Calcium channels in isolated cells and strips of longitudinal muscle of rat myometrium

The properties of Ca2+ channels in strips and single muscle cells of longitudinal muscle of estrogen-dominated rat myometrium were studied under the effects of elevation of K+ concentration, the partial channel agonist Bay K 8644, and nitrendipine. In isolated strips in 0.5 mM Ca2+, Bay K 8644 (pD2 = 7.8-8.0) lowered the threshold for and enhanced the contractions in response to an elevation of K+ concentration, including the maximum response to K+ elevation alone. Bay K 8644 alone in concentrations up through 10(-6) M did not initiate contractions in 0.5 mM Ca2+ solutions. At higher concentrations (10(-5) M), Bay K 8644 behaved as an antagonist to contractions induced by elevation of K+. In isolated cells 10(-7) M Bay K 8644 enhanced the shortenings to elevated K+ and lowered the threshold K+ concentration required. Also no significant contraction occurred with 10(-7) M Bay K 8644 at normal K+ concentration. In contrast with its effect in isolated strips, no significant increase in maximum shortening (to 60 mM K+) was observed, possibly because cells without a mechanical load were maximally shortened by K+ alone. From these studies, we conclude that Ca2+ channels of isolated strips and cells of rat myometrium behave similarly and have similar properties to those of other smooth muscles in their interactions with elevation of K+, nitrendipine, and Bay K 8644.     

Nitrendipine potentiates Bay k 8644-induced contraction of isolated porcine coronary artery: evidence for functionally distinct dihydropyridine receptor subtypes

Bay k 8644 and nitrendipine, dihydropyridines classified as calcium channel agonist and antagonist, respectively, produced concentration-dependent biphasic responses (contraction and relaxation) in porcine coronary artery rings. Nitrendipine relaxed rings (IC50 = 60 nM) that were contracted with 100 nM Bay k 8644. Pretreatment of rings with 60 nM nitrendipine caused paradoxical potentiation of Bay k 8644-induced contraction. The data are consistent with a model that consists of two functionally-distinct dihydropyridine "receptors" with which Bay k 8644 and nitrendipine interact as partial agonists. We propose that these excitatory and inhibitory dihydropyridine receptor subtypes mediate contraction and relaxation, respectively, by dihydropyridines.     

Dual effect of dihydropyridines on 45Ca uptake induced by the K+ -channel blocker, 4-aminopyridin on mast cells

The 1,4-dihydropyridines (DHP) are calcium antagonists and represent a new class of drugs which act by a selective inhibition of Ca++ influx through voltage-operated calcium channels. We report the effect of nifedipine (Bay A 1040), nisoldipine (Bay K 5552) and nitrendipine (Bay E 5009) on the histamine release and on the 45Ca uptake promoted by 4-aminopyridin in mast cells. These cells treated with DHP (10(-12)-10(-3) M) activated the secretory response in a dose-dependent manner in the range of concentrations 10(-6)-10(-3) M, whereas concentrations of 10(-12)-10(-6) M did not significantly inhibit the secretion. 4-Aminopyridin, a known K+ -channel blocker, induced 45Ca uptake. Pretreatment of mast cells with DHP prior to 4-aminopyridin stimulation inhibited or stimulated 45Ca uptake depending on concentration; thus, concentrations of DHP below 10(-12) of nitrendipine and 10(-9) for nisoldipine and nifedipine were inhibitory, while higher doses potentiated 45Ca uptake. These results demonstrate a diversity of pharmacological effects of DHP on mediator secretion and 45Ca uptake in mast cells and throw into question their only properties as Ca++ antagonists.     

Further investigation of a potential calcium channel modulator isolated from rat erythrocytes

The contractile effects of a peptide isolated from rat erythrocytes were further studied in rat aortic rings. Previous data showed that preincubation of aortic tissue with the peptide had no effect on resting tension, but significantly enhanced K+ and norepinephrine (NE) induced contraction. The calcium channel antagonist verapamil noncompetitively blocked the effect of the peptide, whereas nifedipine blockage appeared to be competitive. In the present study the peptide enhanced K+, NE, and phenylephrine (PE) induced contraction in a concentration-dependent manner, with a maximum enhancement at peptide concentrations of 10(-7)-10(-6) M. At a concentration as low as 10(-9) M, the peptide significantly enhanced K(+)-induced, but not NE- or PE-induced, contraction. The magnitude of maximal enhancement was greater for K(+)-induced contraction than that for NE- or PE-induced contraction. Preincubation of the tissues with the peptide caused a leftward shift of cumulative concentration-response curves to K+ and NE. The peptide enhancement of contraction increased with increasing K+ and NE concentration. The peptide potentiated the contractile response to Ca2+ in K(+)-depolarizing medium. It also enhanced the contractile response to NE in intracellular Ca2(+)-pool-depleted tissue following the replenishment of extracellular Ca2+, but had no apparent effect on the mobilization of intracellular calcium. Addition of nifedipine caused a rightward shift of both the peptide and Bay K 8644 concentration-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ channel ligand sensitive responses to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in vascular smooth muscle

The action of a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), on isolated rat aortic and tail artery strips has been characterized. TPA (10(-9)-10(-7) M) produced a graded contraction developing maximum tension over 30-40 min. The contraction was irreversible and was not relaxed by prolonged washing with physiologic saline. Relaxation occurred upon washing with Ca2+-free saline but readdition of Ca2+ restored response. TPA was without significant effect in rat tail arteries in physiologic saline but produced responses in saline containing elevated K+ (15 mM). The protein kinase C inhibitor, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)n-propyl]-4-phenylpiperidine dihydrochloride) (5 X 10(-5) M), blocked the response to TPA but was without effect on responses to Bay K 8644 (2,6-dimethyl-3-carbomethoxy-5-nitro-4-(2-trifluoromethylphenyl) 1,4-dihydropyridine), KCl, phenylephrine, and B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). The calcium channel antagonist nifedipine and its analogue, 2,6-dimethyl-3,5-dicarbomethoxy-4-(3-cyanophenyl)-1,4-dihydr opyridine, inhibited TPA responses with IC50 values of 9.28 X 10(-9) and 1.96 X 10(-7) M, respectively. Responses to Bay K 8644 in rat aorta were maximum in the presence of elevated KCl (10 mM), but TPA at concentrations of 10(-9) and 3 X 10(-9) M potentiated responses to Bay K 8644 in physiologic saline to levels approximating those in elevated K+ saline. TPA similarly potentiated responses to Ca2+ in Ca2+-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).     

Mechanism of contractile action of barium ion on rat aortic smooth muscle

The mechanism of Ba2+-induced contraction has been examined in helical strips of Ca2+-depleted, 60 mM K+-depolarized rat aortae. The concentration-response curves to Ca2+ or Ba2+ were significantly potentiated by exposure to 3 X 10(-8) M Bay K 8644 (a Ca2+ channel agonist) in the order Ca2+ greater than Ba2+, suggesting an action of Ba2+ ions through potential-sensitive membrane Ca2+ channels. Exposure of strips to background concentration of Ca2+ (0.05 mM) enhanced the contractile responses to Ba2+, whereas background exposure to Ba2+ (0.1 mM) attenuated Ca2+ responses. Repeated stimulation with Ba2+ resulted in tachyphylaxis, contrary to the result when Ca2+ was used. The results suggest that Ba2+ ions enter rat aortic smooth muscle cells through Ca2+ channels and mobilize a noradrenaline-insensitive intracellular Ca2+ pool. Ba2+ may also cause a desensitization of some intracellular process.     

Characterization of voltage-sensitive calcium channels in a clonal pituitary cell line

We have pharmacologically characterized voltage sensitive calcium channels (VSCCs) in GH3 cells, an anterior pituitary clonal cell line known to secrete prolactin and growth hormone. Raising the medium K+ concentration from 5 to 50 mM caused an immediate increase in net 45Ca2+ uptake which remained apparent over a 15 minute time course. 45Ca2+ uptake was maximally stimulated nearly 10-fold over basal levels. This K+-induced stimulation of Ca2+ uptake was not prevented by 10-5M tetrodotoxin or by replacing sodium with choline in the assay medium. Ca2+ uptake was, however, inhibited by several VSCC antagonists: nitrendipine, D-600, diltiazem and Cd2+. Further, the novel dihydropyridine VSCC agonists, BAY K8644 and CGP 28392, enhanced 50 mM K+-stimulated 45Ca2+ uptake and these effects were blocked by nitrendipine.     

Hypothyroid state reduces calcium channel function in 18-day pregnant rat uterus

Hypothyroidism significantly reduced the mean amplitude and increased the mean frequency of spontaneous rhythmic contractions in 18 day pregnant rat uterus. Nifedipine (10(-12)-10(-9) M) and diltiazem (10(-10)-10(-6) M) caused concentration related inhibition of the myogenic responses of the uterine strips obtained from both pregnant and hypothyroid state. However, nifedipine was less potent (IC50:2.11 x 10(-11) M) in pregnant hypothyroid state as compared to pregnant control (IC50: 3.1 x 10(-12) M). Similarly, diltiazem was less potent (IC50: 3.72 x 10(-9) M) in inhibiting the uterine spontaneous contractions in hypothyroid than in pregnant rat uterus (IC50:5.37 x 10(-10) M). A similar decrease in the sensitivity to nifedipine and diltiazem for reversal of K+ (100 mM)-induced tonic contraction and K(+)-stimulated 45Ca2+ influx was observed with these calcium channel antagonists in uterus obtained from hypothyroid pregnant rats compared to the controls. Nifedipine-sensitive influx of 45Ca(2+)-stimulated either by K+ (100 mM) or by Bay K8644 (1,4-dihydro-2,6-methyl-5-nitro-4-[2'-(trifluromethyl)phenyl]-3-pyridine carboxylic acid methyl ester) (10(-9) M) was significantly less in uterine strips from hypothyroid rats compared to controls. The results suggest that the inhibition of uterine rhythmic contractions may be attributable to a reduction in rat myometrial Ca2+ channel function in the hypothyroid state.     

Novel 1,4-Dihydropyridine (Bay K 8644) Facilitates Calcium-Dependent [3H]Noradrenaline Release from PC 12 Cells

The effects of the novel 1,4-dihydropyridine Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration-dependent manner evoked 3H-transmitter release with an EC50 of 50-56 mM. Bay K 8644 at 30 nM potentiated the K+-evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644-potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol X mg-1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a Ki of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a "calcium agonist" to mediate calcium-dependent cellular events such as catecholamine release from PC 12 cells.     

45Ca2+ uptake in rat brain neurons: absence of sensitivity to the Ca2+ channel ligands nitrendipine and Bay K 8644

K+-stimulated 45Ca2+ uptake into intact rat brain cells was biphasic, consisting of a fast first phase and a slow second phase; the latter was Na+ dependent. Cobalt and cadmium at 10(-4) and 10(-3) M produced 19-97% block of first phase 45Ca2+ uptake, but nitrendipine (to 10(-6) M) and Bay K 8644 (to 10(-6) M) were without effect on uptake and were similarly without effect in cells prepared in the presence of ATP, cAMP, Mg2+, and protease inhibitors. The second phase of K+-stimulated 45Ca2+ uptake was inhibited by 3,4-dichlorobenzamil (IC50, 29.6 microM). Depolarization-induced 45Ca2+ uptake into intact rat brain cells occurs by at least two different mechanisms. The first phase probably represents uptake through 1,4-dihydropyridine-insensitive Ca2+ channels, while the second phase is probably due to Na+-Ca2+ exchange.     

In vitro secretion of calcitonin from a rat C cell line: effect of repetitive stimulation with the calcium channel agonist BAY K 8644.

Calcium and BAY K 8644 acutely stimulate calcitonin secretion by influx of extracellular calcium (Ca) through voltage-dependent calcium channels, leading to an increase in cytosolic free Ca. Repetitive exposure to BAY K 8644 (10(-6) M) resulted in an increase in calcitonin (CT) secretion in the rat C-cell line (rMTC 6-23) lasting 9 hours, in comparison to that of 3 mM Ca2+ which lasted 6 hours. Equimolar concentration of nifedipine did not inhibit the stimulatory effect of BAY K 8644 as compared to the nifedipine only group. The decrease in stimulated CT secretion during long-term exposure to BAY K 8644 is due to desensitization of cells which may be attributed to down-regulation of dihydropyridine receptors. After 12 h exposures to 3 mM Ca2+ alone, BAY K 8644 (10(-6) M) alone or in combination with nifedipine (10(-6) M), CT content decreased below the control level, indicating a decrease in synthesis. Overall cellular protein content was not affected by the test agents. Repetitive exposure of C-cells to BAY K 8644 revealed a desensitization of the stimulatory effect on CT secretion and a decrease in CT cell content.     

Interaction of steroidal alkaloid toxins with calcium channels in neuronal cell lines

Depolarization with 50 mM K+ increased 45Ca2+ uptake into neuronal clonal cell lines NG108-15, N1E-115 and NH15-CA2. In each cell line this depolarization-induced uptake was blocked by inorganic and organic blockers of voltage sensitive calcium channels. However, tetrodotoxin (10(-6) M) was ineffective. Moreover, in the presence of tetrodotoxin, neither batrachotoxin nor veratridine inhibited the depolarization-induced uptake. The novel dihydropyridine BAY K8644 enhanced depolarization-induced 45Ca2+ uptake into each cell line in a nitrendipine reversible fashion. In the presence of tetrodotoxin, the BAY K8644/50 mM K+ stimulated uptake could be partially inhibited by batrachotoxin (10(-6) M) and veratridine (5 X 10(-5) M). These effects were not altered by the presence of scorpion venom (1 microgram/ml). The results indicate that both batrachotoxin and veratridine can modulate the effects of dihydropyridines on the gating properties of voltage sensitive calcium channels.     

Melatonin inhibits release of luteinizing hormone (LH) via decrease of [Ca2+]i and cyclic AMP

The role of [Ca2+]i and cAMP in transduction of the melatonin inhibitory effect on GnRH-induced LH release from neonatal rat gonadotrophs has been studied, because melatonin inhibits the increase of both intracellular messengers. Treatments increasing Ca2+ influx (S(-) Bay K8644 or KCI) or cAMP concentration (8-bromo-cAMP or 3-isobutyl-1-methylxanthine) potentiated the GnRH-induced LH release and partially diminished the inhibitory effect of melatonin. Combination of the treatments increasing cAMP and calcium concentrations blocked completely the melatonin inhibition of LH release. The combined treatment with 8-bromo-cAMP and S(-) Bay K8644 also blocked the melatonin inhibition of GnRH-induced [Ca2+]i increase in 89 % of the gonadotrophs, while any of the treatments alone blocked the melatonin effect in about 25 % of these cells. These observations suggest that a cAMP-dependent pathway is involved in regulation of Ca2+ influx by melatonin and melatonin inhibition of LH release may be mediated by the decrease of both messengers.     

Dihydropyridine-sensitive L-type Ca2+ channels in human foreskin fibroblast cells. Characterization of activation with the growth factor Lys-bradykinin.

We have previously characterized the calcium response of cultured human fibroblasts (HSWP cells) to stimulation by the mitogen Lys-bradykinin (BK). We have reported a biphasic response which includes a rapid rise to a peak that appears to result from mobilization of internal calcium, and a plateau phase, which is due to influx of external calcium (Byron, K., Babnigg, G., Villereal, M. L. (1992) J. Biol. Chem. 267, 108-118). In this paper we examine participation of L-type voltage operated calcium channels in the calcium entry phase of BK-stimulated HSWP cells. We show that there is an increase in 45Ca2+ uptake and an increase in intracellular free calcium concentration ([Ca2+]i) as measured by fura-2, when HSWP cells are stimulated with the L-channel agonist Bay K 8644 under depolarizing conditions. Furthermore, both of these effects are inhibited by low doses of the dihydropyridine antagonist nitrendipine. We also report that BK stimulation of 45Ca2+ uptake can be significantly inhibited by low doses of nitrendipine, while nitrendipine treatment has no effect on the BK-induced rise in [Ca2+]i, as measured by fura-2. These results suggest that under normal conditions the portion of the BK-stimulated Ca2+ influx which is mediated by a nitrendipine-sensitive entry pathway is invisible to the fura-2 technique used to measure [Ca2+]i. This suggest that the nitrendipine-sensitive influx pathway admits calcium preferentially into an intracellular store that is isolated from fura-2. This idea is supported by the observation that in media where calcium has been replaced by 2 mM Ba2+ nitrendipine inhibits most of the BK-stimulated Ba2+ influx.     

Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

Dihydropyridine Modulation of the Chromaffin Cell Secretory Response

Prolonged perfusion of cat adrenal glands with Krebs-bicarbonate solutions containing nicotine, muscarine, or excess K rapidly increased the rate of catecholamine output proportional to the concentrations of secretagogue used. The secretory responses to nicotine or high K reached a peak and declined to almost basal rates of secretion after about 10 min of stimulation. The dihydropyridine Ca channel agonist Bay K 8644 potentiated markedly the secretory responses to 1 microM nicotine and to 17.7 mM K but not to higher concentrations of these secretagogues. The muscarinic response did not decrease with time and was modestly potentiated by Bay K 8644. Similar curves were obtained with 17.7 mM K plus Bay K 8644 and with 59 mM K alone. CGP28392, another agonist, was about 10 times less potent than Bay K 8644 in potentiating the secretory responses to 17.7 mM K. Bay K 8644 also potentiated the release of [3H]noradrenaline evoked by stimulation of cultured bovine adrenal chromaffin cells with 17.7 mM K or 2 microM nicotine but not with higher concentrations of K or nicotine. Dihydropyridine Ca channel antagonists reversed the effects of Bay K 8644 with the following order of potency: niludipine greater than nifedipine = nimodipine greater than nitrendipine. The secretory rates from intact chromaffin cells treated with the Ca ionophores X537A or A23187, or those evoked by Ca-EGTA buffers from digitonin-permeabilized cells, were not affected by Bay K 8644. These results are compatible with the following conclusions: Bay K 8644 selectively potentiates catecholamine secretory responses mediated through the activation of voltage-sensitive Ca channels; during nicotine or high-K stimulation, Ca gains access to the cell interior through a common permeability pathway, the Ca channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C potentiates stretch-induced cerebral artery tone by increasing intracellular sensitivity to Ca2+

The effects of PMA, an activator of protein kinase C, was studied on Ca2+-induced tone in the rabbit basilar artery. Contractile responses to Ca2+ occurred only in arteries pretreated with PMA; the extent of Ca2+-induced contractions were related to the level of stretch applied to the vessels. Bay K 8644, a Ca2+-channel agonist, at a concentration that was subthreshold for contraction, augmented the extent of Ca2+-induced tone occurring in PMA-treated arteries. Nifedipine, a Ca2+-entry inhibitor, and staurosporine, an inhibitor of protein kinase C attenuated the response to Ca2+ occurring either in the absence or presence of Bay K 8644. Our results suggest that PMA increases myofilament sensitivity to Ca2+, such that levels of Ca2+ previously ineffective for contraction Ca2+-influx, e.g. due to Bay K 8644, is manifest as contraction. Our results also confirm the role of extracellular Ca2+ entry via plasma membrane stretch-dependent Ca2+-channels in the maintenance of vascular tone in the basilar artery.