Effects of S-11701 on accumulation, release and metabolism of norepinephrine in isolated canine saphenous veins.

1. The effects S-11701 ([morpholinyl-2)-methoxy]-8-tetrahydro-1,2,3,4 quinoline) on accumulation, overflow and metabolism of [3H]norepinephrine were investigated in isolated canine saphenous veins. 2. Saphenous veins were incubated with [3H]norepinephrine in the absence or the presence of S-11701; the drug caused a concentration-dependent inhibition of the tissue content of [3H]norepinephrine and its metabolites, except for 3-methoxy-4-hydroxymandelic acid (VMA). 3. In helical strips of canine saphenous veins previously incubated with [3H]norepinephrine and then suspended for isometric tension recording and measurement of the overflow of labelled transmitter and its metabolites, S-11701 (30 microM) significantly increased the spontaneous efflux of total 3H; this effect was almost exclusively due to an augmentation of the efflux of [3H]DOPEG. 4. During electrical stimulation (9 V, 1 Hz), S-11701 at 1 microM slightly increased the overflow of extraneuronal norepinephrine metabolites without affecting the contractile response. At the higher concentration (30 microM) the compound increased the contractive response and the overflow of 3H; the latter was due mainly to an increase in [3H]DOPEG and, to a lesser extent, in [3H]norepinephrine. 5. DMI (1 microM) did not interfere with the effects of S-11701 on DOPEG efflux. 6. These experiments indicate that in the canine saphenous vein, S-11701 causes a concentration-dependent inhibition of neuronal accumulation of [3H]norepinephrine. At higher concentrations, S-11701 enters the adrenergic nerve terminals independently of the neuronal amine carrier and displaces [3H]norepinephrine from its storage sites.     

Comparison of calcium ionophore and receptor-activated inositol phosphate formation in primary glial cell cultures.

The possible role of Ca2+ influx in alpha 1-adrenoceptor-stimulated [3H]inositol phosphate [( 3H]InsP) formation was examined in primary cultures of glial cells from 1-day-old rat brain. The Ca2+ ionophore A23187 caused a concentration- and time-dependent increase in [3H]InsP formation similar in magnitude to that caused by norepinephrine (NE). Responses to A23187 and NE were both completely dependent on extracellular Ca2+, with a similar concentration dependence. However, cadmium was more potent in blocking the response to A23187 than to NE. Lanthanum (1 mM) blocked the response to NE, although cobalt (5 mM) did not. The [3H]InsP response to A23187 was not additive with the response to NE or to the muscarinic agonist carbachol, although responses to NE and carbachol were addictive Both A23187 and ionomycin inhibited the additive stimulation caused by a combination of NE and carbachol, and this inhibition was potentiated by cadmium. Ionomycin stimulated [3H]InsP formation at concentrations lower than those inhibiting receptor-mediated responses, and this stimulation was not additive with responses to NE or carbachol. High-performance liquid chromatography separation showed similar patterns of [3H]InsPs formed in response to both Ca2+ ionophore and receptor agonists. These results raise the possibility that receptor-activated Ca2+ influx may be involved in stimulation of [3H]InsP formation in these cells.     

Increased formation of phosphatidic acid induced with vasopressin or Ca2+ ionophore A23187 in rat hepatocytes

The effects of vasopressin and Ca2+ ionophore A23187 on phospholipid metabolism were investigated in rat hepatocytes. Vasopressin stimulated the incorporation of [32P]Pi into phosphatidic acid within 2 min but then it returned to control level after 10 min. On the other hand, the stimulation of the incorporation of [32P]Pi into phosphatidylinositol continued with incubation times up to 20 min. The Ca2+ ionophore A23187 also increased the 32P-labeling in phosphatidic acid, although it had no effect on [32P]Pi incorporation into phosphatidylinositol. Concerning the incorporation of [3H]glycerol, vasopressin did not enhance its incorporation into phosphatidic acid and phosphatidylinositol. The Ca2+ ionophore A23187 increased the incorporation into phosphatidic acid without significant effects on that into phosphatidylinositol. In the hepatocytes prelabeled with [3H]arachidonic acid, stimulated degradation of phosphatidylinositol with the addition of vasopressin and resultant formation of phosphatidic acid were observed within 5 min. The transient accumulation of diacylglycerol, the product of phosphatidylinositol hydrolysis, also occurred within 5 min with vasopressin. On the other hand, with the Ca2+ inophore A23187, stimulated degradation of triacylglycerol to diacylglycerol and the consequent formation of phosphatidic acid were observed. The Ca2+ ionophore A23187 caused a significant release of free [3H]arachidonic acid, although vasopressin had no effect.     

Celiprolol has no direct or indirect relaxing effects in isolated arteries and veins.

Experiments were designed to study the potential mechanisms underlying the vasodilator effect of celiprolol. Rings of canine left circumflex coronary artery and rat mesenteric artery, with and without endothelium, were suspended in organ chambers for isometric tension recording. In both blood vessels, celiprolol (10(-9)-10(-4) M) failed to produce relaxation in rings with and without endothelium; these same tissues relaxed in an endothelium-dependent manner to acetylcholine (10(-6) M). All tissues relaxed completely in the presence of papaverine (10(-4) M). In the coronary artery, isoproterenol (10(-9)-10(-4) M) produced endothelium-independent relaxations which were inhibited in a competitive fashion by celiprolol (pA2 = 7.52 +/- 0.14; slope = 0.98, 95% confidence limits = 0.80-1.15). In other experiments, strips of canine saphenous veins were incubated with [3H]norepinephrine [( 3H]NE) and suspended for superfusion. Electrical stimulation (2 Hz, 4 V, 2 ms for 6 min) produced an increase in [3H]NE overflow. Isoproterenol (2 X 10(-6) M) augmented the evoked release of [3H]NE. Treatment of the strips with celiprolol (up to 5 X 10(-6) M) did not inhibit isoproterenol-induced facilitation of [3H]NE release. Thus, although celiprolol is a potent antagonist of postjunctional beta-adrenoceptors in the coronary artery, no evidence was obtained for a direct or indirect vasodilator effect of celiprolol on isolated blood vessels.     

Atrial natriuretic factor inhibits norepinephrine release evoked by sympathetic nerve stimulation in isolated perfused rat mesenteric arteries

The effect of atrial natriuretic factor (ANF) on [3H]norepinephrine release evoked by sympathetic nerve stimulation was examined in the isolated perfused rat mesenteric arteries. ANF (1 nM to 0.1 microM) caused a dose-dependent inhibition of [3H]norepinephrine release during nerve stimulation. The present result indicates that ANF inhibits noradrenergic neurotransmission in the rat mesenteric arteries through a prejunctional mechanism. This prejunctional effect of ANF may in part contribute to its vasodilation action.     

Effects of endothelial impairment by saponin on the responses to vasodilators and nitrergic nerve stimulation in isolated canine corpus cavernosum.

1. Responsiveness to EDRF-releasing substances and inhibitory nerve stimulation of canine isolated penile corpus cavernosum with and without saponin treatment were investigated. 2. Histological studies demonstrated that saponin did not detach endothelial cells from underlying tissues, but induced degenerative changes in the endothelial cells selectively. 3. In the cavernous strips contracted with phenylephrine, addition of acetylcholine, sodium nitroprusside, ATP and Ca2+ ionophore A23187 induced relaxations, but substance P and bradykinin did not change the muscle tone. 4. Acetylcholine-induced relaxation was significantly attenuated but not abolished by NG-nitro-L-arginine (L-NOARG). L-arginine restored the response inhibited by L-NOARG. The L-NOARG resistant relaxation was not influenced by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) but was suppressed in the strips contracted with K+. Treatment with saponin abolished the relaxation elicited by acetylcholine and A23187 but did not influence the response to nitroprusside and ATP. The ATP-induced relaxation was attenuated by aminophylline. 5. Transmural electrical stimulation at 2-20 Hz produced endothelium-independent relaxations which were abolished by tetrodotoxin and L-NOARG but unaffected by treatment with saponin. In saponin-treated cavernous strips, the neurogenic relaxation was not affected by acetylcholine, physostigmine, atropine and vasoactive intestinal peptide (VIP) but was abolished by ODQ. 6. It is concluded that acetylcholine-induced relaxations are endothelium-dependent and mediated partly by NO and also by other substances from the endothelium. The endothelium-independent relaxation to ATP is likely to be mediated by P1 purinoceptors. The function of nitrergic nerve does not seem to be prejunctionally modulated by acetylcholine and VIP.     

Receptor-mediated inositol phospholipid hydrolysis in astrocytes

Astrocyte-enriched cultures of the neonatal rat cortex were incubated for 24 h with [3H]inositol to prelabel the membrane inositol phospholipids. Exposure of the cultures to either noradrenaline or carbachol in the presence of Li+ produced a time- and dose-dependent accumulation of intracellular [3H]inositol phosphates. The separation of the individual inositol phosphates formed in response to receptor stimulation revealed that the major 3H-metabolite accumulated under these conditions was inositol monophosphate but that at least some of this was due to the initial formation of inositol trisphosphate. The use of selective receptor antagonists showed that noradrenaline- and carbachol-induced [3H]inositol phosphate accumulation was the result of the activation of alpha 1-adrenoceptors and muscarinic acetylcholine (probably of the M1 subtype) receptors respectively. Agonist-evoked [3H]inositol phosphate accumulation were found to be additive but the simultaneous addition of agonists and the Ca2+ ionophore A23187, which also promoted inositol phospholipid hydrolysis, was not. Agonist-induced [3H]inositol phosphate accumulation was only partially dependent on extracellular Ca2+, whilst that elicited by A23187 was entirely Ca2+-dependent. The results suggest that alpha 1-adrenoceptors and muscarinic acetylcholine receptors in these cultures are present either on the same cells and linked to separate inositol lipid pools or associated with different subpopulations of astrocytes in these cultures. Moreover, inositol lipids other than phosphatidylinositol 4,5-bisphosphate may be hydrolysed in response to agonist stimulation.     

Effects of calcium depletion on norepinephrine- and A23187-induced stimulation of inositol phosphate formation

The role of calcium in the stimulation of phosphoinositide (PIn) hydrolysis by norepinephrine and the calcium ionophore A23187 was investigated in chopped cerebral cortex in which the PIns had been labeled previously with ([3H]myo-inositol. The accumulation of the newly formed [3H]inositol phosphate ([3H]IPs) was used as an index of PIn hydrolysis. Norepinephrine produced a concentration-related increase in the accumulation of [3H]inositol-phosphates ([3H]IP), and this effect was only partially antagonized by omission of calcium from the incubation medium. Norepinephrine had relatively little effect on the accumulation of [3H]inositol 1,4-bisphosphate [3H]IP2 and inositol 1,4,5-triphosphate and/or inositol 1,3,4-trisphosphate ([3H]IP3). A23187 also increased the accumulation of [3H]IP but this effect was not antagonized by omission of calcium from the incubation medium. When the tissue had been washed extensively with EGTA, the basal levels of [3H]IP, [3H]IP2 and [3H]IP3 were decreased markedly, and the effects of both norepinephrine and A23187 were antagonized. Addition of calcium back to the depleted tissue led to an increase in the basal level of [3H]IPs as well as restoration of the stimulation produced by norepinephrine and A23187. The calcium threshold for the PIn effect was 0.1 microM. Additional calcium did not affect markedly the stimulation of accumulation of [3H]IP produced by norepinephrine and A23187. The results suggest that there is an absolute calcium requirement for PIn phosphodiesterase which is attained at 0.1 microM Ca2+. A23187 can stimulate the accumulation of [3H]IP perhaps by providing access of another form of the enzyme to artificially high concentrations (millimolar range) of calcium.     

Effects of S9977 on adrenergic neurotransmission

1. Experiments were designed to determine whether or not the putative promnesic drug S9977 (1,3,7-trimethyl 8-[3-(4-diethylaminocarbonyl-1-piperazinyl) 1-propyl]-3,7-dihydro (1H)2,6-purinedione hydrochloride) affects peripheral adrenergic neurotransmission.2. Rings of canine saphenous veins (without endothelium) were suspended for isometric tension recording in conventional organ chambers filled with modified Krebs-Ringer bicarbonate solution. The adrenergic nerve endings were activated with electrical impulses (9 V, 2 msec, 0.25–8 Hz).3. At 10⁻⁵ M, S9977 significantly reduced the contraction to 0.25, 0.5 and 1 Hz. The compound did not affect the response to higher stimulation frequencies or to exogenous noradrenaline. The inhibitory effect of S9977 was prevented by methiothepin, and not affected by atropine or 8-phenyltheophylline.4. Helical strips of canine saphenous veins were incubated with [³H]noradrenaline and suspended for superfusion and isometric tension recording. Under basal conditions, S9977 (10⁻⁴ M) augmented, the total ³H-overflow which was due mainly to an augmented overflow of [³H]deoxyphenylglycol (DOPEG); the extraneuronal metabolites 3,4-dihydromandelic acid (DOMA) and 3-methoxy-4-hydroxymandelic acid (VMA) were reduced.5. During electrical stimulation of the adrenergic nerves, S9977 (10⁻⁴ M) augmented the total ³H-overflow but reduced the contractile response; the evoked overflow of [³H]noradrenaline was not significantly affected.6. These experiments suggest that S9977 the displacement of noradrenaline from the adrenergic varicosities; most of the displaced transmitter is metabolized by intraneuronal monoamine oxidase before reaching the junctional cleft. In addition, S9977 exerts an inhibitory effect on the extraneuronal metabolism of catecholamines. S9977 does not inhibit the exocytotic release of the adrenergic neurotransmitter.     

Stimulation of endothelial prostacyclin production plays no role in endothelium-dependent relaxation of the pig aorta.

Stimulation of prostacyclin production by pig aortic endothelial cells adhering to microcarrier beads superfused in columns, or 3H release from cells prelabelled with [3H]-arachidonate, was studied in response to a range of agents that induce endothelium-dependent vascular relaxation. Bradykinin, adenosine triphosphate (ATP) and ionophore A23187 each stimulated release of prostacyclin from unlabelled cells and of 3H from prelabelled cells but acetylcholine did not. Bradykinin induced a parallel, dose-dependent increase in 3H release and 86Rb efflux, measured simultaneously from columns of aortic endothelial cells preloaded with 86Rb and [3H]-arachidonate. The rank-order of effectiveness at inducing both 3H and 86Rb release, measured simultaneously from columns of aortic endothelial cells prelabelled with 86Rb and [3H]-arachidonate and challenged with maximal doses of each agonist, was: A23187 greater than bradykinin greater than ATP. The similarity between agonist-induced 3H release (from cells prelabelled with [3H]-arachidonate) and 86Rb efflux indicates that a common mechanism may be responsible, and the effectiveness of ionophore A23187 suggests that a rise in the intracellular level of calcium may be involved. The lack of effect of acetylcholine on release of prostacyclin from unlabelled cells or of 3H from cells prelabelled with [3H]-arachidonate provides further evidence that acetylcholine acts on endothelial cells by a mechanism that does not involve calcium mobilisation. Although bradykinin, ATP and ionophore A23187 each induced release of prostacyclin from aortic endothelial cells, prostacyclin did not relax the pig aorta. Furthermore, endothelium-dependent relaxation was unaffected by pretreating aortic strips with aspirin. It therefore appears that neither prostacyclin nor any other cyclo-oxygenase product mediates endothelium-dependent relaxation of the pig aorta.     

Adenosine enhancement of adrenergic neuroeffector transmission in guinea-pig pulmonary artery.

1. Adenosine and its derivatives N6-[(R)-1-methyl-2-phenylethyl]adenosine (R-PIA) or 5'-N-ethyl-carboxamideadenosine (NECA) enhanced nerve-induced contractile responses and augmented the basal smooth muscle tone in transmurally stimulated isolated strips of the guinea-pig pulmonary artery. 2. Adenosine, R-PIA and NECA enhanced contractile responses induced by noradrenaline, whereas N6-[(S)-1-methyl-2-phenylethyl]-adenosine (S-PIA) was virtually inactive. 3. Adenosine, R-PIA and NECA inhibited the nerve stimulation evoked release of [3H]-noradrenaline. However, the total release of [3H]-noradrenaline during the periods of NECA application was increased. 4. The nucleoside effects were blocked by the adenosine receptor antagonist 8-p-sulphophenyltheophylline. 5. 8-p-Sulphophenyltheophylline inhibited nerve-induced contractions and lowered basal muscle tone in preparations not having received any exogenous purines. 6. It is suggested that the observed stimulatory effects on muscle tone and on contractile responses to transmural nerve stimulation are mainly due to action at postjunctional stimulatory A1 adenosine receptors. In addition, actions at prejunctional inhibitory A1 and stimulatory A2 adenosine receptors are evident in this preparation.     

Influences of verapamil, X-537A, A-23187 and adenosine 3',5'-cyclic monophosphate on release of 5-hydroxytryptamine from rat brain slices.

Influences of verapamil, X-537A, A-23187 and cyclic-AMP on the release of [3H]-5-HT taken up into rat brain slices, were examined. Incubation with 40 mM KCl induced tritium release which was dependent on the presence of Ca2+. Verapamil, which blocks Ca2+ influx in excitable tissues, decreased potassium-induced release of 5-HT. Tritium release induced by ionophore X-537A was not dependent on extracellular Ca2+ while that induced by A-23187 required Ca2+. Cyclic-AMP, dibutyryl cyclic-AMP and theophylline did not significantly stimulate 5-HT release either in the presence or absence of Ca2+.     

Altered characteristics of [3H]dopamine release from superfused slices of corpus striatum obtained from rats receiving ethanol in vivo

Ethanol, 50 mM, in vitro inhibited the release of [3H]dopamine ([3H]DA) induced by depolarisation with 40 mM K+ from slices of corpus striatum of the rat. In contrast, the release of [3H]DA induced by the Ca2+ ionophore (A23187) was enhanced by the presence of ethanol in vitro. When similar preparations were obtained from brains of rats which had received ethanol in vivo chronically by inhalation for 5-7 days the characteristics of release of [3H]DA were altered. Thus, the inhibitory effect of ethanol in vitro on release induced by K+-depolarisation was lost, as was the enhancing effect of ethanol on the release induced by A23187. When release of [3H]DA was studied in the absence of added ethanol the fraction of stored 3H released either by K+-depolarisation or by A23187 was increased in the preparations from animals which had received ethanol in vivo. Similar changes in release induced by A23187, though of lesser magnitude, could be seen in rats which had received ethanol acutely (3 g kg-1 i.p.; 30 min). An even greater fraction of [3H]DA was released by A23187 in preparations from rats which had been made physically dependent on ethanol. These changes in the release characteristics of [3H]DA were still apparent in animals undergoing a physical syndrome of withdrawal from ethanol. The results are discussed in relation to the cellular basis for the development of tolerance to and dependence on ethanol.     

Effects of adenosine on 45Ca uptake and [3H]acetylcholine release in synaptosomal preparation from guinea-pig ileum myenteric plexus

The effects of adenosine on acetylcholine (ACh) release and calcium uptake were examined in a synaptosomal fraction prepared from guinea-pig ileum myenteric plexus-longitudinal muscle. A high concentration of potassium (40 mM) and electrical pulses (ES:10Hz) caused a marked increase in the output of [3H]ACh from [3H]choline-preloaded crude synaptosomes. This [3H]ACh output was calcium- and temperature-dependent. Adenosine reduced the high potassium-induced release significantly, and the electrically stimulated release completely. When the preparation was depolarized by high potassium or electrical pulses, the 45Ca uptake by synaptosomes was significantly enhanced. The uptake of 45Ca induced by high potassium was significantly reduced and that induced by electrical stimulation was completely abolished by adenosine. From these results, it may be suggested that adenosine inhibits neurotransmitter release by suppressing the presynaptic influx of calcium ion during depolarization of the cholinergic nerve terminals in guinea-pig ileum.     

Adenosine modulation of adrenergic neurotransmission in the human fallopian tube

The effects of adenosine and adenosine analogues on nerve-induced contractile responses and [3H]noradrenaline ([3H]NA) release, were studied in the isthmic part of human oviducts. Adenosine and L-N6-phenylisopropyladenosine (L-PIA) could enhance neurogenic contractile responses in preparations obtained mainly in the proliferative phase. At higher concentrations, adenosine derivatives inhibited contractile responses to nerve stimulation in both proliferative and secretory phase, with the potency order: 5'-N-ethylcarboxamideadenosine (NECA) greater than or equal to L-PIA much greater than D-PIA. This indicated actions at both stimulatory A1- and inhibitory A2-receptors. Adenosine, L-PIA and NECA but not D-PIA inhibited [3H]NA release during nerve stimulation. The relative potency order for the prejunctional inhibition was compatible with an action at A1-receptors. Furthermore, adenosine was found to modulate nerve-induced contractions via postjunctional stimulatory A1- and inhibitory A2-like receptors. The postjunctional effects may be influenced by cyclic hormonal changes. The adenosine antagonist 8-p-sulfophenyltheophylline (PS?T) reversibly antagonized the stimulatory and inhibitory effects by adenosine and analogues.     

Prejunctional modulation of acetylcholine release from the skeletal neuromuscular junction: link between positive (nicotinic)- and negative (muscarinic)-feedback modulation.

1. Presynaptic receptor-mediated modulation of stimulation-evoked [3H]-acetylcholine[( 3H]-ACh) release from the neuromuscular junction was studied in the region of the mouse hemidiaphragm which contains the motor endplates, and which can easily be loaded with [3H]-choline. This method made it possible to detect exclusively the [Ca2+]0-dependent, quantal release of [3H]-ACh in response to axonal stimulation. 2. Atropine enhanced, and non-depolarizing muscle relaxants [+)-tubocurarine, pancuronium and pipecuronium) reduced, the release of [3H]-ACh evoked by high frequency trains of stimulation (50 Hz, 40 shocks) of the phrenic nerve. The effect of (+)-tubocurarine was frequency-dependent as at 5 Hz (40 shocks) it was less effective than at 50 Hz. The resting release of [3H]-ACh was not affected by these compounds. These findings indicate that ACh released into the synaptic gap by axonal firing reaches a concentration sufficient to influence its own release by a prejunctional effect. 3. The anticholinesterase, physostigmine sulphate, enhanced the release of [3H]-ACh in a concentration-dependent manner. This effect was mediated via prejunctional nicotinic receptor stimulation: (+)-tubocurarine, pancuronium and pipecuronium completely prevented the effect of physostigmine. 4. When the prejunctional nicotinic and muscarinic receptors were stimulated by a high concentration of extracellular ACh which had accumulated in the junctional gap in the presence of physostigmine, atropine did not influence the evoked release of [3H]-ACh. However, when the effect of endogenous ACh on nicotinic receptors was prevented by (+)-tubocurarine, atropine enhanced the release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prejunctional modulation of the nitrergic innervation of the canine ileocolonic junction via potassium channels.

1. The effects of different K+ channel blockers were studied on nitric oxide (NO)-mediated non-adrenergic non-cholinergic (NANC) relaxations in the canine ileocolonic junction. 2. The non-selective blockers of K+ channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA) and the blocker of large conductance Ca(2+)-activated K+ channels, charybdotoxin, potently enhanced the NANC relaxations induced by low frequency stimulation. The blocker of small conductance Ca(2+)-activated K+ channels, apamin, had no effect on electrically-induced NANC relaxations. 3. NANC nerve-mediated relaxations induced by adenosine 5'-triphosphate (ATP), acetylcholine (ACh) and gamma-aminobutyric acid (GABA) were significantly enhanced by 4-AP and charybdotoxin but not by apamin. TEA significantly enhanced the NANC relaxations in response to GABA and ATP while that in response to ACh was abolished. 4. None of the K+ channel blockers had an effect on the dose-response curve to NO, on the noradrenaline-induced contraction or on the relaxation to nitroglycerine (GTN). 5. From these results we conclude that inhibition of prejunctional K+ channels increases the nitrergic relaxations induced by electrical and chemical receptor stimulation of NANC nerves and thus suggests a regulatory role for these prejunctional K+ channels in the release of NO from NANC nerves in the canine ileocolonic junction.     

Differential effects of Ca antagonists on the noradrenaline release and contraction evoked by nerve stimulation in the presence of 4-aminopyridine.

We previously reported that verapamil, nicardipine and diltiazem inhibited both neurotransmitter release and contraction evoked by transmural nerve stimulation (TNS) in the canine saphenous vein. To evaluate whether the three Ca antagonists act on the nerve endings by inhibiting Ca2+ influx, the effects of the three antagonists were studied in the presence of 4-aminopyridine (4-AP) 3 X 10(-4) M on the TNS-evoked tritium overflow and contraction of canine saphenous veins preloaded with [3H]-noradrenaline. 4-AP increased both tritium overflow and contraction evoked by TNS, but did not enhance the contraction induced by exogenous noradrenaline (10 nmol). In the veins pretreated with 4-AP, verapamil (3 X 10(-5) M) and nicardipine (10(-5) M and 3 X 10(-5) M) caused no significant effects on the TNS-evoked tritium overflow, but they still inhibited the contraction. Diltiazem (10(-5) M and 3 X 10(-5) M) significantly inhibited both responses to TNS in the veins pretreated with 4-AP, the effects being nearly equipotent to those in the absence of 4-AP. The (-)-cis isomer of diltiazem (10(-5) M and 3 X 10(-5) M), which is about 100 times less potent than diltiazem in inhibiting Ca2+ influx, inhibited both responses to TNS in the presence of 4-AP to almost the same degree as diltiazem. When 4-AP was added after the Ca antagonists, it reversed the TNS-evoked tritium overflow inhibiting actions of verapamil (3 X 10(-5) M) and nicardipine (3 X 10(-5) M) much more effectively than that of diltiazem (3 X 10(-5) M). Tetracaine (4 X 10(-6) M) significantly inhibited the TNS-evoked tritium overflow and contraction, which were unaffected by 4-AP. Sodium salicylate (10(-2) M) failed to modify the inhibition of TNS-evoked tritium overflow following diltiazem (3 X 10(-5) M), but it enhanced that of tetracaine (4 X 10(-6) M). Verapamil but not diltiazem and nicardipine significantly increased the spontaneous tritium overflow from veins pretreated with 4-AP. The present study together with previous results suggests that diltiazem but not verapamil and nicardipine may inhibit the TNS-evoked neurotransmitter release through an action other than inhibition of Ca2+ influx into the adrenergic nerve endings, allowing an inhibition of the resulting contraction.     

Influence of stimulation on Ca(2+) recruitment triggering [3H]acetylcholine release from the rat motor-nerve endings

The influence of rat phrenic nerve stimulation frequency (5-50 Hz) and of pulse duration (0.04-1 ms) on Ca(2+) mobilization triggering [3H]acetylcholine release was investigated. The P-type voltage-dependent Ca(2+) channel (VDCC) blocker, omega-agatoxin IVA (100 nM), decreased [3H]acetylcholine release evoked by pulses of 0. 04-ms duration delivered at 5 Hz frequency. When the stimulus pulse duration was increased to 1 ms (5 Hz frequency) or the stimulation frequency to 50 Hz (0.04-ms duration), inhibition of [3H]acetylcholine release became evident after blockade of L-type VDCC, with nifedipine (1 microM), and/or depletion of thapsigargin-sensitive internal stores. The inhibitory effect of thapsigargin (2 microM) was still observed in Ca(2+)-free medium. Neither omega-conotoxin GVIA (1 microM) nor omega-conotoxin MVIIC (150 nM) modified neurotransmitter release. The results suggest that, depending on the stimulus paradigm, both internal (thapsigargin-sensitive) and external (either P- or L-type channels) Ca(2+) pools can be mobilized to promote acetylcholine release from motor nerve terminals.