Differential effects of nifedipine,verapamil, and diltiazem on noradrenaline-induced contractions,adrenergic transmitter release,and alpha-adrenoceptor binding in the female rabbit urethra

Summary In order to study differences in action between Ca²⁺-entry blockers on smooth muscle and peripheral nerves, the effects of nifedipine, verapamil, and diltiazem on noradrenaline (NA)-induced contractions and electrically evoked release of ³H-NA were investigated in the female rabbit urethra. In addition, possible influences of Ca²⁺-entry blockers on alpha-adrenoceptors were studied with radioligand binding technique. Exposure to Ca²⁺-free medium completely abolished the contractile response to 1 M NA in the rabbit urethra, indicating that the contraction was entirely dependent on influx of extracellular Ca²⁺. The Ca²⁺-entry blockers inhibited the NA-induced contractions in the following order of potency: nifedipine>verapamildiltiazem. In contrast to nifedipine and diltiazem, which produced a maximum inhibition of between 50 and 60%, verapamil was able to abolish the contractile responses to NA. The electrically evoked efflux of ³H-NA was decreased by diltiazem and increased by verapamil, whereas nifedipine failed to alter the ³H-NA efflux. Only verapamil was effective in inhibiting specific ³H-DHE binding to a crude membrane preparation of the rabbit bladder base and urethra, and the inhibition appeared to be of the competitive type. It is suggested that the effects of verapamil on electrically evoked efflux of ³H-NA and on NA-induced contractions can be partly explained by blockade of pre- and post-junctional alpha-adrenoceptors. The failure of nifedipine and diltiazem to abolish the NA-induced contraction might indicate the existence of different Ca²⁺-entry pathways in urethral smooth muscle.     

Unrepeatable extracellular Ca-dependent contractile effects of cyclopiazonic acid in rat vascular smooth muscle

Cyclopiazonic acid (CPA), a specific reversible inhibitor of Ca²⁺-pumps in sarcoplasmic reticulum, causes a slowly developing and subsequently diminishing characteristic contraction in endothelium-denuded rat vascular smooth muscle. We recently found that CPA-induced contractions were not completely repeatable in endothelium-denuded rat aorta and superior mesenteric artery. 10 µM CPA-induced contractions expressed as a percentage of 80 mM KCl-induced contraction were significantly decreased from 51.4 ± 5.7% to 11.8 ± 2.6% (P < 0.0001) upon the second application in endothelium-denuded rat aorta, and this was not due to any irreversible cytotoxic effects of CPA. The decrease of CPA-induced contractile responses upon the second application was dependent on both types of blood vessels and doses of CPA upon the first application. CPA upon the second application in Ca²⁺-containing solutions did induce its characteristic contractions in the rings pretreated with Ca²⁺-free solutions or Ca²⁺ entry blockers before and during its first application, suggesting that capacitative mode of Ca²⁺ influx during the application of CPA might be responsible for the diminishment of contractions upon the second application. These data suggest that CPA by inducing a transient rise in cytosolic Ca²⁺ level might cause a long-lasting upregulation of Ca²⁺ extrusion across the plasma membrane in vascular smooth muscle cells and thus accelerate Ca²⁺ efflux over a prolonged period, leading to unrepeatable contractile effects of CPA. Such long-lasting upregulation of Ca²⁺ extrusion may contribute to the regulation of excitability of vascular smooth muscle cells and protect the cells against excitotoxic injury.     

Role of extra- and intracellular calcium in the contractile action of agonists in the guinea-pig ileum

Summary The effects of Ca²⁺-channel blockers (nifedipine and verapamil), EGTA, caffeine or the removal of external Ca²⁺ on the contractile action of different agonists and transmural electrical stimulation were examined in isolated segments of the proximal and terminal part of the guinea-pig ileum. The effects of agonists and nerve stimulation on membrane potential were also studied by means of the sucrose gap method. Acetylcholine-elicited contractions in both parts and noradrenaline-as well as histamine-induced contractions in the terminal part of the ileum were composed of an initial phasic and a sustained tonic component. Single pulse transmural nerve stimulation elicited smooth muscle twitches, whereas addition of CaCl₂ to the tissue bath containing Ca²⁺-free and high-K⁺ medium elicited a sustained contraction. Both verapamil and nifedipine were more potent in inhibiting the tonic phase of the responses to the agonists or CaCl₂ than inhibiting the phasic contractions elicited by transmural nerve stimulation, acetylcholine or noradrenaline. The excitatory junction potentials (e j.p.s.) as well as smooth muscle twitches were reduced only by high nifedipine concentrations. The effects of acetylcholine on membrane potential and input membrane resistance were affected minimally by the omission of extracellular Ca²⁺, while the contractions gradually disappeared on repetitive agonist application in the absence of external Ca²⁺ and were blocked by caffeine preexposure. In Ca²⁺-free solution noradrenaline and histamine partially reduced each other's motor effect, while neither of them changed the contractile action of acetylcholine, yet the contraction induced by noradrenaline was prevented and that of histamine significantly reduced by preexposure to acetylcholine. These results suggest that the potency of acetylcholine to release Ca²⁺ from its caffeine-sensitive intracellular stores is much higher than that of histamine and noradrenaline. Send offprint requests to V. Bauer at the above address     

Lead Induces Endothelium‐ and Ca2+‐Independent Contraction in Rat Aortic Rings

Abstract: The contractile effect of lead on rat aortic rings was examined. Lead (0.1–3.1 mM) elicited concentration‐dependent but endothelium‐independent contractions, which were unaffected by prazosin (1 μM). The contractile effects of lead were similar when the aortic rings were bathed either in the absence or presence of external Ca²⁺. Lanthanum (1 mM) but not verapamil (1 μM) inhibited the lead contractions; hence non‐L‐calcium channels are involved in such effect. In addition, lead induced contractions on aortic rings incubated in Ca²⁺‐free EGTA‐containing solution for 70 min., an experimental condition in which intracellular Ca²⁺‐stores are depleted. Finally, the contractile effect of lead was not modified by calphostin C (an inhibitor of protein kinase C). In conclusion, the present results suggest that in rat aorta, the lead‐induced contraction is independent of extra‐ and intracellular calcium stores. In addition, the effect of lead is independent of either catecholamines or protein kinase C. It is likely that in rat aorta, lead enters into the smooth muscle cells through non‐L‐calcium channels, and when acting like calcium on the contractile machinery it produces contraction. The differences observed between our results and those obtained by other authors may indicate that the mechanism of the contractile effect of lead varies among the different blood vessels.     

The involvement of protein kinase C and tyrosine kinase in vanadate-induced contraction

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM LaCl₃ and significantly inhibited by 10 μM verapamil and 1 μM nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular Ca²⁺ concentration, and the influx of extracellular Ca²⁺ was mediated through voltage-dependent Ca²⁺ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin (1 μM) and sodium nitroprusside (1 μM) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosine kinase are involved in the vanadate-induced contraction which required the influx of extracellular Ca²⁺ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.     

A possible effect of sulfhydryl reagents on the contractile activity of the rat detrusor muscle

We aimed to investigate the effect of sulfhydryl (SH) inactivating agents, ethacrynic acid and N-ethylmaleimide, on the contractile activity of rat detrusor muscle. Wistar Kyoto rats weighing 150-250 g were anaesthetized with ketamine and bled to death. The urinary bladders were surgically removed and detrusor strips were mounted under 0.5 g tension in organ baths. The responses were recorded with isotonic transducers on polygraph paper. After an equilibrium period, the tissues were contracted by electrical field stimulation, acetylcholine, ethacrynic acid or N-ethylmaleimide and the effects of l-cysteine, glutathione, verapamil, Ca²⁺-free solution, sodium nitroprusside or atropine were then examined on these contractions. Verapamil, Ca²⁺-free solution or atropine significantly reduced the contractions elicited by electrical field stimulation and acetylcholine whereas l-cysteine, glutathione or sodium nitroprusside had no effect on the contractions in response to these stimuli. l-Cysteine, glutathione, verapamil or Ca²⁺-free solution significantly inhibited the contractions induced by ethacrynic acid or N-ethylmaleimide. Sodium nitroprusside slightly inhibited only the contraction induced by ethacrynic acid but not that with N-ethylmaleimide. Atropine has no action on the contractions in response to these SH reagents. These findings suggest that SH reagents may play a role in the contractile activity of rat detrusor muscle and this action seems to be related to the gating of Ca²⁺ channels. Further experiments are needed to determine the cellular mechanism(s) of action by which these SH reagents act on the detrusor smooth muscle.     

Effects of synthetic analogues of poly-APS on contractile response of porcine coronary arteries

APS12-2 and APS3 are synthetic analogues of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. The aim of the present study was to determine the possible direct contractile effects of these two synthetic molecules on coronary arteries, in order partly to explain hemodynamic and cardiotoxic effects of APS12-2 previously observed in in vivo studies and to reveal possible adverse effects on the organism in the case of their clinical use. In contrast to APS3, APS12-2 caused a concentration-dependent vascular smooth muscle contraction of isolated porcine coronary ring preparations in a concentration-range from 1.36 to 13.60 μM. Lanthanum chloride (5 mM) and verapamil (10 μM) completely abolished the APS12-2 evoked contraction of the coronary rings. Pre-incubation with indomethacin (10 μM) had no effect on the contractile responses of coronary ring preparations. These results indicate that APS12-2 contracts vascular smooth muscle in a concentration-dependent manner, due to an increase of Ca²⁺ influx through the voltage-gated Ca²⁺ channels. Our data show for the first time that APS12-2 induces concentration-dependent contraction of coronary ring preparations, which may contribute to the cardiotoxic effects of APS12-2, in addition to hyperkalemia.     

Inhibition of calcium influx in rabbit aorta by nicardipine hydrochloride (YC-93)

The Ca²⁺ antagonistic effects of a potent vasodilator, nicardipine hydrochloride [2-(N-benzyl-N-methylamino)ethyl methyl 2,6-dimethyl-4-(m-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate hydrochloride], were investigated. Both nicardipine and verapamil, neither of which exhibited a significant effect on cellular ⁴⁵Ca²⁺ uptake by the rabbit aorta in normal buffer containing 2.68 mM KCl as measured by the “lanthanum method”, inhibited the enhancement of ⁴⁵Ca²⁺ uptake induced by equimolar replacement of 120 mM NaCl by KCl. Nicardipine with an ic₅₀ (the concentration required for 50 per cent inhibition) of 10^?9 M was sixty-eight times more potent than verapamil in the inhibition of KCl-induced ⁴⁵Ca²⁺ uptake. Nicardipine also repressed KCl-induced contraction of the aorta and its ic₅₀ was about 2 × 10^?9M, eighty-five times smaller than that of verapamil. These comparative studies on the relation between cellular ⁴⁵Ca²⁺ uptake and KCl-induced contraction of the rabbit aorta suggest that nicardipine causes relaxation of smooth muscle mainly by interfering with Ca²⁺ influx.     

Different Mechanism of Relaxation Induced by Aporphine Alkaloids in Rat Uterus

Abstract— We have examined the uterine relaxant action of three aporphine molecules (S-glaucine, S-boldine and R-apomorphine) in two experimental conditions, with and without calcium in the bathing solution, and compared these effects with those obtained with the calcium antagonists verapamil and diltiazem. The present study shows that the alkaloids relax the uterine muscle but with different mechanisms of action. In Ca²⁺-containing solution all three alkaloids relaxed the uterus previously contracted by KCl or acetylcholine, but in Ca²⁺-free medium only R-apomorphine was able to relax oxytocin-induced contraction. The calcium antagonists, verapamil and diltiazem, relaxed KCl- or acetylcholine-induced contraction in Ca²⁺-containing solution, whereas they only relaxed oxytocin-induced contraction in Ca²⁺-free medium at much higher doses. These results suggest that glaucine and boldine behave as specific calcium entry blockers without affecting the contractile machinery or intracellular Ca²⁺ levels as apomorphine does. The absolute configuration (S-glaucine and S-boldine vs R-apomorphine) may account for this different action.     

阿托品对兔胸主动脉平滑肌收缩和细胞增殖的影响

用兔胸主动脉条研究Atr,Ver对CaCl₂,Atr对KCI量—效反应的影响。观察到Atr和Ver能抑制2种激动剂所致兔主动脉条的收缩,量一效曲线右移,最大反应降低,其pD₂值分别为4.4和5.8。两药也能明显抑制NE依内Ca²⁺性收缩,Atr对NE依外Ca²⁺性收缩影响较小,说明Atr主要对细胞外Ca²⁺经PDC所致的收缩有抑制作用。在兔ASMC培养中,有Ca²⁺时,Atr抑制ASMC增殖,无Ca²⁺时,Atr 20.6～185.2 μmol/L表现刺激增殖,555.7～1666.7 μmol/L则抑制MSMC增殖,说明Atr对ASMC作用也与Ca(2+)有关。     

Participation of both intracellular free Ca2+ and protein kinase C in tonic vasoconstriction induced by prostaglandin F2α

The roles of intracellular free Ca²⁺ and protein kinase C in the tonic contraction induced by prostaglandin were studied. Prostaglandin F_2α induced tonic contraction of rat thoracic aorta in both control and Ca²⁺-free solution. Close correlations were observed between the contractile response of aortic strips and the changes in intracellular free Ca²⁺ concentration in vascular smooth muscle cells assessed with the fluorescent Ca²⁺ indicator fura 2, both in control and Ca²⁺-free solutions. Prostaglandin F_2α also enhanced the production of inositol 1,4,5-triphosphate in vascular smooth muscle cells before the rise of the intracellular free Ca²⁺ concentration. Moreover, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, inhibited the tonic contractions induced by PGF_2α and 12-O-tetradecanoyl phorbol-13-acetate, a direct activator of protein kinase C, at similar concentrations. These results suggest that both intracellular free Ca²⁺ and protein kinase C participate in prostaglandin F_2α-induced tonic contraction.     

In vitro pharmacological profile of SK-896, a new human motilin analogue

SK-896 ([Leu(13)]motilin-Hse) is a new human motilin analogue synthesized by Escherichia coli using a biotechnological method. We investigated the binding of SK-896 to motilin receptors and the contractile effect of SK-896 on smooth muscle preparations isolated from the gastrointestinal tract and various regional organs in order to clarify its in vitro pharmacological profile. SK-896 inhibited the binding of (125)I-human motilin to rabbit gastroduodenal motilin receptors with the same potency as unlabeled human motilin. The IC(50) values of SK-896 and human motilin were 3.5 +/- 1.5 and 3.1 +/- 1.8 nmol/l, respectively. The K(d) of human motilin was 3.0 +/- 1.5 nmol/l, and the Ki of SK-896 was 3.4 +/- 1.5 nmol/l. SK-896 induced contraction of smooth muscle preparations isolated from rabbit duodenum in a concentration-dependent manner. However, there was no effect of SK-896 on duodenal preparations isolated from the dog and the rat. SK-896 thus exhibited species specificity in its contractile effect. We next investigated the effect of SK-896 on various smooth muscle preparations isolated from rabbit gastrointestinal tract, trachea, bladder, gallbladder, uterus, vas deferens and artery. Results showed that SK-896 induced contraction of smooth muscle preparations isolated from gastrointestinal tract, with potencies in the order duodenum > gastric pylorus = jejunum = descending colon > ascending colon >/= ileum. However, there was no effect of SK-896 on smooth muscle preparations from gastric fundus and other regional organs. SK-896 thus exhibited regional specificity in its contractile effect. Moreover, the effects of SK-896 on smooth muscle preparations from rabbit duodenum were the same as those of human motilin, and were not inhibited by pretreatment with tetrodotoxin and atropine but were inhibited by verapamil. These findings indicate that SK-896 has the same pharmacological profile as human motilin. They suggest that SK-896 acts on gastrointestinal smooth muscle isolated from rabbit directly and specifically.     

Facilitation of the vasorelaxant action of calcium antagonists by basal nitric oxide in depolarized artery

We investigated the effect of NO/cyclic GMP pathway on the action of calcium antagonists (isradipine, nisoldipine, lacidipine, verapamil, diltiazem) in rat aorta exposed to 100 mM KCl. For this purpose constitutive NO synthase was blocked by using 100 M N-nitro-l-arginine (l-NNA).The steady-state contractile response evoked by 100 mM KCl was enhanced when the basal NO release had been blocked. The combined effects of basal NO release and calcium antagonists resulted in an inhibition greater than additive. Concentrations of calcium antagonists producing 50% inhibition of contraction were about 3-fold lower in the presence of the basal NO release than in its absence (P < 0.01). ⁴⁵Ca^2– influx stimulated by 100 mM KCl was not affected by the basal NO release, but was inhibited by isradipine and verapamil regardless of NO blockade. Thus, the facilitation of the action of calcium antagonists by NO/cyclic GMP pathway seemed not to be accompanied by a modification of their action on L-type calcium channels. To confirm this, we measured the contractile tension and the calcium signal in fura-2 loaded rings, pretreated with either verapamil or verapamil plus 8-bromo cyclic GMP (BrcGMP), and further exposed to increasing concentrations of extracellular Ca^2– ([Ca²⁺]_o) in 100 mM KCl solution. The increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) evoked by increasing ([Ca²⁺]_o) in rings pretreated with verapamil alone was not different from rings pretreated with verapamil plus BrcGMP. In contrast, the [Ca²⁺]_o-contraction curve was significantly shifted to the right in rings pretreated with verapamil plus BrcGMP.These results show that the NO/cyclic GMP pathway facilitates the inhibitory effect of calcium antagonists on 100 mM KCl-evoked contraction. This phenomenon is not related to a modification of calcium channel blockade, but could result from the reduction of the sensitivity of contractile machinery to Ca²⁺ by cyclic GMP.     

High K+-induced contraction requires depolarization-induced Ca2+ release from internal stores in rat gut smooth muscle

Aim:

Depolarization-induced contraction of smooth muscle is thought to be mediated by Ca²⁺ influx through voltage-gated L-type Ca²⁺ channels. We describe a novel contraction mechanism that is independent of Ca²⁺ entry.

Methods:

Pharmacological experiments were carried out on isolated rat gut longitudinal smooth muscle preparations, measuring isometric contraction strength upon high K⁺-induced depolarization.

Results:

Treatment with verapamil, which presumably leads to a conformational change in the channel, completely abolished K⁺-induced contraction, while residual contraction still occurred when Ca²⁺ entry was blocked with Cd²⁺. These results were further confirmed by measuring intracellular Ca²⁺ transients using Fura-2. Co-application of Cd²⁺ and the ryanodine receptor blocker DHBP further reduced contraction, albeit incompletely. Additional blockage of either phospholipase C (U 73122) or inositol 1,4,5-trisphophate (IP₃) receptors (2-APB) abolished most contractions, while sole application of these blockers and Cd²⁺ (without parallel ryanodine receptor manipulation) also resulted in incomplete contraction block.

Conclusion:

We conclude that there are parallel mechanisms of depolarization-induced smooth muscle contraction via (a) Ca²⁺ entry and (b) Ca²⁺ entry-independent, depolarization-induced Ca²⁺-release through ryanodine receptors and IP₃, with the latter being dependent on phospholipase C activation.     

Involvement of Calmodulin Kinase II in the Action of Sulphur Mustard on the Contraction of Vascular Smooth Muscle

Abstract: Sulphur mustard (SM) is an alkylating agent whose mechanism is not fully understood. To investigate the early action of SM, we examined the effect of SM on contraction of vascular smooth muscles. Phenylephrine (PE)‐induced contraction was reduced by SM, but only marginally by 70 mM KCl^‐. Additional reduction was induced by nifedipine in SM‐treated arteries. In the absence of extracellular Ca²⁺, contraction of arteries by PE was reduced, which was fully recovered by addition of 2 mM Ca²⁺. However, recovery was attenuated by pre‐treatment with SM. The effect of SM on contraction by PE was not influenced by pre‐ and post‐treatment with Phorbol 12, 13‐dibutyrate. Calmodulin kinase II (CaMKII) was implicated as being responsible for the action of SM, because the contractile mechanisms of vascular smooth muscle via both Ca²⁺‐calmodulin‐myosin light chain kinase axis and protein kinase C‐proline‐rich tyrosine kinase axis were not related to the action of SM. Elevation of phosphorylated CaMKII level by Ionomycin or PE was attenuated by treatment of SM on western blot. CaMKII may be a candidate target molecule of SM in early stage contraction of vascular smooth muscle.     

Effects of the calcium channel facilitator, CGP 28,392, on different modes of contraction in smooth muscle of rabbit and rat aortae and guinea-pig taenia caeci.

Effects of a Ca2+ channel facilitator, CGP 28,392, on smooth muscle contractions were examined in order to delineate characteristics of Ca2+ channels in rabbit and rat aortae and guinea-pig taenia caeci. Application of increasing concentrations of KCl induced contractile responses in these smooth muscles and CGP 28,392 shifted the concentration-response curve for KCl to the left. The maximum response was also increased in rat aorta and guinea-pig taenia. CGP 28,392 also shifted the concentration-response curves for noradrenaline in rat aorta and for histamine in taenia to the left and increased the maximum response in rat aorta. However, the corresponding curve for noradrenaline in rabbit aorta was not affected by CGP 28,392. The sustained contractions induced by KCl were inhibited by cumulative application of verapamil in these smooth muscles. Pretreatment of the muscle with CGP 28,392 decreased the inhibitory effect of verapamil. The noradrenaline-induced contraction in rat aorta and the histamine-induced contraction in taenia were also inhibited by verapamil, and CGP 28,392 antagonized the effect of verapamil. The noradrenaline-induced contraction in rabbit aorta was only slightly inhibited by verapamil, and CGP 28,392 did not modify the effect of verapamil. In these smooth muscles, cumulative application of Ca2+ to the Ca2+-depleted, KCl-treated muscle induced contraction, and the concentration-response curve for Ca2+ was shifted to the left by CGP 28,392 and to the right by verapamil. The concentration-response curves for Ca2+ in Ca2+-depleted, noradrenaline-treated rabbit and rat aortae and in Ca2+-depleted, histamine-treated taenia were also shifted to the left by CGP 28,392 and to the right by verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of spontaneous contraction in the circular smooth muscles of cat ileum

We investigated the characteristics of spontaneous contraction in feline ileal circular smooth. Smooth muscle contractions were recorded by an isometric force transducer. The neurotoxin tetrodotoxin did not alter the spontaneous contraction of the circular muscles. Atropine or guanethidine also did not affect on the contraction. In Ca²⁺-free Krebs, the spontaneous contraction completely disappeared in the muscles. An L-type Ca²⁺ channel blockade with nimodipine decreased the amplitude of spontaneous contraction. A selective inhibitor of the sarcoplasmic reticulum Ca²⁺-ATPase, thapsigargin, had not no effect on spontaneous contraction within incubation time for 10min. However, phosphoinositide-specific phopholipase C inhibitor, U-73122, decreased the frequency of the contraction in circular smooth muscle These results suggest that the neuronal component, adrenergic or cholinergic innervation was not involved in spontaneous activity and that extracellular Ca²⁺ influx via an L-type Ca²⁺ channel may mediate the contractile amplitude in circular smooth muscles of cat ileum.     

Mode of action of cholecystokinin octapeptide on smooth muscles of stomach, ileum and gall bladder

Mechanical activity of preparations isolated from canine and guinea-pig stomach, ileum and gall bladder was recorded. At least 2 strips were cut out from each organ and investigated simultaneously in thermostatically controlled organ baths. Responses to acetylcholine (ACH) were used for comparison. Octapeptide of cholecystokinin (CCK-OP) at concentrations of 5 X 10(-11) M to 10(-8) M produced dose-dependent tonic concentrations in all muscle strips and showed a higher affinity but lower efficacy as compared to ACH. Atropine (10(-7) M 10(-5) M) had no effect on the CCK-OP responses in stomach and gall bladder muscle strips but it significantly decreased the CCK-OP responses in ileum muscle strips. Dibutyryl cyclic GMP (dbc GMP) at concentrations of 10(-5) M-5 X 10(-4) M did not change the ACH dose-response curves but shifted to the right in parallel to the control the dose-response curves for CCK-OP in all muscle preparations with pA2 values 5, 5.3, 7.2, and 6 for canine stomach and guinea-pig stomach, ileum and gall bladder, respectively. Michaelis-Menten's analysis suggested a competitive type of interaction of dbc GMP on CCK-OP contractile responses of guinea-pig ileum and gall bladder; at a higher concentration (5 X 10(-4) M) the antagonistic effect of dbc GMP in canine and guinea-pig stomach appeared to be a mixed or uncompetitive type. The data suggest that the contractile effects of CCK-OP in the iliac smooth muscle are caused by cholinergic and direct mechanisms whereas on the gall bladder and gastric smooth muscles by direct myogenic mechanisms only.     

Angiotensin II-induced increase in slowly exchanging 45Ca2+ in relation to contractile responses of rat and guinea-pig aorta

Summary To gain more information about sources of activator Ca²⁺ involved in the contraction of rat and guinea-pig aorta evoked by angiotensin II and their sensitivity to Ca ²⁺ entry blockers, measurement of slowly exchanging ⁴⁵Ca²⁺ was established. A more physiological procedure was used, replacing La³⁺- and EGTA- containing solutions by a normal Ca²⁺-containing buffer. It was demonstrated that the angiotensin 11-induced increase in slowly exchanging ⁴⁵Ca²⁺ in rat aorta was incompletely (by approximately 60%–70%) inhibited by the organic Ca²⁺ entry blockers nifedipine, verapamil and diltiazem and by other Ca⁺ entry blocking compounds like CoCl₂ and chlorpromazine. 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) was able to inhibit the angiotensin II-induced increase in ⁴⁵Ca²⁺ content completely, but this may be an intracellular storage effects. By contrast, the organic Ca²⁺ entry blockers completely inhibited that part of the angiotensin II-induced contraction of rat aorta which was dependent upon extracellular Ca²⁺.In guinea-pig aorta, the increase in ⁴⁵Ca²⁺ content elicited by angiotensin 11 could be completely suppressed by all compounds under study. The results of these experiments correlated well with data from the functional experiments in guinea-pig aorta. In both preparations the release of Ca ²⁺ from a rapidly as well as a slowly exchanging intracellular pool appears to contribute to the contractile response elicited by angiotensin 11. Offprint requests to P. N. M. van Heiningen at the above address     

Contraction Induced Either by Iso-osmolar or Hyper-osmolar Potassium-rich Solutions Influences Relaxant Responses to Pinacidil and Verapamil in Rat Isolated Aorta

Abstract— Relaxant responses of pinacidil and verapamil were studied in rat isolated aorta contracted by either iso-osmolar or hyper-osmolar potassium-rich solutions. Relaxant response profiles of pinacidil and verapamil in rat isolated aorta contracted by 124 mm K⁺ hyper-osmolar Krebs solutions showed marked reductions in inhibiting E_max values and substantial increases in corresponding IC50 values in comparison with results obtained at iso-osmolar conditions. Changes in the slopes of the fitted log concentration-relaxation curves were also observed, whereas pinacidil relaxation curves obtained after initial contraction induced by 30 mm K⁺ Krebs solutions were only slightly influenced by osmolarity, a definite decrease in E_max occurred for verapamil at hyper-osmolar conditions. Initial tension development was much slower and maximal tension lower when induced by 124 mm K⁺ in hyper-osmolar compared with iso-osmolar Krebs solutions. After incubation in Ca²⁺-deprived EGTA-containing Krebs solutions the maximal tension produced by 124 mm K⁺ iso-osmolar Krebs solution in rat aorta was nearly 95% reduced, whereas it was only reduced by 50% at hyper-osmolar conditions. Hyper-osmolarity as established by direct addition of potassium chloride to Krebs solutions in order to induce contraction in vascular smooth muscle could influence the in-vitro action profile, potency and intrinsic activity of the two vascular relaxant drugs.