentration-dependently enhanced the stimulation-evoked tritium overflow. The 1,9-dideoxy derivative of forskolin, which does not stimulate adenylate cyclase, was ineffective. Exposure to the cyclic AMP phosphodiesterase inhibitor rolipram 30 μmol/l produced a moderate increase (about 20%) in tritium overflow. However, in the presence of rolipram the facilitatory effect of forskolin was significantly more pronounced than in its absence. Whereas 8-Br-cAMP produced a slight concentration-dependent enhancement of the stimulation-induced vasoconstriction, forskolin and rolipram depressed it. The α₂-adrenoceptor agonist B-HT 933 (3–30 μmol/l) concentration-dependently inhibited the tritium overflow. The effect of B-HT 933 30 μmol/l was slightly, but significantly reduced in the presence of 8-Br-cAMP 100 and 300 μmol/l, but was not changed in the presence of forskolin 3 μmol/l The facilitatory effect of rauwolscine 1 μmol/l was enhanced in the presence of 8-Br-cAMP 100 μmol/l. During perfusion with 8-Br-cAMP 100 μmol/l, the current strength and frequency were decreased to 150 mA and 0.2 Hz, respectively in order to obtain similar amounts of tritium overflow to those observed in the absence of the cyclic AMP analogue with the initial stimulation parameters. Under these conditions, the inhibition of the overflow by B-HT 933 30 μmol/l and the facilitation by the α₂-adrenoceptor antagonist rauwolscine 1 μmol/l were unaltered as compared to controls under initial stimulation conditions. It is concluded that, in the rat tail artery, the terminals of perivascular sympathetic nerves are endowed with an adenylate cyclase system. Cyclic AMP is able to modulate noradrenaline release, but does not appear to play a role in the initiation of the release process itself. In addition, the results do not support the hypothesis that prejunctional α₂-adrenoceptors depress noradrenaline release through the inhibition of adenylate cyclase. Send offprint requests to B. Bucher at the above address     

Calcium channel subtypes and exocytosis in chromaffin cells: a different view from the intact rat adrenal

In the intact rat adrenal gland perfused with an oxygenated Krebs-bicarbonate solution at 37°C, the electrical field stimulation of splanchnic nerves (100 V, 0.5 ms duration, 10 Hz during 10 s) produced transient catecholamine release peaks that were reproduced in subsequent stimuli, applied at 8-min intervals. ω-Conotoxin GVIA (0.3 μM) caused only a modest inhibition of the secretory response, suggesting that the N-subtype of voltage-dependent Ca²⁺ channels are scarcely involved in such a response. Both ω-conotoxin MVIIC (1 μM) and furnidipine (1 μM) halved the secretion, suggesting that the L- and P/Q-types of Ca²⁺ channels were involved. N-type Ca²⁺ channels appear to be involved in the maintenance of secretion in response to sustained stimulus since ω-conotoxin GVIA (0.3 μM) reduced the catecholamine output to 28%. When secretion was elicited by acetylcholine (10 μM), furnidipine reduced the catecholamine release by 50% and ω-conotoxin MVIIC by 40%, whereas ω-conotoxin GVIA did not modify the response. The K⁺-induced secretory responses (23.6 mM K⁺, 15 s) were reduced 75% by furnidipine and 45% by ω-conotoxin MVIIC, indicating that this type of stimulation preferentially recruited L-type channels. These data show that electrical stimulation recruits Ca²⁺ channel subtypes different from those recruited by direct depolarization of chromaffin cells. Received: 2 December 1998 / Accepted: 21 April 1999 / Published online: 21 June 1999     

Na+ channel modulation and force-frequency relationship in human myocardium

The enhancement of force of contraction (FOC) following increasing frequencies of stimulation is an important mechanism of positive inotropy in human myocardium. The present study aimed to investigate the influence of alterations in Na⁺ influx on FFR in human myocardium. Isometric FOC of electrically stimulated right auricular trabeculae (AUT, n=12) from human nonfailing hearts (n=8) was measured at different stimulation rates (0.5-3 Hz) under control conditions, after increasing Na⁺ influx by the addition of (±)BDF 9148 (BDF, 3 μmol l^-1) and after decreasing Na⁺ influx by the addition of lidocaine (LIDO, 10 μmol l^-1). Additionally, the rate dependent changes in diastolic tension (DT) were measured in all experiments. Under control conditions FOC increased with increasing frequencies of stimulation. The rate at which maximal FOC was observed (SFmax) was 2.0±0.2 Hz and maximal increase in FOC (PIEmax) by increasing frequency of stimulation was +1.5±0.5 mN. After increase of Na⁺ influx by BDF (3 μmol l^-1) SFmax was decreased to 0.8±0.1 Hz (p<0.05 versus control) and PIEmax was +0.1±0.3 mN (p<0.05). When Na⁺ influx was diminished by LIDO (10 μmol l^-1) SFmax and PIEmax were increased compared to control (2.4±0.1 Hz and +4.1±0.9 mN, p<0.05 versus control). The diastolic tension (DT) of AUT at 3 Hz was not changed at higher rates in the control group and after application of LIDO (10 μmol l^-1), whereas after enhancement of Na⁺ influx by BDF there was an increase in DT of +0.7±0.2 at 3Hz (p<0.05 versus control and LIDO). An enhanced Na⁺ influx leads to a decrease in the optimal frequency and to a smaller force potentiation by higher stimulation rates which could be at least partly due to incomplete relaxation at higher frequencies, whereas a reduced Na⁺ influx is followed by opposite alterations. It is concluded that besides Ca²⁺ handling also Na⁺ influx and Na⁺ homeostasis might determine the frequency-induced force potentiation in human myocardium. Thus, the negative FFR in diseased human myocardium might result from changes in cellular Ca²⁺ or Na⁺ regulatory sites. Received: 20 November 1996 / Accepted: 21 February 1997     

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

In vitro studies on the mechanism by which (+)-norfenfluramine induces serotonin and dopamine release from the vesicular storage pool

(+)-Norfenfluramine is the main metabolite of the serotoninergic anorectic agent (+)-fenfluramine. Both compounds inhibit 5-HT reuptake and stimulate its release, although they induce release from different pools, with (+)-norfenfluramine acting primarily on the cytoplasmic pool. Moreover, (+)-norfenfluramine was more potent than the parent drug in inducing dopamine release. In order to investigate whether (+)-norfenfluramine induces a Ca²⁺-dependent vesicular release, like some amphetamine derivatives, in the present study we preloaded synaptosomes with the [³H]neurotransmitter ([³H]5-HT or [³H]dopamine), superfused (washed) them for 47 min in the absence of pargyline and then exposed them to the releasing stimulus. With this protocol, the cytoplasmic pool should be absent and the [³H]neurotransmitter should mainly be stored in synaptic vesicles, where (+)-norfenfluramine should act to induce release. This was confirmed by a significant decrease of (+)-norfenfluramine-induced [³H]5-HT and [³H]dopamine release after reserpine pretreatment. The dose-response curves of (+)-norfenfluramine-induced [³H]5-HT release were superimposable in hippocampus and hypothalamus, and also superimposable on the curve for (+)-fenfluramine-induced [³H]5-HT release; the dopamine releasing potency of (+)-norfenfluramine in the striatum was more than ten times lower. The [³H]5-HT release induced by (+)-norfenfluramine was partly (about 50%) but significantly Ca²⁺-dependent, and it was also markedly (68%) inhibited by Cd²⁺, a non-specific blocker of voltage-dependent Ca²⁺ channels, suggesting that the Ca²⁺-dependent release is mediated by entry of Ca²⁺ into the synaptosomes through these channels. The [³H]dopamine release induced by 5 μM (+)-norfenfluramine was completely Ca²⁺-independent whereas at higher concentrations (10 and 20 μM) it was only slightly (20%) Ca²⁺-dependent. We have no clear explanation why (+)-norfenfluramine has these different effects on serotoninergic and dopaminergic synaptosomes. Received: 6 April 1998 / Accepted: 9 June 1998     

Endothelin receptors and calcium translocation pathways in human airways

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 μM), the L-type calcium channel inhibitor, or incubation in Ca²⁺-free medium, produced marked inhibition of contractions to the ET_B receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca²⁺-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ET_A and ET_B receptor agonist. In Ca²⁺-free medium, ryanodine (10 μM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni²⁺; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCl concentration-response curves. The mixed ET_A/ET_B receptor antagonist SB 209670 (3 μM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 μM), the ET_A receptor antagonist, was without effect on responses induced by either agonist. ET-1 (1 nM–0.3 μM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM–0.1 μM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 μM), and also by BQ-123 (3 μM). This is consistent with linkage of ET_A receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca²⁺ mobilization mechanisms elicited by ET_A and ET_B receptor activation. Thus, sarafotoxin S6c-induced, ET_B receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca²⁺, although a significant component of the response was also mediated by intracellular Ca²⁺ release, including from ryanodine-sensitive stores. ET_A receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca²⁺. Received: 21 July 1998 / Accepted: 26 January 1999     

Inhibitory prejunctional muscarinic receptors at sympathetic nerves do not operate through a cyclic AMP dependent pathway

Summary In mouse atria previously incubated with [³H]-noradrenaline, carbachol (1.0 μmol/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the α-adrenoceptor antagonist phentolamine (1.0 μmol/l), which by itself significantly increased the S-I outflow of radioactivity. In both cases the inhibitory effect of carbachol was blocked by atropine (0.3 μmol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 μmol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX,100 μmol/l), was used to maximally enhance the S-I outflow of radioactivity through the cyclic AMP mechanism. The inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was not reduced in the presence of 8-bromo cyclic AMP and IBMX. Similar results with carbachol in the presence of 8-bromo cyclic AMP and IBMX were also found in rat right atrial strips which had been incubated with [3H]-noradrenaline. These results suggest that the effects through inhibitory prejunctional muscarinic receptors are not mediated by cyclic AMP. The protein kinase inhibitor, staurosporine (0.1 μmol/l), significantly blocked the enhancing effects of 8-bromo cyclic AMP (270 μmol/l) plus IBMX (100 μmol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 μmol/l) however, was not reduced in the presence of staurosporine, suggesting that protein kinases affected by staurosporine (protein kinase A, protein kinase C) are not involved in the postreceptor mechanism for inhibitory prejunctional muscarinic receptors. This finding further rules out the involvement of cyclic AMP in muscarinic inhibition. The inhibitory effect of carbachol either by itself or in the presence of phentolamine, was not reduced in atria from mice that had been pretreated with pertussis toxin (1.5 or 3.0 μg). Furthermore, in rat atrial strips, the inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was also not altered by pretreating the rats with pertussis toxin (8.4 μg). The results suggest that in both tissues the major mechanism for inhibition of noradrenaline release through muscarinic receptors does not involve a pertussis toxin sensitive G protein. Send offprint requests to M. Costa at the above address     

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 μM), and by ryanodine (10 μM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca²⁺ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 μM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-d-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 μM) and ryanodine (10 μM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of –90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential. Received: 6 July 1998 / Accepted: 30 November 1998     

Effect of the Na+-channel modulator BDF 9148 on Ca2+-sensitivity and force of contraction of hypertrophic myocardium from transgene rats harboring the mouse Renin gene (TG(mREN2)27)

The present study aimed to investigate the inotropic effect of the Na⁺-channel modulator BDF 9148 in hypertrophic myocardium compared to control tissue. Thus, TG(mREN2)27 rats (TGR), a model with hypertension induced cardiac hypertrophy, was compared with age matched Sprague-Dawley rats (SPDR). The effect of BDF 9148 (0.01–10 μM) on force of contraction (1 Hz, 37°C), the force-frequency relationship (0.5–7 Hz) and the frequency-dependent diastolic tension (0.5–7 Hz) was studied on leftventricular papillary muscles from SPDR and TGR. Chemically skinned muscle fibers of the same hearts were used to examine the influence of BDF 9148 on the Ca²⁺-sensitivity of the contractile proteins. For control the Ca²⁺-sensitizer EMD 57033 was examined. In addition the Na⁺/K⁺-ATPase activity was measured in both, SPDR and TGR. BDF 9148 showed a concentration dependent positive inotropic effect in SPDR and TGR cardiac preparations. Comparing SPDR and TGR, a higher effectiveness of BDF 9148 on TGR was found, while the potency was unchanged. With increasing stimulation rates a significant higher decrease in force of contraction in TGR compared to SPDR was observed. In addition, a significant higher increase in diastolic tension was found in TGR. After exposure to 1 μM BDF 9148 the decrease in force of contraction was significantly reduced in both SPDR and TGR, while only in TGR the increase in diastolic tension was reduced. BDF 9148 had no effect on the Ca²⁺-sensitivity or maximal developed tension of skinned fiber preparations from SPDR or TGR. In contrast, the Ca²⁺-sensitizer EMD 57033 increased the Ca²⁺-sensitivity. The activity of the Na⁺/K⁺-ATPase was significantly reduced in TGR compared to controls. Conclusions: The Na⁺-channel modulator BDF 9148 was more effective in hypertrophic compared to control myocardium in increasing force of contraction, enhancing frequency-dependent force generation and reducing diastolic tension. These effects were not mediated via interaction with the contractile apparatus. The enhanced effectiveness of Na⁺-channel modulation in hypertrophic myocardium could result from alterations of the Na⁺ homeostasis, i. e. a reduced Na⁺/K⁺-ATPase activity. Received: 13 August 1997 / Accepted: 4 February 1998     

Influence of alpha- and beta-adrenoceptor antagonists on ventricular fibrillation in ischemic rat hearts

Although it is generally accepted that adrenergic influences contribute to arrhythmias during myocardial ischemia, it is still a matter of debate whether these arrhythmogenic effects are mediated via alpha- or beta-adrenergic receptors and which particular receptor subtype is involved. Controversial results may be due to ancillary properties of the adrenergic receptor antagonists used to resolve this question. Therefore, we compared the influence of various, structurally different alpha- and beta-adrenoceptor blocking agents on the occurrence of ventricular fibrillation in rat isolated hearts. Regional ischemia was induced by ligature of the left coronary artery and ECG was monitored over 30 min of coronary occlusion. Myocardial ischemia precipitated ventricular fibrillation in a well reproducible manner with an incidence of about 80% in control hearts. Racemic propranolol (0.1-1 μmol/l) concentration-relatedly reduced the incidence of ventricular fibrillation from 71% in controls to 10%, whereas the beta-adrenoceptor blocking agents atenolol (10 μmol/l) and timolol (1 μmol/l) did not influence the occurrence of arrhythmias. Moreover, both stereoisomers of propranolol were equipotent in suppressing ischemia-induced ventricular fibrillation, indicating an action of propranolol independent of its beta-adrenoceptor blocking properties. Unselective antagonism of alpha-adrenoceptors by phentolamine decreased the incidence of ventricular fibrillation from 90% to 58% at 0.1 μmol/l and totally suppressed ventricular fibrillation at 1 μmol/l. The alpha₁-selective antagonists prazosin and HEAT concentration-dependently (0.1-10 μmol/l) reduced the incidence of ventricular fibrillation from 83% to 0%, whereas the alpha₂-selective antagonist RX 821002 revealed no antiarrhythmic effect. Furthermore, subtype specific antagonism of alpha_1A-adrenoceptors by WB 4101 clearly reduced the occurrence of ventricular fibrillation in a concentration-dependent manner (0.01-1 μmol/l) from 66% to 17%. Conversely, CEC, known to block the alpha_1B-adrenoceptor subtype, possessed no antifibrillatory effect. In conclusion, the contribution of catecholamines to ischemia-induced arrhythmias in rat isolated heart is primarily mediated via WB 4101-sensitive alpha₁-adrenergic activation. Beta- and alpha₂-adrenoceptor blockade did not affect ventricular fibrillation in this model. The antifibrillatory action of propranolol was rather due to ancillary properties of this agent. Received: 5 June 1996 / Accepted: 14 March 1997     

Heterogeneous characteristics of imidazoline-induced insulin secretion

Imidazolines are regarded as a pharmacological group of insulin secretagogues with one uniform mechanism of action, namely closure of ATP-dependent K⁺ channels (K_ATP channels) and, in consequence, depolarization of the plasma membrane, Ca²⁺ influx and stimulation of secretion. This assumption was investigated by measuring insulin secretion from perifused pancreatic islets in response to three imidazoline compounds and comparing the characteristics of secretion with changes in membrane potential and cytosolic Ca²⁺ concentration [Ca²⁺]_i of single β-cells. Phentolamine (32 μM) stimulated insulin secretion from perifused mouse islets in the presence of stimulatory (10 mM and 30 mM) and substimulatory (5 mM) glucose concentrations and even in the absence of glucose. Idazoxan in concentrations up to 500 μM was virtually ineffective in the presence of 5 mM glucose. At 10 mM glucose, there was a moderate but significant increase of secretion by idazoxan, 20 μM being nearly as effective as 100 μM. The effect of phentolamine was of slow onset and irreversible in the time frame of the experiments, while the effect of idazoxan was of fast onset and reversible. Alinidine also stimulated secretion in the presence of 10 mM glucose with fast and reversible kinetics, but in contrast to idazoxan, 100 μM was clearly more effective than 20 μM. These heterogeneous characteristics of secretion were reflected by changes of [Ca²⁺]_i: the increase of [Ca²⁺]_i by phentolamine was slow and only partially reversible, whereas idazoxan led to a smaller, but faster and reversible response. The increase of [Ca²⁺]_i by phentolamine and idazoxan was abolished by the Ca²⁺ channel blocker D 600. Surprisingly, all three compounds depolarized the β-cell plasma membrane from a resting potential of –71 mV to about –36 mV. Again, the effect of phentolamine was slow and that of idazoxan and alinidine fast. Thus, the characteristics of phentolamine-induced secretion appear to be attributable to the consequences of K_ATP channel closure. It is unclear, however, why all three test compounds achieved the same degree of depolarization in spite of their known different efficiency to close K_ATP channels. Apparently, there are additional mechanisms involved in the action of idazoxan and alinidine, which may contribute to the obvious differences in the characteristics of secretion. Received: 2 October 1998 / Accepted: 21 December 1998     

Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery

The interaction of superoxide anions (O₂ ^–) generated by menadione with the synthesis and/or action of nitric oxide (NO), prostacyclin (PGI₂) and endothelium-derived hyperpolarizing factor (EDHF) was investigated in segments of the left anterior descending coronary artery (LAD) isolated from bovine hearts. EDHF and NO release were monitored by superfusion bioassay in segments pre-constricted with the thromboxane mimetic, U46619, PGI₂ release in addition by enzyme immunoassay for 6-keto-prostaglandin F_1α, and generation of O₂ ^– by lucigenin-enhanced chemiluminescence. Bradykinin (1–1,000 pmol) elicited a prominent, endothelium-dependent, relaxant response, 50–60% of which was insensitive to combined blockade of cyclooxygenase with diclofenac (1 μM) and NO synthase with N ^G-nitro-l-arginine (50 μM). This diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response was completely abrogated in the presence of tetrabutylammonium (3 mM), a non-selective inhibitor of Ca²⁺-dependent K⁺ channels, or when the segments were pre-constricted with potassium chloride (60 mM) instead of U46619, and thus most likely mediated by EDHF. Despite causing a two- to fourfold increase in the concentration of O₂ ^– in or at the vessel wall, menadione (30 μM) did not affect the diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response to bradykinin. When administered in the absence of N ^G-nitro-l-arginine, however, menadione significantly inhibited the relaxant response to bradykinin, presumably by attenuating the NO-mediated relaxation. Menadione also abolished the bradykinin-stimulated release of PGI₂ from luminally perfused segments of the LAD. This effect was more pronounced in the absence of N ^G-nitro-l-arginine, indicating that PGI₂ release in this preparation may be more sensitive to inhibition by peroxynitrite, the reaction product of NO and O₂ ^–, than to O₂ ^– alone. These findings suggest that, in contrast to NO and PGI₂, the release, and presumably also the mechanism of action, of EDHF in the bovine LAD is resistant to an increase in the local concentration of O₂ ^– or peroxynitrite which is thought to play an important role in coronary heart disease. Received: 27 August 1998 / Accepted: 18 November 1998     

MEN 11,420, a peptide tachykinin NK2 receptor antagonist, reduces motor responses induced by the intravesical administration of capsaicin in vivo

This study investigates the role of tachykinin NK₁ and NK₂ receptors in motor responses induced by the intravesical instillation of capsaicin in urethane-anaesthetized rats. SR 140,333 (1 μmol/kg, i.v.), a non-peptide NK₁ receptor antagonist, abolished urinary bladder contractions induced by the selective NK₁ receptor agonist [Sar⁹]SP-sulfone (0.1-100 nmol/kg, i.v.) without affecting those induced by the NK₂ receptor agonist [?Ala⁸]NKA(4-10). MEN 11,420 (100 nmol/kg, i.v.), a cyclic peptide NK₂ receptor antagonist, abolished bladder contractions induced by [?Ala⁸]NKA(4-10) (0.3-300 nmol/kg, i.v.) without modifying those induced by [Sar⁹]SP-sulfone. Intravesical instillation of capsaicin (6 nmol/0.6 ml/rat) produced a motor response consisting in a primary contraction followed by a series of high amplitude phasic contractions. The intravesical instillation of saline (0.6 ml/rat) produced a primary contraction of lower amplitude with respect to that induced by capsaicin and the total area under the curve was also lower in saline-instilled rats, however the number and the amplitude of phasic contractions was similar to that induced by capsaicin. MEN 11,420 (100 nmol/kg, i.v.) did not modify motor responses induced by the intravesical administration of saline. In contrast, in capsaicin-instilled rats, MEN 11,420 (100 nmol/kg, i.v.) reduced the primary contraction, the area under the curve and also the number of phasic contractions. SR 140,333 (1 μmol/kg, i.v.) reduced the primary contraction but not other parameters. The combination of SR 140,333 (1 μmol/kg, i.v.) and MEN 11,420 (100 nmol/kg, i.v.) produced an additive inhibitory effect on the primary contraction but not a further inhibition on other parameters with respect to that observed with MEN 11,420 alone. In hexamethonium (110 μmol/kg, i.v.)-pretreated animals the intravesical instillation of capsaicin produced a tonic contraction having greater amplitude and area than that induced by saline. MEN 11,420, but not SR 140,333, significantly reduced the bladder response to capsaicin in hexamethonium-pretreated rats. Again, the combined administration of MEN 11,420 and SR 140,333 did not produce further inhibitory effect in comparison to MEN 11,420 alone. It is concluded that the motor responses induced by the intravesical instillation of capsaicin are mediated by the activation of peripheral tachykinin NK₂ receptors. Received: 11 February 1997 / Accepted: 17 April 1997     

Phorbol ester-induced contractions of mouse detrusor muscle are inhibited by nifedipine

The effects of phorbol esters on contractions of detrusor strips isolated from mouse urinary bladder were studied. β-Phorbol-12,13-dibutyrate (β-PDBu, 10 nM) significantly enhances both the neurogenic and myogenic detrusor contractions to a similar extent. By contrast, an inactive isoform of protein kinase C (PKC) stimulation, α- phorbol-12,13-dibutyrate (100 nM) has no such enhancing effect on the muscle contraction. The effect of β-PDBu was dependent on the extracellular Ca²⁺ concentration. Nifedipine (0.3 μM, a L-type Ca²⁺ channel blocker), staurosporine (1 μM) and bisindolylmaleimide I ( μM, a selective PKC inhibitor) but not ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) abolished the enhancing effect of β-PDBu. In other words, β-PDBu failed to augment the nifedipine-insensitive component of the muscle contraction. Moreover, β-PDBu not only enhances the muscle response induced by exogenous agonists (acetylcholine or ATP) and KCl but also increases the resting tone of detrusor muscle, an effect which is also inhibited by nifedipine and bisindolylmaleimide I. From these findings, it is concluded that the enhancing effect of β-PDBu is due to activation of the L-type Ca²⁺ channel through phosphorylation by protein kinase C. This allows more Ca²⁺ influx from the extracellular medium, leading to an increase in the contractions of the mouse detrusor muscle. Received: 29 October 1997 / Accepted; 17 February 1998     

Involvement of different Ca2+ pools during the canine bronchial sustained contraction in Ca2+-free medium: lack of effect of PKC inhibition

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca²⁺-free medium and the possibility that each agonist uses a different Ca²⁺ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca²⁺) and in Ca²⁺-free medium. Cch and histamine induced an SBC in bronchial tissues in Ca²⁺-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca²⁺-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 μM cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SBC. The SBC induced by Cch or histamine in Ca²⁺-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca²⁺ from two different sources during the SBC in Ca²⁺-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca²⁺ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only. Received: 12 March 1998 / Accepted: 2 September 1998     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

Effect of a long-acting somatostatin analogue (octreotide) on circulating tachykinins and the pentagastrin-induced carcinoid flush

Summary Cutaneous flushing was provoked in seven patients with metastatic carcinoid tumours and the carcinoid syndrome by an intravenous injection of pentagastrin (0.6 μg·kg⁻¹ body weight). The patients were studied before and 1 h after a subcutaneous injection of the long-acting somatostatin analogue octreotide 50 μg (Sandostatin). The severity of the carcinoid flush in all the patients was reduced by administration of the analogue. The rise in facial temperature was 1.3 (0.3)° C before and 0.8 (0.2)° C after octreotide. Six patients responded to pentagastrin with a rise in the circulating neurokinin A-like immunoreactivity (NKA-LI) and five patients with a rise in circulating substance P-like immunoreactivity (SP-LI). No cutaneous flushing or rise in tachykinin concentration was observed in healthy subjects (n=6) after injection of pentagastrin. The rise in NKA-LI in the patients was decreased by 61 (14)% and the rise in SP-LI by 54 (13)% after octreotide. Although flushing still occurred, the tachykinin response in two patients was completely abolished. The data demonstrate that the release of tachykinins from carcinoid tumours during pentagastrin-induced flushing is subject to partial inhibition by octreotide. However, the occurrence of a flush in some patients in the absence of a detectable rise in circulating tachykinins indicates that the latter peptides cannot be the sole causative agent of the carcinoid flush.     

A K+ single channel and whole-cell clamp study on the effects of levcromakalim in guinea pig portal vein cells

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K⁺ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K⁺ in bath/pipette), 10 μM levcromakalim activated K⁺ channels with a chord conductance of 23.2 pS (K_KCO), which were sensitive to the blocker of ATP-dependent K⁺ channels (K_ATP), glibenclamide. Voltage steps from –80 mV to +20 mV activated 4-aminopyridine-sensitive K⁺ channels of 6.5 pS with properties of delayed rectifier K⁺ channels (K_v). In patches which upon a previous voltage step had revealed the existence of K_v, levcromakalim reduced the open-probability of K_v, but it did not concomitantly activate K_KCO. During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K⁺ channels (BK_Ca) appeared which could be inhibited by iberiotoxin, a selective blocker of BK_Ca, and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to –100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 μM levcromakalim, the KCO-induced K⁺ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K⁺]_i versus low [K⁺]_o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of K_v to K_ATP (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK_Ca (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K_ATP in other tissues. Received: 1 August 1997 / Accepted: 7 June 1998     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

Histamine H1 receptors in C6 glial cells are coupled to calcium-dependent potassium channels via release of calcium from internal stores

We investigated the action of histamine on C6-astroglioma cells using patch clamp recording and intracellular calcium measurement. Application of 100 μM histamine hyperpolarized the resting membrane potential and increased free intracellular calcium. Membrane hyperpolarization was accompanied by a decrease in input resistance. The effect of histamine was reversible and responses persisted following repeated applications. In voltage clamp experiments histamine elicited an outward current associated with a conductance increase and a reversal potential near the Nernst potential for potassium. The action of histamine was blocked by mepyramine but not by cimetidine or thioperamide suggesting that a H₁ receptor mediated the response. Quinidine and charybdotoxin, but not apamin, blocked the hyperpolarization. Buffering internal calcium with BAPTA diminished the activation of the potassium channel, suggesting a calcium-dependent K⁺-channel, which was also found to be regulated by protein kinase C and phosphatases. The increase in intracellular calcium was not dependent on external calcium or sensitive to pertussis toxin, cholera toxin, forskolin or 8-bromo-cAMP. Both the hyperpolarization and the increase in intracellular calcium were blocked by thapsigargin or the phospholipase C inhibitor U73122. These results indicate that histamine liberates calcium from internal stores by activation of phospholipase C which in turn leads to an increase of intracellular Ca²⁺ and thereby to the activation of a calcium-dependent potassium channel in C6 glial cells. Received: 8 August 1996 / Accepted: 22 January 1997