Guanine nucleotide binding proteins may modulate gating of calcium channels in vascular smooth muscle. I. Studies with fluoride

Fluoride (F-), a known stimulator of G-proteins, was used to examine the relationship between G-proteins and calcium channels (CaC) in rat vascular smooth muscle (VSM). Treatment of isolated rat tail artery helical strips with F- (2.5-20 microM) produced a Ca++-dependent contraction. In the absence of added AlCl3, subthreshold NaF shifted the KCl, as well as the arginine vasopressin and norepinephrine concentration-related tension curves to the left. Nifedipine and verapamil, known CaC blockers, inhibited the NaF-related contraction. AlCl3 (20 microM), which is required for G-protein stimulation by F-, strikingly potentiated the contractile response to F-. The NaF-induced contraction was relaxed by 3-isobutyl-1-methylxanthine as well as by forskolin and by dibutyryladenosine-cyclic AMP, and the effect therefore may be independent of cAMP. 45Ca-uptake was elevated by NaF, and partially blocked by nifedipine and verapamil. NaF also inhibited the basal and forskolin-stimulated cAMP production, suggesting that F- stimulated the putative Gi in the intact VSM cells. NaF stimulated accumulation of IP in a concentration-dependent manner, indicating that F- stimulated the putative G-protein Gp which couples various receptors to hydrolysis of phosphoinositides and mobilization of Ca++. These results indicate that NaF-induced vasoconstriction is related to the opening of the CaC in the plasma membrane and perhaps a subsequent entry of the extracellular Ca++ into the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura 2

In rat aortic strips, muscle contraction was recorded simultaneously with cytosolic Ca++ level, which was indicated by the 500 nm fluorescence of Ca++ indicator, fura 2, due to excitation at either 340 nm (F340) or 380 nm (F380) and the ratio of F340 to F380 (R340/380). On the addition of 72.7 nM K+ or 1 microM norepinephrine, muscle contraction followed the increase in R340/380 (resulted from the increased F340 and the decreased F380). Cytosolic Ca++ concentrations of resting, 72.7 mM K+-stimulated and 1 microM norepinephrine-stimulated aortas were tentatively calculated as 228 +/- 25, 1784 +/- 154 and 1528 +/- 180 nM, respectively. Cumulative addition of K+ or norepinephrine induced concentration-dependent increase in both muscle tension and R340/380. However, norepinephrine induced greater contraction than K+ when both of these stimulants induced similar increase in R340/380. Addition of 10 mM tetraethylammonium and 1 microM Bay k8644 caused rhythmic contractions which followed the rhythmic changes in R340/380. EGTA decreased the muscle contraction and decreased R340/380. In Ca++-free solution, addition of 10 microM norepinephrine or 20 mM caffeine induced transient increase in both muscle tension and R340/380. Tension changes always were preceded by the fluorescent changes. Verapamil (10 microM) decreased both tension development and R340/380 in high K+- and norepinephrine-stimulated tissues. Sodium nitroprusside (1 microM) also decreased both tension and R340/380 in norepinephrine-stimulated tissues, whereas it decreased tension more strongly than R340/380 in high K+-stimulated tissues. These results indicate that vasoconstrictors and vasodilators may modulate smooth muscle contraction by changing the cytosolic Ca++ concentrations and also by changing the sensitivity of contractile elements to Ca++.     

Ca++ utilization in the constriction of rat aorta to stimulation of protein kinase C by phorbol dibutyrate

To determine the role of activated protein kinase C in vascular smooth muscle contraction, phorbol dibutyrate was used to stimulate this enzyme in order to evaluate the source(s) of Ca++ (10(-8) to 3 X 10(-6) M) elicited a concentration-dependent sustained contraction which was slow in onset but progressive in developed tension. The maximal contractile response induced by phorbol dibutyrate was only partly dependent on influx of extracellular Ca++ as shown by similar reductions (40%) produced by Ca++-free buffer, LaCl3 (1 mM) or nifedipine (10(-6) M). These data suggest that phorbol dibutyrate is able to open Ca++ channels which are sensitive to nifedipine blockade. However, unlike norepinephrine or K+-depolarization, phorbol dibutyrate evoked a slow 45Ca++ influx which occurred only after extended contact time. Pretreatment with nifedipine again abolished this response. In contrast to norepinephrine, phorbol dibutyrate did not cause 45Ca++ efflux indicating that intracellular Ca++ was not mobilized. It is concluded that the residual 60% contraction to phorbol dibutyrate most likely occurs via a mechanism independent of the Ca-calmodulin pathway.     

Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol diesters alter the contractile responses of porcine coronary artery

We have studied the effects of activators of the Ca++- and phospholipid-dependent enzyme protein kinase C on isometric tension development by both intact and skinned coronary artery strips. The intact strips contracted upon incubation with 12-O-tetradecanoylphorbol-13-acetate. 12-O-tetradecanoylphorbol-13-acetate produced a leftward shift in the concentration-response relationship for contraction of the tissues by K+, histamine and norepinephrine. Phorbol-12,13-dibutyrate elicited contraction of detergent-skinned artery strips when the free Ca++ concentration in the bathing media was 0.1 microM or greater. This effect was diminished greatly in the presence of polymyxin B, a putative inhibitor of protein kinase C. Phorbol-12,13-dibutyrate shifted the Ca++ concentration-tension response relationship for the skinned tissue to the left. These results are consistent with a role for protein kinase C in regulating the contractile responses of coronary arterial smooth muscle to a variety of stimuli, at least in part by increasing the sensitivity of the contractile apparatus to Ca++.     

Factors affecting rabbit mesenteric artery smooth muscle sensitivity to calcium antagonists

The sensitivity of rabbit isolated superior mesenteric artery to Ca++ antagonists was examined under various conditions. Relaxation dose-response curves for D600 or nifedipine were generated, and IC50 values were calculated. In the first series of experiments, D600 or nifedipine IC50 was found to be 20-25-fold greater for norepinephrine (NE, 5 microM) contraction than for 80 nM K+ contraction. Even when the tissues were depolarized with 80 mM K+ before NE contraction, D600 or nifedipine IC50 still remained significantly greater compared with 80 mM K+ alone and remained closer to that during NE alone. Also a protocol was designed to study NE-induced phasic contraction in EGTA-physiological salt solution (a functional indicator of intracellular Ca++ release) as well as NE-induced sustained contraction after readdition of Ca++. The effects of varying [K+]ex (0-80 nM range) on NE-induced [Ca++]i release as well as on the D600 IC50 for NE contraction was studied. Increasing [K+]ex was found to enhance NE-sensitive [Ca++]i release and lower the D600 IC50 for NE contraction. Thus, conditions causing an increase in the ability of NE to cause [Ca++]i release were associated with an increase in the sensitivity of NE contraction to D600. These data provide functional evidence that the receptor-agonist sensitive Ca++ influx process in vascular smooth muscle is not solely regulated by changes in membrane potential. Additional mechanisms, such as a modulatory role of [Ca++]i release, in this process are implicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetic aspects of the regulation of Ca++ sensitivity of permeabilized rabbit mesenteric artery: possible involvement of a second Ca++ regulatory system in smooth muscle contraction

The effects of phosphagen concentrations and adenosine-5'-O-(2-thiodiphosphate)(ADP beta S), a nonhydrolyzable ADP analog, on the pCa++ tension relationships were investigated, using alpha-toxin permeabilized rabbit mesenteric artery. The removal of creatine phosphate (CP) greatly affected the Ca++ sensitivity and induced a leftward shift of the pCa++ tension curve. Addition of ADP beta S (10-300 microM) also caused a leftward shift of the pCa++ tension curve. Ca++ solutions (0.3-10 microM) containing 0.1 mM ATP did not induce contraction. However, the addition of CP in the presence of 0.1 mM ATP dose-dependently increased force development which reached a maximum around 3 mM CP. A 10 microM Ca++ solution containing 0.1 mM ATP and 1 mM CP was much more effective in inducing contraction than a 10 microM Ca++ solution containing 1.1 mM ATP alone, although the total concentration of phosphagen (ATP + CP) was the same. Application of 0.1 mM ATP solution containing various concentrations of Ca++ after the maximal Ca+(+)-induced contraction relaxed the tissue, with the higher Ca++ concentrations inducing the faster relaxation. The same pattern of the relaxation was seen when the tissue was pretreated with adenosine-5'-O-(3-thiotriphosphate) beforehand. The contractile state observed in the Ca+(+)-free solution containing 0.1 mM ATP and 0.1 mM CP was completely relaxed by 1 mM vanadate, consistent with the idea that the sustained contraction was due to accumulation of the actomyosin-ADP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular and cardiac effects of a new dihydropyridine derivative, YC-170: a comparison with Bay K 8644

The pharmacological effects of YC-170, a new dihydropyridine derivative, were studied in the rabbit aortic strips and guinea pig cardiac preparations and compared with those of Bay K 8644. In the rabbit aortic strips, YC-170 produced contraction in normal physiological saline solution ([K+]0 = 5.9 mM) in a concentration-dependent manner. Increasing the [K+]0 of the medium to 15 mM enhanced the contractile response. The maximum contraction produced by YC-170 at [K+]0 of 15 mM was comparable to that by Bay K 8644. However, YC-170 induced relaxation when the strip was contracted by 60 mM K+. In guinea pig left atrium, YC-170 produced a positive inotropic effect in a concentration-dependent manner, but its extent was far less than that of Bay K 8644. Like Bay K 8644, however, YC-170 increased the time to peak tension and relaxation time of the isometric tension, and prolonged the action potential duration. YC-170 failed to produce a positive inotropic action in the papillary muscle in which Bay K 8644 was a potent positive inotropic agent. In spontaneously beating right atria, YC-170 caused a negative chronotropic effect, whereas Bay K 8644 a positive one. The positive inotropic and vasoconstrictor effects of YC-170 were antagonized competitively by a Ca++ antagonist nicardipine. When the left atria were depolarized with high-K+ medium, the positive inotropic effect of YC-170 was attenuated progressively with increasing [K+]0 and at 13.2 mM K+ a negative inotropic effect was induced by YC-170.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Bay k 8644 and nifedipine on isolated dog cerebral, coronary and mesenteric arteries

Vasoconstrictor effects of Bay k 8644, a dihydropyridine Ca++ agonist, and vasorelaxant effects of nifedipine were investigated in helical strips of dog cerebral (basilar, posterior cerebral and middle cerebral) and peripheral (coronary and mesenteric) arteries. The addition of Bay k 8644 produced a dose-dependent contraction in the absence of any contractile agent in the basilar artery with a pD2 value of 8.53. Similar sensitivity to Bay k 8644 was observed in the posterior cerebral, middle cerebral or coronary artery. Bay k 8644 was much less effective in producing a contraction in the mesenteric artery. An elevation of the concentration of extracellular K+ eliminated the difference between the responses to Bay k 8644 in the basilar and mesenteric artery. Contractile responses of the basilar artery to Bay k 8644 were antagonized competitively by nifedipine (pA2 = 8.17), but non-competitively by diltiazem. The pA2 values for nifedipine antagonism of Bay k 8644 responses with the elevated K+ were the same between the basilar and mesenteric arteries. Increased sensitivity to exogenously added K+ also was observed in cerebral and coronary arteries when compared with the mesenteric artery. The addition of nifedipine to an unstimulated strip produced a dose-dependent relaxation in cerebral and coronary arteries, but not in the mesenteric artery. When the cerebral and peripheral arteries were contracted with K+ to the same magnitude, nifedipine produced similar relaxations among these arteries. Nifedipine was less efficacious in antagonizing the contractile response to Bay k 8644 compared with the contractile response to K+ in cerebral arteries. These results suggest that 1) the voltage-dependent Ca++ channels in the cerebral and coronary arteries are in different states of activation from those in the mesenteric artery, 2) Bay k 8644 contracts the cerebral and coronary arteries by acting primarily on the same site, presumably dihydropyridine receptors of the voltage-dependent Ca++ channels at which nifedipine acts, 3) the dihydropyridine receptors were the same between the basilar and mesenteric arteries and 4) there may be a difference in the state of the Ca++ channel in the arteries between the stimulation with Bay k 8644 and K+-depolarization.     

On the mechanism of vasodilating action of berberine: possible role of inositol lipid signaling system.

We have studied the effect of the alkaloid berberine on the contraction of guinea pig aortic strips induced by various stimuli. Berberine (25-200 microM) inhibited the response of the strips to norepinephrine and histamine, but did not decrease the high K(+)-elicited contraction. The antagonism of berberine was not competitive because in the presence of the alkaloid, maximum response to agonists could not be obtained. Analysis of the drug's effect on the time course of norepinephrine-induced contraction showed that berberine reduced both the rate and the relative contribution to developed tension of the initial, rapid phase, whereas the slow, later component was less affected. Berberine inhibited the response of aortic strips incubated in 0 mM Ca++ to norepinephrine, but did not reduce caffeine-induced contraction and also inhibited phospholipase C-activated contractile response, which has been ascribed to production of inositol phosphate-3 in smooth muscle cells. In cultured arterial smooth muscle cells (A7r5 line), the alkaloid did not significantly decrease the production of inositol phosphates activated by Arg8-vasopressin. The pattern of berberine action is difficult to reconcile with an involvement of the contractile machinery and suggests that the drug has no effect on the voltage-operated calcium channels. Although an antagonism at the receptors or an increase of cyclic AMP or cyclic GMP cannot be completely excluded, we suggest that at least one component of the berberine inhibitory effect may be due to its action on some step of the chain of events linking receptors to contractile response.     

Actions of nifedipine on calcium fluxes and contraction in isolated rat arteries

Experiments were performed on isolated rat aorta and superior mesenteric artery in order to study the action of nifedipine on norepinephrine and K-depolarization-evoked contractions and transmembrane calcium fluxes. Concentration-dependent contractions were obtained with norepinephrine in physiological solution and with Ca++ in K-depolarizing solution. Nifedipine caused a concentration-dependent depression of the maximum response. When aorta was depolarized by 40 mM KCI (instead of usual 100 mM KCI concentration), high concentrations of Ca++ evoked a relaxation that was also blocked by nifedipine. The action of nifedipine has been examined on Ca influx and efflux in arteries stimulated by norepinephrine and K-depolarization. Norepinephrine-evoked Ca influx, but not Ca efflux, was reduced by nifedipine. Concentration inhibitory curves for Ca influx and contraction could be superimposed. K-depolarization-dependent Ca entry and Ca efflux were blocked by nifedipine at concentrations lower than those required to antagonize norepinephrine actions. The results suggest that the action of nifedipine on artery contractility can be related to blockade of calcium entry through channels opened during depolarization or receptor-response coupling.     

Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle

The Ca dependence of contraction and myosin phosphorylation was investigated in canine tracheal smooth muscle stimulated with carbachol, K or serotonin. Previous studies of tracheal muscle showed carbachol concentration-response curves for contraction and myosin phosphorylation were superposable. In contrast, there was a striking difference in the Ca++ sensitivities of tension and myosin phosphorylation when Ca++ concentration-response curves were constructed in the presence of 10(-7) M carbachol. Significant phosphorylation (greater than 0.3 moles phosphate/mole 20,000 dalton myosin light chain) was observed in the absence of active tension. In the present study, carbachol (10(-7) and 10(-6) M) and serotonin (10(-5) M) also induced significant myosin phosphorylation in low Ca++ solutions (0-0.025 mM CaCl2) without proportional increases in tension. K+ depolarization in Ca++-free physiological salt solution (60 mM KCl, 10(-6) M atropine) yielded phosphorylation not significantly different from basal levels. All stimulants induced active stress after readmission of Ca. The Ca++ dependence curve for myosin phosphorylation in muscles stimulated with carbachol was shifted up and to the left of the force curve. Atropine (10(-6) M) significantly reduced phosphorylation induced by carbachol in Ca++-free solutions, as did 3 X 10(-6) M nifedipine and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Phorbol 12-myristate, 13-acetate or phorbol 12,13-dibutyrate did not increase basal phosphorylation or phosphorylation in low Ca++ solutions, suggesting that protein kinase C did not phosphorylate myosin in this case. Myosin phosphorylation under these conditions is not sufficient to support contraction, and is reduced by treatments that decrease Ca++ entry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of forskolin on cytosolic Ca++ level and contraction in vascular smooth muscle

The effects of forskolin, an activator of adenylate cyclase, on cytoplasmic Ca++ level ([Ca++]cyt) measured simultaneously with muscle tension using fura-2-Ca++ fluorescence were examined in isolated smooth muscle of rat aorta. Forskolin decreased muscle tension and [Ca++]cyt in resting aorta whereas both norepinephrine and high K+ solution produced sustained increase in muscle tension and [Ca++]cyt. Addition of forskolin during the sustained contractions decreased muscle tension more strongly than [Ca++]cyt. Norepinephrine-induced contraction was more sensitive to forskolin than high K+-induced contraction. The inhibitory effect of forskolin was attenuated when the concentration of norepinephrine or K+ was increased. Cumulative addition of norepinephrine or K+ induced a concentration-dependent increase in both [Ca++]cyt and muscle tension and a positive [Ca++]cyt-tension correlation was observed. In the presence of 0.1 microM forskolin, the norepinephrine-induced increments in [Ca++]cyt and muscle tension were inhibited without changing the [Ca++]cyt-tension relationship. In the presence of a higher concentration (1 microM) of forskolin, muscle tension was inhibited more strongly with only a small additional decrease in [Ca++]cyt resulting in a shift of the [Ca++]cyt-tension relationship. Norepinephrine induced transient increments in [Ca++]cyt and muscle tension in Ca++-free solution and forskolin inhibited these changes. These results suggest that forskolin has concentration-dependent inhibitory effects on vascular contractility to decrease [Ca++]cyt at lower concentrations and to decrease the sensitivity of contractile elements to Ca++ at higher concentrations.     

Mode of vasorelaxing action of 5-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]amino]-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate (KT-362), a new intracellular calcium antagonist

In rabbit aorta, pretreatment with KT-362 (KT; 10(-6) and 10(-5) M) inhibited contractile responses to norepinephrine (NE; 3 X 10(-9)-10(-5) M) and methoxamine (10(-7)-10(-4) M) but failed to affect responses to potassium (10-70 mM). KT (10(-5) M) partially inhibited Ca++-induced contractions in K+-depolarized aorta pre-equilibrated in a Ca++-free medium. After incubation of tissues for 30 min in a Ca++-free medium containing EGTA (0.2 mM), residual responses to NE and methoxamine were inhibited by KT (10(-6)-10(-4) M) and nitroglycerin (10(-5) M), but not by nifedipine, verapamil or diltiazem (all 10(-5) M). The inhibitory action of a combined treatment with KT and nitroglycerin (both 10(-5) M) on the residual response to NE was also much greater than that of either agent alone. In a Ca++-free medium, the residual caffeine-induced contraction of rabbit iliac artery was inhibited by KT (10(-5)-10(-4) M) but not by nifedipine (10(-5) M). The inhibitory action of KT on the residual responses to methoxamine and caffeine in a Ca+-free medium was much greater than that of nitroglycerin. In a Ca++-free medium with low EGTA (0.01 mM), D600 (10(-5) M) and NE (3 X 10(-7) M), the addition of Ca++ (2 mM) resulted in a tonic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

RP 49356 and cromakalim relax airway smooth muscle in vitro by opening a sulphonylurea-sensitive K+ channel: a comparison with nifedipine

RP 49356 is a novel compound which relaxes airway smooth muscle in vitro. Like cromakalim, RP 49356 reduced contractility in guinea pig isolated trachealis under basal conditions or when challenged with low (less than 20 mM) but not high K+. These effects were antagonized by the sulphonylureas glibenclamide and glipizide. This spectrum of action is typical of the class of compounds known as potassium channel openers (KCOs). Unlike RP 49356 and cromakalim, nifedipine had no effect on basal tone, relaxed tissues contracted with low or high K+ and was not antagonized by the sulphonylureas. These data suggest that the KCOs are not acting directly at the voltage-gated Ca++ channel in this tissue. RP 49356 and cromakalim were similar to nifedipine by being more potent at relaxing tissues precontracted with carbachol or histamine (spasmolytic effects) than they were at preventing initiation of the response to these spasmogens (antispasmogenic effects). Because the maintained phase of contraction in airway smooth muscle may be associated with some Ca++ influx, the data presented here suggests that, like nifedipine, the KCOs are more active smooth muscle relaxants under conditions of Ca++ influx. In summary, RP 49356, like cromakalim, is a compound which relaxes airway smooth muscle in vitro by opening a sulphonylurea-sensitive K+ channel which may be similar to the ATP-sensitive K+ channel found in other tissues.     

Regulation of phosphorylase A formation and calcium content in aortic smooth muscle and smooth muscle cells: effects of atrial natriuretic peptide II

Atrial natriuretic peptide II (ANP II) raises cyclic GMP and relaxes vascular smooth muscle in vitro. The manner in which ANP II relaxes vascular smooth muscle is unknown but may involve alterations in the concentration of free intracellular Ca++. To examine this possibility, changes in intracellular Ca++ were monitored in rat aortic strips using the Ca++-dependent conversion of phosphorylase b to a, while Ca++ levels and phosphorylase were measured in cultured rat aortic smooth muscle cells. ANP II produced time- and concentration-dependent decreases in phosphorylase a and tension in norepinephrine-contracted aortic strips. The decrease in the formation of phosphorylase a was accompanied by an increase in cyclic GMP content. ANP II also decreased phosphorylase a formation in K+-depolarized tissues but to a lesser extent. Agonists such as angiotensin II and arginine vasopressin, and depolarizing concentrations of K+ elevated Ca++ levels in cultured aortic cells. ANP II inhibited Ca++ accumulation to either agonists or K+, but was more effective against agonists. Phosphorylase a formation which was increased by agonists and K+ in cultured cells was also inhibited by ANP II. We conclude that phosphorylase a formation can be a useful indicator of intracellular Ca++ concentrations in smooth muscle preparations and that ANP II regulates Ca++ levels in agonist and depolarized smooth muscle, suggesting that ANP II affects mainly Ca++ removal from the cytoplasm.     

Effects on calmodulin of bepridil, an antianginal agent

Using biopharmacological techniques, we determined the effect on calmodulin of bepridil, a Ca++ channel blocker. We used two Ca++/calmodulin-dependent enzymes, Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain and myosin light chain kinase from chicken gizzard. Bepridil inhibited the calmodulin-induced activation of Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase and the concentration of this drug producing 50% inhibition (IC50) of this enzyme was 8 microM. There was no significant effect on unactivated Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase (in the absence of Ca++-calmodulin), up to a concentration of 100 microM. Bepridil inhibited specifically Ca++/calmodulin-dependent phosphorylation of chicken gizzard myosin light chain with an IC50 value of 18 microM. Moreover, this agent produced a marked displacement of [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, an antagonist that interacts selectively with calmodulin. The influence of bepridil on the dose-response curves of mesenteric arterial strips for CaCl2, norepinephrine and serotonin differed from the influence seen with nifedipine, another Ca++ blocker. Bepridil (100 microM) suppressed the Ca++-induced contraction of saponin-skinned mesenteric arteries and calmodulin (26 microM) reversed partly the relaxant effect of this agent. These results suggest that the effect of bepridil on the cardiovascular system is due not only to its Ca++ channel blocking action but also to a calmodulin antagonistic action.     

Mechanism of action of minoxidil sulfate-induced vasodilation: a role for increased K+ permeability

The mechanism of smooth muscle relaxing effect of minoxidil sulfate (MxSO4) was investigated in isolated rabbit superior mesenteric artery. MxSO4 (5 X 10(-6) M) was found to effectively relax maximal norepinephrine (NE; at 5 X 10(-6) M) contraction, but failed to relax 80 mM K+-induced contraction. MxSO4-induced relaxation was endothelium independent. When the tissues were exposed to increased extracellular K+ (10-25 mM), and then contracted with NE, the relaxation response to MxSO4 was significantly attenuated. Tetraethylammonium (5-10 mM) pretreatment caused pronounced inhibition of MxSO4-induced relaxation. Pretreatment with ouabain (0.5-5 microM) also significantly inhibited MxSO4 relaxation. This effect of ouabain was found to be due to its effect on K+ gradient. These data suggested a role of K+ permeability during MxSO4 relaxation which was further confirmed when it was found that MxSO4 can cause a significant stimulation of 42K efflux from the mesenteric artery preloaded with 42K. It is suggested that MxSO4 may act as a K+ channel agonist to affect the plasmalemmal Ca++ permeability during agonist activation. Consistent with this, MxSO4 was demonstrated to cause an inhibition of NE-stimulated 45Ca influx in this tissue. Such a strong dependence on K+ permeability makes MxSO4 a unique vasodilator among the clinically used vasodilators.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of hydralazine and hydrazone derivatives with contractile mechanisms in rabbit aortic smooth muscle

The mechanism of action and relative potency of hydralazine (H) and tow hydrazone derivatives were investigated using isolated rabbit aortic strips. H, hydralazine acetone hydrazone (HA) and hydralazine butanone hydrazone (HBH) relaxed established K+ and norepinephrine (NE) contractures, and inhibited the development of contractures to these two agents on preincubation. H, HA and HBH increased the threshold to Ca++ and decreased the maximum tension responses during K+-Ca++-contractures (HA greater than H, P less than .05; HBH greater than H P less than .01). The Ca++-dependent and Ca++-independent components of NE contractures were both inhibited by H, HA and HBH. NE contractures were more sensitive to the effects of H than K+ contractures. These results are consistent with the conclusion that H and hydrazone derivatives produce effects on vascular muscle both by interactions with the fluxes of Ca++ from the extracellular space and effects on release from cell stores. However, other possibilities need to be assessed experimentally.