Presynaptic inhibition of acetylcholine release

High potassium (51 mM) has been shown to evoke release of acetylcholine ([3H]ACh and endogenous ACh) from cholinergic nerves in rat bronchial smooth muscle. The release of [3H]ACh was reduced by 85% when the Ca2+ concentration was changed from 2 to 0.1 mM. The veratridine-induced release was completely inhibited by tetrodotoxin, but tetrodotoxin did not reduce the potassium-evoked release. The muscarinic agonist, oxotremorine, reduced the potassium stimulated release of [3H]ACh, without affecting the basal release. In contrast, scopolamine substantially potentiated the potassium-evoked release. Adenosine had a dual effect in the rat bronchi. Adenosine inhibited the potassium-evoked release of [3H]ACh and this presynaptic effect of adenosine was antagonized by 8-phenyltheophylline. Adenosine also induced contraction of the bronchial smooth muscle and there was potentiation by adenosine of the ACh-induced contraction. The results indicate that cholinergic nerve terminals in the rat bronchi possess muscarinic receptors which inhibit the release of ACh. Adenosine may have analogous effects, e.g. presynaptic inhibition of transmitter release in addition to postsynaptic enhancement of bronchial smooth muscle contraction.     

Light–dependent effects of zinc protoporphyrin IX on endothelium–dependent relaxation resistant to NoM–nitro–L–arginine

Acetylcholine (ACh) induces an Nω–nitro–L–arginine (L–NOARG)–resistant relaxation and hyperpolarization in the rat isolated hepatic artery. The possibility that carbon monoxide (CO) produced by haem oxygenase (HO) is an endogenous mediator of this response was investigated. Exogenously applied CO evoked a concentration–dependent relaxation, and the CO ‘scavenger’ oxyhaemoglobin (10μM) reduced the maximum ACh–induced relaxation by 25%. The HO inhibitor zinc protoporphyrin IX (ZnPP, 10 μM virtually abolished the ACh–induced relaxation in experiments carried out under ordinary light conditions. However, ZnPP did not affect the ACh–induced relaxation under dark conditions, even after exposure of ZnPP to intense light before the preincubation period. Biliverdin (0.1 mM), a feedback inhibitor of HO, was also inactive under dark conditions, and the HO substrate haematin (0.1 mM) did not facilitate the ACh–induced relaxation. The relaxation induced by the nitric oxide (NO) donor 3–morpholino–sydnonimin was not affected by ZnPP in the presence of light. However, ZnPP inhibited the relaxation evoked by the potassium channel opener levcromakalim and the tonic component of the contractile response to 60 mM potassium, indicating that ZnPP has effects distinct from HO inhibition in the presence of light. ZnPP should therefore be protected from light when used to inhibit HO–mediated CO formation. The results do not suggest that CO generated by HO mediates the endothelium–dependent, L–NOARG–resistant relaxation induced by ACh in the rat hepatic artery.     

Effects of calmodulin antagonist (W-7) on phorbol ester (PMA)-induced contractile response in isolated rat aorta.

The aim of this study was to investigate effects of calmodulin antagonist (W-7) on the contractile response of the rat aorta induced by activation of protein kinase C (PKC) by phorbol ester. Phorbol 12-myristate 13-acetate (PMA) produced biphasic contraction i.e., a sustained contraction (initial contraction) and 17.9 +/- 1.7 min later, this progressively developed contraction was changed to a delayed contraction superimposed on the initial contraction. The delayed contraction was completely inhibited by treatment with nicardipine. The onset of the delayed contraction was significantly delayed by treatment with W-7, whereas same concentration of W-7 showed a weak relaxant effect (10%) on the PMA-induced maximal contraction of aorta. Higher concentration of W-7 strongly inhibited PMA-induced sustained contraction. These results suggest that PMA-induced biphasic contractile response may be regulated by calmodulin.     

Contribution of intracellular stored calcium to contractile activation in contractures of stomach circular muscle of Bufo vulgaris formosus

Contractile responses of stomach circular muscle of Bufo to high-K, to acetylcholine (ACh) in normal Ringer or in high-K solution, and to calcium in Ca-free high-K solution showed a phasic contraction which relaxed completely in 30-45 sec. K-induced contracture was abolished in Ca-free solution containing 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) within 20 sec, while ACh-induced contracture was not abolished and 10-25% of control tension was kept up to 40 sec. This response increased to 40-50% when all extracellular Na was replaced with tris(hydroxymethyl)-aminomethane (Tris). K-induced contracture was inhibited completely by 1 mM La. ACh-induced contracture in the muscle depolarized by high-K solution was dependent on the depolarization time, 0-10, 60-70, and nearly 100% of control after 1, 3, and 10 min depolarization, respectively. These ACh-induced contractures were not inhibited by 1 mM EGTA or La. All contractures mentioned above were markedly inhibited by 5 mM procaine. These results suggest that activation of both contractures induced by high-K and ACh were, at least partly, dependent on the Ca at the intracellular Ca storage sites. Ca-induced contracture was dependent on depolarization time as was ACh-induced contracture, when the muscle was depolarized by Ca-free high-K solution without pre-treatment with Ca-free Ringer solution. These results suggest that activation of Ca-contracture is also dependent on intracellular stored Ca.     

Mechanisms of the relaxant and contractile responses to bradykinin in rat duodenum

The signal pathway for bradykinin-induced relaxation followed by contraction in the isolated rat duodenum was investigated by comparing the effect of blocking agents on the response to bradykinin and acetylcholine. The phospholipase C inhibitor U-73122 inhibited the relaxation induced by bradykinin, but had no effect on the contraction to either bradykinin or acetylcholine. The same response pattern was observed when the tissues were pre-treated with thapsigargin, a selective inhibitor of microsomal Ca2+ pumps. An inhibitor of non-voltage-dependent Ca2+ influx, SK&F 96365, inhibited the relaxant response to bradykinin and the contraction induced by acetylcholine, but not the contraction induced by bradykinin. In Ca2+-free Krebs-Henseleit buffer, the tissues failed to respond when they were exposed to either bradykinin or acetylcholine. When the tissues were partly depolarized (30 mM KCI), both bradykinin and acetylcholine induced contraction, while the relaxant response to bradykinin was almost completely abolished. Apamin (an antagonist of low-conductance calcium-activated K+ channel) together with charybdotoxin (CTX, an antagonist of large-conductance calcium-activated K+ channel) and CTX alone inhibited the relaxant but not the contractile response to bradykinin. We conclude that the biphasic response in isolated rat duodenum to bradykinin involves two distinct pathways. We propose that the relaxant component is induced indirectly via inositol-mediated increase in cytosolic Ca2+ in non-muscle cells with subsequent signals to the smooth muscle cells, whereas the contractile response is induced by direct effect on the smooth muscle cells.     

Accumulation of cAMP evoked by acetylcholine stimulation in rat submandibular acinar cells: observation of exocytosis, fluid secretion and [Ca2+]i

The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 microM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50 %). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 microM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 microM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.     

Effects of neuropeptide Y (NPY) on mechanical activity and neurotransmission in the heart, vas deferens and urinary bladder of the guinea-pig

The effects of preincubation for 10 min with synthetic porcine neuropeptide Y (NPY) on muscle tone and autonomic transmission in the guinea-pig right atrium, vas deferens, urinary bladder, portal vein and trachea were analysed in vitro. NPY induced a metoprolol-resistant, long-lasting, positive inotropic and chronotropic effect per se in the spontaneously beating right atrium. Furthermore, NPY caused a reversible inhibition of both the metoprolol and atropine-sensitive auricle responses to field stimulation (2 Hz or 4 Hz for 2 s) without affecting the response to exogenous noradrenaline (NA) or acetylcholine (ACh). NPY did not induce any contraction of the vas deferens, but inhibited both the rapid twitch response and the sustained tonic contraction induced by field stimulation. The NPY-induced inhibition of the tonic contraction was more long-lasting than that of the twitch response. The tonic contraction was blocked by phentolamine and the twitch response by alpha-, beta-methylene ATP tachyphylaxis. NPY did not inhibit the contractile effects of NA, ATP or alpha-, beta-methylene ATP. NPY also induced a reversible reduction of the non-cholinergic, non-adrenergic contractile response to field stimulation of the urinary bladder. In the portal vein, NPY (up to 5 X 10(-7) M) did not inhibit the spontaneous motility or the phentolamine-sensitive contractile responses to field stimulation and NA. The atropine-sensitive contraction of the trachea or the non-adrenergic, non-cholinergic relaxation induced by field stimulation were not significantly influenced by NPY in doses up to 5 X 10(-7) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of sympathetic neurotransmission by neuropeptide Y Y2 receptors in rats and guinea pigs

We have investigated the effect of the Y₂ receptor agonist (Y₂ agonist; N-acetyl [Leu^28,31] NPY 24-36), on contractions evoked by transmural electrical stimulation of sympathetic nerves of isolated arteries from a range of vascular beds in rats and guinea pigs. Contractions evoked by transmural stimulation of the rat renal, mesenteric and femoral arteries were significantly attenuated in the presence of the Y₂ agonist. In these arteries, contractions were significantly inhibited in the presence of an α-adrenoceptor antagonist (76–97%). So we conclude that these responses were primarily mediated by noradrenaline and that the Y₂ agonist attenuates the release of noradrenaline via presynaptic Y₂ receptors. Contractions of the rat carotid artery were not attenuated by the Y₂ agonist but were completely abolished in the presence of an α-adrenoceptor antagonist suggesting that in this artery the Y₂ agonist has no effect on release of noradrenaline. In the guinea pig, carotid arteries contractions evoked by transmural nerve stimulation were attenuated in the presence of the Y₂ agonist and inhibited by an α-adrenoceptor antagonist 75–87% suggesting that the Y₂ agonist attenuates the release of noradrenaline via presynaptic Y₂ receptors in this vessel. In the guinea pig femoral artery contractions evoked by transmural stimulation were not modified in the presence of the Y₂ agonist but were completely abolished in the presence of an α-adrenoceptor antagonist. This suggests that the Y₂ agonist does not modify noradrenaline release in this vessel. Contractions of the guinea pig mesenteric artery were significantly potentiated by the Y₂ agonist, possibly by potentiation of neuropeptide Y (NPY) at the Y₁ receptor. The Y₁ antagonist inhibited more than 70 % of the response, indicating that the majority of the contraction was mediated by NPY. The current study demonstrates heterogeneity of neurotransmitter substances in sympathetic nerves supplying vascular beds within and across species and in subsequent functional response.Supported by the National Health and medical Research Council of Australia.     

Enhanced Ca2+ sensitization of the bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats

To investigate the alteration in acetylcholine (ACh)-induced increase in Ca2+ sensitization of bronchial smooth muscle contraction concurrent with the airway hyperresponsiveness (AHR), the ACh-induced increases in cytosolic Ca2+ ([Ca2+]) level and contractile response were simultaneously determined by using Fura-2 loaded bronchial smooth muscle. The left main bronchi were isolated from AHR rats which were sensitized and repeatedly challenged with DNP-Ascaris antigen. The tissue ring preparations were incubated in loading solution containing 10 microM Fura-2AM for 3 hr at room temperature. Then the isometrical contraction and [Ca2+]i (F340/F380) were monitored. Although the ACh (10(-3) M)-induced contractile response in AHR group (322 +/- 60 % of 60 mM K+ induced contraction) was significantly greater than that in control animals (173 +/- 15 %, p<0.05), the ACh (10(-3) M)-induced increase in [Ca2+]i was without significant difference between the two groups (128 +/- 15 and 171 +/- 29% of 60 mM K+ -induced increase in [Ca2+]i, respectively). These findings suggest that an augmentation of ACh-induced Ca2+ sensitization may occur in bronchial smooth muscle of the rats with antigen-induced AHR.     

Low potency of Ca antagonists in smooth muscle from different levels of the respiratory tract

Respiratory smooth muscle at different levels of the respiratory tract was isolated from rabbit, rat and guinea-pig and incubated with the Ca antagonists, verapamil, D-600, felodipine, and nifedipine. There was no effect of the Ca antagonists when added on top of an already developed contraction. When added before the contractile agent at high concentrations (10-100 microM) the Ca antagonists reduced the contractions. Lanthanum (2 mM) was considerably more effective to prevent contractions than the Ca antagonists. Preparations from the trachea and bronchus responded in all animals, whereas the third generation bronchus from rat and guinea-pig failed to contract and that from rabbit showed very weak contractions. The failure to contract was not specific for the agonist, as depolarization with isotonic potassium solution (124 mM) did not evoke contractions either. Microscopic investigation showed well-developed smooth muscle in the trachea and main bronchus of the rat, whereas the muscle of the third generation bronchus was inhomogenous with scattered groups of muscle cells.     

Reversal of alpha 1-receptor mediated relaxation in intestinal smooth muscle

The alpha 1-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba2+ concentration was increased to 10-15 mM the response to phenylephrine was a contraction, and at Ba2+ concentrations in between the high and low range this response was biphasic--a relaxation followed by a contractile phase. The alpha 2-receptor agonist clonidine did not affect the tone of the Ba2+ contracted preparation. When the muscle preparation was contracted by Sr2+ (1-20 mM) in the presence of Ca2+ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca2+, 5 mM Ba2+ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca2+ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca2+ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ could be reverted to the normal relaxation by the addition of 20 mM Mg2+. Increasing the K+ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba2+ could reverse the response of alpha 1 receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca2+.     

Non-quantal release of acetylcholine in guinea-pig airways: role of choline transporter

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ～75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.     

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.     

Inhibitory effects of potassium channel blockers on carbachol-induced contraction in rat detrusor muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (<2 mM) of TEA and 4-AP inhibited phasic and tonic contractions induced by 5 micro M carbachol, whilst the relatively higher concentrations of TEA and 4-AP (>5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 microM to 5 microM. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 microM thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potassium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.     

Positive inotropic, negative chronotropic, and coronary vasoconstrictor effects of acetylcholine in isolated rat hearts: role of muscarinic receptors, prostaglandins, protein kinase C, influx of extracellular ca2+, intracellular Ca2+ release, and endothelium

The involvement of nitric oxide (NO), muscarinic receptors, prostaglandins, calcium influx via slow calcium channels, Ca2+ release from intracellular stores, protein kinase C, and endothelium in the positive inotropic, negative chronotropic, and coronary vasoconstrictor effects of acetylcholine (ACh) has been investigated in isolated rat hearts. The perfusion of hearts with ACh (10(-7), 5 x 10(-7), and 10(-6) M) produced marked decreases in heart rate and coronary flow and a marked increase in contractile force. Similar effects have been observed during the perfusion of hearts with ACh in the presence of Nomega-nitro-L-arginine methyl ester (L-NAME), which is an inhibitor of NO synthesis. The positive inotropic, negative chronotropic, and coronary vasoconstrictor effects of ACh were abolished by muscarinic receptor blocker atropine. In hearts pretreated with cyclooxygenase inhibitor indomethacin, ACh significantly decreased heart rate but did not significantly affect coronary flow and contractile force. In the presence of calcium channel antagonist verapamil or protein kinase C inhibitor staurosporine, ACh produced a significant drop in heart rate but did not significantly affect coronary perfusion pressure and force of contraction. In the presence of the inhibitor of the release of Ca2+ from intracellular stores dantrolene sodium, ACh produced a significant increase in coronary perfusion pressure and a marked decline in heart rate, but did not significantly affect force of contraction. Furthermore, the disruption of endothelium by perfusing the hearts with saponin abolished the vasoconstrictor effect of ACh but did not alter negative chronotropic and positive inotropic effect. Our results suggest that ACh causes vasoconstrictor, negative chronotropic, and positive inotropic effects in isolated rat hearts. Cardiac effects of ACh are related to muscarinic receptor activation, and prostaglandins modulate ACh-induced vasoconstriction and positive inotropy. Our data also suggest that protein kinase C and calcium influx from extracellular source may be responsible for the vasoconstrictor and positive inotropic effect of ACh. The calcium release from intracellular stores may mediate the positive inotropic effect, and the vasoconstrictor effect of ACh depends on an intact endothelium.     

Enhanced contractile response to noradrenaline and calcium influx in thoracic aorta isolated from dietary magnesium deficient rats

Dependency on extracellular calcium influx in contractile response to noradrenaline has been investigated in the thoracic aorta isolated from dietary magnesium (Mg) deficient rats. Adult male Wistar rats were fed with a Mg deficient diet (0.001% Mg) for 30 days, together with control groups (0.07% Mg). The contractile response to noradrenaline in the thoracic aortas from Mg deficient rats was significantly greater than that in the controls. However, there were no significant differences between control and Mg deficient rats in the contractile response to high potassium. The rate of 45Ca uptake induced by noradrenaline was greater in Mg deficient rat aortas than in the controls, but that induced by high potassium showed no significant differences between control and Mg deficient rats. Calcium entry blockers (verapamil and nifedipine) caused a concentration-dependent depression of the noradrenaline-induced contraction. The aortas from Mg deficient rats were more sensitive to these drugs than were those from the controls. These results suggest that the noradrenaline-induced contraction depends much more on extracellular calcium influx in Mg deficient rats than in the controls.     

Effects of nimodipine, Bay K 8644 and pinacidil on alpha 1- and alpha 2-adrenoceptor-mediated vasoconstriction in human hand veins

The effects of nimodipine, Bay K 8644 and pinacidil, three drugs interfering with transmembrane Ca2+ fluxes in different ways, were investigated in isolated human hand veins. Their ability to influence the concentration-response relationship for noradrenaline (NA) was assessed in the absence and presence of prazosin or rauwolscine. The contractile response to NA was almost abolished in Ca2+ -free medium. Nimodipine and pinacidil depressed the NA concentration-response curve both in the absence and presence of alpha-adrenoceptor blockers. The NA response was only partially inhibited by nimodipine, indicating that NA may activate nimodipine-insensitive influx pathways, presumably receptor-operated calcium channels. Pinacidil inhibited the contractile response to 124 mM K+ and reduced the NA-induced contraction in the presence of nimodipine, suggesting that pinacidil has actions other than the opening of potassium channels and subsequent membrane hyperpolarization. Bay K 8644 increased the NA potency fourfold in the presence of rauwolscine, whereas it had no effect on the NA response in the presence of prazosin and in the absence of alpha-adrenoceptor blockade. Such an action of Bay K 8644 can be reconciled with alpha 1-adrenoceptor activation causing membrane depolarization and opening of potential-operated calcium channels. It may be concluded that both alpha 1- and alpha 2-adrenoceptor-mediated contractions in human hand veins are highly dependent on Ca2+ influx, although the mechanisms utilized to bring about this influx partly differ between the two receptor subtypes.     

Influence of extracellular calcium and nifedipine on alpha 1- and alpha 2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca2+ and nifedipine on contractile responses to 10 microM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly alpha 2-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly alpha 1-adrenoceptors) and CMA (sensitive to both alpha 1- and alpha 2-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca2+-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K+ was more sensitive to Ca2+ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca2+, can also release Ca2+ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, alpha 1-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca2+ influx and intracellular Ca2+ release, whereas alpha 2-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca2+ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca2+-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca2+, whereas in the other types of artery, Ca2+ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca2+ influx and intracellular Ca2+ release to the contractile activation, but also in the nifedipine sensitivity of the Ca2+ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of alpha-adrenoceptor stimulated by NA and to the type of vessel studied.     

The functional significance of sodium channels in pancreatic beta-cell membranes

1. The existence and functional significance of Na channels in pancreatic beta-cell membranes were investigated by studying the effects of the plant alkaloid veratridine on the temporal release of insulin from perfused isolated rat islets of Langerhans.2. 100 muM veratridine evoked a sustained threefold increase in insulin release which was almost completely inhibited by 3 muM tetrodotoxin (TTX). This action of TTX was rapidly reversible.3. The simultaneous presence of 100 muM propranolol, 100 muM phenoxy-benzamine and 10 muM atropine did not alter the magnitude of the response to 100 muM veratridine, indicating that the action of veratridine on the beta-cells was direct and was not mediated via the release of neurotrans-mitters from nerve endings within the islets.4. (45)Ca uptake by isolated islets in static incubation was increased almost threefold by 100 muM veratridine. This increase was completely inhibited by the simultaneous presence of 3 muM TTX.5. Replacement of Na(o) by choline caused a transient fourfold increase in insulin release which was associated with an increase in the uptake of (45)Ca from the extracellular space of similar magnitude. Subsequent exposure of islets to 100 muM veratridine still evoked some insulin release but this only achieved 32% of that secreted by islets exposed to veratridine in medium of normal [Na](o).6. The addition of 2.5 mM CoCl(2) to the medium caused a 62.5% inhibition of veratridine-mediated insulin release.7. In Ca-free medium supplemented with 1 mM EGTA, 100 muM veratridine evoked insulin release of equal magnitude and of similar temporal relationship to that obtained in the presence of normal [Ca](o).8. A twofold increase in insulin release that occurred in the 15 min period immediately following exposure to 1 mM ouabain was completely independent of [Ca](o). Subsequent ouabain-evoked release became increasingly dependent on [Ca](o).9. Tetrodotoxin (3 muM) inhibited the first phase of insulin release evoked by 16.7 mMd-glucose by 37% and the second phase by 20%.10. Both Na and Ca appear capable of entering through Na channels opened in the beta-cell membrane by veratridine. The increase in [Na](i), resulting from the veratridine mediated increase in P(Na+), causes depolarization of the beta-cell membrane with a consequent opening of voltage-sensitive, Co(2+)-blockable channels for additional Ca entry. An increase in [Na](i) also increases [Ca](i) by altering the equilibria of intracellular Ca-sequestering mechanisms. The small but significant reduction of glucose-mediated insulin release by TTX indicates that glucose has a rather weak action on the Na channel and a more pronounced effect on the voltage-dependent Co(2+)-blockable Ca channel.     

小鼠Ⅱ型前庭毛细胞ACh-敏感性BK通道特性及其钙调制

目的： 研究小鼠球囊Ⅱ型前庭毛细胞乙酰胆碱 (ACh)-敏感性钾电流的药理学特性以及钙离子对ACh-敏感性钾电流的调制作用。方法： 应用全细胞膜片钳技术研究新鲜分离的小鼠球囊Ⅱ型前庭毛细胞ACh-敏感性钾电流的药理学特性以及细胞内外钙离子对ACh-敏感性钾电流的影响。结果： (1)细胞外ACh激活一缓慢持久的外向性电流。ACh-敏感性电流对钾通道阻断剂四乙胺 (TEA,tetraethylammonium) 和卡律布德蝎毒素 (CTX，charybdotoxin) 敏感，而对4-氨基吡啶(4-AP，4-aminopyride)不敏感。(2)细胞内应用钙离子螯合剂EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra-acetic acid)，ACh-敏感性钾电流的幅值逐渐被抑制；细胞内预先应用肝素(heparin)阻断三磷酸肌醇-依赖性钙离子释放，ACh-敏感性钾电流的幅值不受影响。(3)ACh-敏感性钾电流对钙通道阻断剂Cd2+和Ni2+敏感。结论： 细胞外ACh激活小鼠球囊Ⅱ型前庭毛细胞产生大电导钙依赖性钾电流(BK)。ACh-敏感性BK电流的活动依赖于细胞外钙离子通过膜上钙通道的内流，而三磷酸肌醇-依赖性细胞内钙离子释放机制不参与ACh-敏感性BK电流的激活过程。