The regulation of L-type Ca2+ currents in cardiac myocytes of hibernating animals

The action of isoproterenol and BAY K 8644 on voltage-dependent Ca2+ currents in isolated ground squirrel cardiac myocytes was studied in two (active and hibernating) states of the animal. In cardiac myocytes of active animals the effect of both drugs was shown to depend on the holding potential. At Vh of about -50 mV both isoproterenol and BAY K 8644 increased the Ca2+ current and their action was additive. At Vh of about -20 mV, both drugs inhibited the Ca2+ current. In cardiac myocytes from hibernating animals, isoproterenol increased the Ca2+ current at any holding potentials, while the effect of BAY K 8644 did not differ significantly from its effect on active animals. The combined action of the two drugs caused the inhibition of the Ca2+ current at high holding potentials. In terms of the two-site Ca2+ channel model, this means that one of the two pathways of channel phosphorylation is blocked in hibernating animal cardiac cells, and BAY K 8644 restores this pathway.     

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

Effects of the calcium channel agonist, BAY K 8644, on electrical activity in mouse pancreatic B-cells.

We studied the effects of the dihydropyridine derivative BAY K 8644 on the membrane potential of B-cells in mouse pancreatic islets. BAY K 8644, in a dose-dependent manner, decreased the spike frequency but increased the duration of the spikes elicited by glucose with or without quinine or tetraethylammonium (TEA). These effects were antagonized by cobalt and nifedipine but not by tetrodotoxin. The interval between spikes was proportionate to the duration of the spikes and the ratio of the interval to the spike duration was constant at all concentrations of BAY K 8644 tested. Peak inward current, estimated from the derivative of the action potential recorded in the presence of TEA, was increased by BAY K 8644 and decreased by nifedipine. BAY K 8644 elicited spike activity when the membrane was moderately depolarized by either 5.6 mM glucose or 15 mM K+, but did not change the membrane potential of the resting hyperpolarized B-cell. These results suggest that BAY K 8644 acts on the open Ca2+-channels. The threshold occurs at a membrane potential of -50 mV. Also, the modifications of the shape of the spikes appear to reflect specific changes in Ca2+ entry. We propose the existence of a Ca2+-induced Ca2+-channel inactivation process in the pancreatic B-cell.     

Calcium channel activation in vascular smooth muscle by BAY K 8644

BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate) and CGP 28 392 (ethyl-4(2-difluoromethoxyphenyl)-1,4,5,7-tetrahydro-2-methyl-5-++ +oxofuro- [3,4-b]pyridine-3-carboxylate) are closely related in structure to nifedipine and other 1,4-dihydropyridine Ca2+ channel antagonists. However, both BAY K 8644 and CGP 28 392 serve as activators of Ca2+ channels. In the rat tail artery, responses to BAY K 8644 are dependent upon Ca2+ext and prior stimulation by K+ or by the alpha-adrenoceptor agonists, phenylephrine and BHT 920 (6-allyl-2-amino-5,6,7,8,-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). Responses are blocked noncompetitively by the Ca2+ channel antagonists D-600 [-)-D-600 greater than (+)-D-600) and diltiazem, but competitively by nifedipine (pA2 = 8.27). This suggests that activator and inhibitor 1,4-dihydropyridines interact at the same site. BAY K 8644 potentiates K+ responses and Ca2+ responses in K+-depolarizing media. The leftward shift of the K+ dose--response curve produced by BAY K 8644 suggests that this ligand facilitates the voltage-dependent activation of the Ca2+ channel. The pA2 value for nifedipine antagonism of BAY K 8644 responses is significantly lower than that for nifedipine antagonism of Ca2+ responses in K+ (25-80 mM) depolarizing media (9.4-9.6), suggesting that the state of the channel may differ according to the activating stimulus.     

Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.     

Adrenergic-cholinergic interactions in left atria. II. Comparison of the antagonism of inotropic responses to alpha- and beta-adrenoceptor stimulation and BAY K 8644 by carbachol, D-600, and nifedipine

The muscarinic agonist carbachol has previously been shown to reverse positive inotropic responses of rabbit left atrial strips to equiactive doses of the beta-adrenoceptor agonist isoproterenol and to the alpha-adrenoceptor agonist phenylephrine. Responses to phenylephrine were measured in the presence of the beta-blocker timolol. However, carbachol was not able to reverse the increase in tension produced by elevating the extracellular Ca2+ concentration. To gain more information about the nature of the functional interaction of carbachol with alpha- and beta-receptor stimulants in left atria, the interaction of carbachol with these agonists, as well as with elevated Ca2+ and the Ca2+ activator compound BAY K 8644, was compared with that of the Ca2+ antagonists D-600 and nifedipine. The results demonstrate that the Ca2+ antagonists exhibit a selectivity similar to that of carbachol, in that responses to both isoproterenol and phenylephrine plus timolol were blocked by low concentrations of D-600 and nifedipine, which had no effect on positive inotropic responses to elevated Ca2+. Higher concentrations of these antagonists shifted the Ca2+ dose-response curve to the right. In addition, although phenylephrine and BAY K 8644 increased tension to a similar extent, responses to phenylephrine were more sensitive than responses to BAY K 8644 to inhibition by both carbachol and D-600. These similarities between the effects of low concentrations of D-600 and nifedipine and those of carbachol are consistent with the hypothesis that carbachol antagonizes responses to alpha- and beta-receptor stimulation in left atria primarily by blocking increases in Ca2+ influx produced by these agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.     

Modulation of calcium channels in arterial smooth muscle cells by dihydropyridine enantiomers

The actions of the optical enantiomers of BAY K 8644 and Sandoz 202,791 were studied on barium inward currents recorded using the whole-cell configuration of the patch clamp technique from enzymatically isolated smooth muscle cells from the rabbit ear artery. The enantiomers were applied by bath perfusion or rapidly by a concentration jump technique, which enabled the study of drug action under equilibrium and nonequilibrium conditions. A larger effect of agonists was seen on peak inward current in 110 mM Ba when small rather than large depolarizations were applied. The midpoint voltage of the steady-state inactivation curve of IBa was -12.8 +/- 1.9 mV (n = 4) in the absence of drug, -16.4 +/- 2.5 mV (n = 4) in 1 microM (+)202,791, and -31.4 +/- 0.4 mV (n = 4) in 1 microM (-)202,791. The rate of onset of action of the agonist and antagonist enantiomers of BAY K 8644 and Sandoz 202,791 was studied by rapid application during 20-ms depolarizing steps from different holding potentials to +30 mV at 1 or 0.2 Hz. The drugs were applied as concentration jumps between two single pulses of a pulse train. The rates of onset of drug action on peak IBa during a 1-Hz pulse train were concentration dependent over the range of 100 nM-3 microM for both (+) and (-)202,791. The rate of onset of inhibition of peak current by antagonist enantiomers was not significantly influenced by the test pulse frequency. At a holding potential of -60 mV, the onset rate of the increase in peak IBa on application of 1 microM of agonist enantiomers (+)202,791 or (-)BAY K 8644 during a train of pulses occurred with mean time constants of 2.1 +/- 0.7 s (n = 7) and 2.3 +/- 0.2 s (n = 4), respectively. The onset of current increase on application of 1 microM (+)202,791 during a single voltage clamp step to 20 mV was faster, with a mean time constant of 380 +/- 80 ms (n = 3).     

Effect of BAY K 8644 on cytosolic free calcium in isolated rabbit gall-bladder epithelial cells

Rabbit gall-bladder epithelial cells were isolated by a combination of Ca2+ omission, enzymatic treatment, and mechanical detachment and had a viability of 96-98% and well preserved morphology. Measurements of cytosolic free Ca2+ concentration ([Ca2+]i) in these cells with the Ca2+-fluorescent indicator fura-2 demonstrated a resting [Ca2+]i level of 115 +/- 12 nM. When used in concentrations which inhibit rabbit gall-bladder isosmotic NaCl absorption (1-100 microM), the Ca2+-channel activator BAY K 8644 caused a dose-dependent increase in the epithelial [Ca2+]i to a maximal value of 850 nM. The effect was dependent on extracellular Ca2+, and was not altered by 1 microM L-verapamil. Depolarization of the epithelial cells with KCl had no effect on [Ca2+]i. The results suggest that BAY K 8644 activates a Ca2+ influx which is not dependent on voltage-gated channels. Cytosolic Ca2+ may be involved in the regulation of isosmotic NaCl absorption in the mammalian gall-bladder.     

Voltage-dependent calcium current and the effects of adrenergic modulation in rat aortic smooth muscle cells.

Smooth muscle cells from rat aorta were cultured in defined, serum-free medium and studied using whole-cell patch-clamp techniques. Under conditions designed to isolate currents through Ca channels, step depolarizations produced inward currents which were fast in onset and inactivated rapidly, with little sustained inward current being observed. Both Ni and Cd blocked these currents, with Ni being effective at 50 microM. Removal of external Na or addition of 1 microM tetrodotoxin had no effect. Peak inward currents were attained at about -15 mV, with half-maximal activation at -41 mV using -80 mV holding potentials. The transient inward currents were reduced by depolarized holding potentials, with half-maximal steady-state inactivation at -48 mV. In three of the 98 cells studied, small maintained inward currents were observed with a -40 mV holding potential. The Ca channel antagonist nicardipine (5 microM) blocked the transient inward current while neither of the dihydropyridine Ca channel agonists S(+)202 791 and (-)BAY K 8644 produced a significant augmentation of sustained inward current. At 10 microM, both noradrenaline and adrenaline but not phenylephrine decreased the peak inward current. This inhibition was unaffected by a variety of adrenoceptor antagonists and was also observed when internal solutions having high Ca buffering capacity were used, but was absent when GDP-beta-S instead of GTP was included in the pipette solution. The main conclusions from this study are that under our cell culture conditions, rat aortic smooth muscle cells possess predominantly a transient, low-threshold-activated inward Ca current and that this Ca current is inhibited by certain adrenoceptor agonists but with a quite atypical adrenoceptor antagonist pharmacology.     

BAY K 8644, a 1,4-Dihydropyridine Ca2+ Channel Activator: Dissociation of Binding and Functional Effects in Brain Synaptosomes

K+-stimulated 45Ca2+ uptake into rat brain and guinea pig cerebral cortex synaptosomes was measured at 10 s and 90 s at K+ concentrations of 5-75 mM. Net increases in 45Ca2+ uptake were observed in rat and guinea pig brain synaptosomes. 45Ca2+ uptake under resting or depolarizing conditions was not increased by the 1,4-dihydropyridine BAY K 8644, which has been shown to activate Ca2+ channels in smooth and cardiac muscle. High-affinity [3H]nitrendipine binding in guinea pig synaptosomes (KD = 1.2 X 10(-10) M, Bmax = 0.56 pmol mg-1 protein) was competitively displaced with high affinity (IC50 2.3 X 10(-9) M) by BAY K 8644. Thus high-affinity Ca2+ channel antagonist and activator binding sites exist in synaptosome preparations, but their relationship to functional Ca2+ channels is not clear.     

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.     

Voltage-Sensitive Calcium Channels in Differentiated Neuroblastoma × Glioma Hybrid (NG108–15) Cells: Characterization by Quin 2 Fluorescence

Depolarization of differentiated neuroblastoma X glioma (NG108-15) cells with KCl (50 mM) or veratridine (50 microM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 +/- 7 to 595 +/- 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage-dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose-dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (-) stereoisomers of 202-791 showed agonist and antagonist properties, respectively. (+)-202-791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage-sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KCl treatment and also by divalent metal cations (micromolar range). High [K+]o-induced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 microM) and tetraethylammonium (10 microM). In contrast, veratridine-induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 microM). Veratridine-induced Ca2+ accumulation was slower (approximately 45 s), smaller in magnitude (approximately 30% of [K+]o-induced Ca2+ entry), and also enhanced by BAY K 8644 (approximately 50%). VSCC were identified in neuronal hybrid (NG108-15 and NCB-20) cells, but not in glial (C6BU-1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108-15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.     

Calcium channel currents in isolated smooth muscle cells from human bronchus

An electrophysiological study was carried out on smooth muscle cells that were enzymatically dissociated from bundles of muscle fibers dissected out of human bronchi obtained at thoracotomy. These cells that retain the contractile properties of intact bundles were voltage-clamped by means of the whole-cell patch-clamp technique. Upon voltage steps from a holding potential of -60 mV to more positive levels, the initial inward current was followed by large outward currents that inactivated slowly. These were subsequently reduced by substituting Cs+ for K+ in the internal solution and by using Ba2+ instead of Ca2+ as a charge carrier in the external solution. Under these conditions, the inward current did not completely inactivate in the course of 300-ms voltage steps. Inward current measured after leak subtraction was activated at a membrane potential of -25.8 +/- 5 mV, was maximum at +18 +/- 4 mV, and had an apparent reversal potential of +52.5 +/- 5.5 mV (n = 5). The potential at which steady-state inactivation was half-maximum was -28 mV (n = 5). This inward current was identified as a calcium current on the following basis: 1) it was not altered by 10 microM tetrodotoxin (TTX) or by lowering to 10 mM external Na+ concentration; 2) it was blocked by 2.5 mM Co2+ or 1 microM PN 200-110; 3) it was enhanced by 1 microM BAY K 8644, which in addition suppressed the PN 200-110 blockade.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opposing actions of Bay K 8644 enantiomers on calcium current, prolactin secretion, and synthesis in pituitary cells.

Dihydropyridine (DHP) Ca2+ channel modulators were used to explore the relationship between voltage-gated Ca2+ channels and PRL secretion, synthesis, and mRNA in PRL-secreting pituitary cells. Optical isomers of the Ca2+ channel agonist Bay K 8644 produced stereospecific and opposing effects on L-type Ca2+ current, PRL release, and synthesis in GH3 and GH4C1 cells. (-)-Bay K 8644 (R5417) behaved as a pure agonist, enhancing Ca2+ current several-fold while shifting the current-voltage curve 10-15 mV in the hyperpolarizing direction. The agonist effect was independent of holding potential, but decreased during prolonged Ba2+ or Ca2+ entry. R5417 produced a concentration-dependent increase in acute PRL release and enhanced PRL production by GH cells several-fold during a 72-h period. (+)-Bay K 8644 (R4407) behaved as a weak Ca2+ channel antagonist, inhibiting L-type Ca2+ current, KCl-stimulated PRL secretion, and PRL production at concentrations of 0.5-5 microM. These two isomers produced similar effects on PRL production by normal rat pituitary cells in dispersed culture. R5417 (500 nM) increased PRL produced in 72 h to 233 +/- 8% of the control value. R4407 reduced this quantity by 36 +/- 9%. The effects of the DHPs on PRL mRNA levels were consistent with the effects observed for acute secretion and hormone production. The agonist R5417 increased PRL mRNA 147 +/- 5% over a 30-h period, and the potent DHP Ca2+ channel blocker nimodipine inhibited PRL mRNA production 2-fold. These results demonstrate that racemic Bay K 8644 interacts with L-type Ca2+ channels in normal and transformed pituitary cells as a mixed agonist-antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation.

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells [published erratum appears in J Gen Physiiol 1996 Jun;107(6):755]

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)- containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.     

Platelet-derived growth factor activation of gastric mucosal calcium channels.

Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H]PN200-100 was reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhancement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channels showed an increase in protein tyrosine phosphorylation of 55 and 170kDa subunits of calcium channel. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results point towards the importance of PDGF in the regulation of gastric mucosal calcium homeostasis.     

PK 11195, an antagonist of peripheral type benzodiazepine receptors, modulates Bay K8644 sensitive but not beta- or H2-receptor sensitive voltage operated calcium channels in the guinea pig heart

In a partially depolarized guinea pig papillary muscle preparation, BAY K8644 stimulated voltage-operated calcium channels, promoting slow action potentials; this effect was dose-dependent over a concentration range of 3 X 10(-7) M to 3 X 10(-6) M. Isoproterenol and histamine also induced slow action potentials by stimulating beta or H2 receptors, respectively. PK 11195, the antagonist of peripheral type benzodiazepine receptors, inhibited the effect of BAY K8644, but not those of histamine or isoproterenol. Moreover, PK 11195 "dose-dependently" antagonized the ability of RO5-4864 to inhibit the slow action potentials elicited by barium chloride. Thus, in the heart, PK 11195, an antagonist of peripheral type benzodiazepine receptors, can modulate voltage-operated calcium channels when they are activated directly, but not when they are activated by stimulation of neurotransmitter receptors.     

Voltage-clamp study of calcium currents during differentiation in the NCB-20 neuronal cell line

1. Calcium currents (ICa) were studied in voltage-clamped NCB-20 cells. In undifferentiated cells, voltage steps from hyperpolarized potentials (-80/-100 mV) essentially revealed transient ICa showing characteristics classically described for "T-type" channels. In about 50% of the cells, there was a residual current at the end of the step; no ICa was elicited from a holding potential of -50 mV. 2. In contrast, 100% of the cells differentiated with dibutyryl cyclic AMP (cAMP) displayed a residual current in addition to the transient one, and depolarizing steps from a holding potential of -50 mV induced a sustained current. In these cells, Bay K 8644 elicited both a negative shift in voltage dependence and a moderate increase of the sustained component. 3. Although these changes in Ca2+ channel physiology result from chemically induced differentiation, they might not be directly related to the concomitant morphologic differentiation. 4. In undifferentiated NCB-20 cells, T-type Ca2+ currents can be elicited in relative isolation.