Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.     

Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea

Contractions of guinea pig trachea in the absence and presence of indomethacin to LTD4 greater than LTC4 greater than K+ greater than histamine greater than acetylcholine were reduced following a 45 minute exposure of the tissues to calcium-free Krebs' solution (Ca2+-free Krebs' solution), were further reduced by a transient exposure to EGTA (1.25 mM) in Ca2+-free Krebs' solution and were virtually abolished when tested in the presence of EGTA (0.125 mM) in Ca2+-free Krebs' solution. In normal Krebs' solution (2.5 mM Ca2+) the Ca2+ entry blockers nifedipine (N) much greater than D-600 greater than verapamil (V) greater than diltiazem (D) almost completely abolished the contractions to K+ but blocked only a component of the maximum response to the other agonists. After exposure to Ca2+-free Krebs' solution for 45 minutes, any residual contractions to LTC4 & LTD4, were reversed by low concentrations of N (0.3 microM) or D-600 (2.1 microM). Leukotrienes appear to mobilize a superficial and a bound store of Ca2+ which gains entry through at least two types of Ca2+ channels (or mechanisms), one of which is blocked by N and D600. K+-induced contractions appear to be dependent on superficial and tightly bound Ca2+ but entry is solely through channels which are blocked by the Ca2+ entry blockers studied. Contraction to histamine and acetylcholine persisted following exposure of the tissues to Ca2+ free Krebs' solution but contractile activity was virtually abolished in Ca2+ free Krebs' solution containing EGTA. Residual contractions to histamine and part of the residual contractions to acetylcholine in Ca2+-free Krebs' solution were blocked by low dose N (0.3 microM) or D600 (2.1 microM). These findings suggest a major role for extracellular Ca2+ during spasmogen-induced contraction in this tissue.     

Effects of verapamil on lipolysis due to dibutyryl cyclic AMP.

The effects of verapamil, a calcium antagonist, on lipolysis in isolated rat adipocytes were studied. Verapamil (100 microM) potentiated lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) at submaximal concentrations, with or without extracellular Ca2+. Lipolysis due to 0.5 mM-Bt2cAMP was potentiated by verapamil in a dose-dependent manner up to 200 microM, whereas at concentrations higher than 100 microM the stimulatory effect of verapamil was progressively diminished with or without extracellular Ca2+. Verapamil showed only an inhibitory effect on lipolysis due to adrenaline (0.1-10 microM) or 3-isobutyl-1-methylxanthine (IBMX; 25-200 microM). The stimulatory effect of verapamil on lipolysis due to Bt2cAMP was not blocked by alpha-adrenergic antagonists. These results suggest (i) that verapamil has a biphasic effect on lipolysis due to Bt2cAMP and only an inhibitory effect on that due to adrenaline or IBMX, and (ii) that extracellular Ca2+ or alpha-adrenergic receptors are not involved in the action of verapamil.     

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

The Effect of Antibodies to Gangliosides on Ca2+ Channel-Linked Release of γ-Aminobutyric Acid in Rat Brain Slices

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.     

Methylxanthines stimulate calcium transport and inhibit cyclic nucleotide phosphodiesterases in abalone sperm

Experiments were designed to determine the mechanism by which methylxanthines elevate abalone sperm cAMP concentrations and induce the acrosome reaction (AR). Theophylline or, more effectively, 1-methyl-3-isobutylxanthine (MIX) inhibit the cyclic nucleotide phosphodiesterase activities of abalone sperm homogenates. 45Ca2+ uptake by sperm is also stimulated by theophylline, and more effectively by MIX, and this stimulatory effect is blocked by KCN. Verapamil, a compound known to antagonize Ca2+ conductance, has no effect on the Ca2+ or MIX-induced cAMP elevation at concentrations up to 200 microM. However, verapamil reduces the sperm cAMP elevation caused by the addition of Ca2+ plus MIX. This inhibition is not complete, even at 200 microM verapamil. The AR induced by Ca2+ plus MIX is completely inhibited by 200 microM verapamil. The data suggest that these methylxanthines elevate abalone sperm cyclic nucleotide concentrations by inhibiting cyclic nucleotide phosphodiesterase activities. Furthermore, since sperm cAMP metabolism is modulated by Ca2+ flux, methylxanthines also appear to elevate abalone sperm cAMP concentrations by their effects on Ca2+ transport. The Ca2+-induced cAMP elevation occurs through a verapamil-insensitive mechanism, whereas the potentiation by MIX of the Ca2+ effect to elevate cAMP occurs through both verapamil-insensitive and -sensitive mechanisms. The methylxanthine-induced AR is mediated by a primary effect on Ca2+ transport and occurs through a verapamil-sensitive mechanism. Cyclic AMP may play a role in the methylxanthine-induced AR, but does not appear to act as the primary mediator of this exocytotic event.     

Excitatory Amino Acid Receptors and Depolarization-Induced Ca2+ Influx into Hippocampal Slices

Hippocampal brain slices were incubated with depolarizing agents or excitatory amino acids either alone or in the presence of excitatory amino acid antagonists [omega-phosphonic alpha-aminocarboxylic acids--2-amino-4-phosphonobutyric acid (AP4), 2-amino-5-phosphonovaleric acid (AP5), or 2-amino-7-phosphonoheptanoic acid (AP7)--or gamma-D-glutamylaminomethylsulphonic acid (GAMS)] or a calcium-channel blocker, (S)-1-(3-methoxyphenyl)-3-methylaza-7-cyano-7-(3,4-dimethoxyphenyl )-8-methyl- nonane hydrochloride [(-)-D888]. The uptake of 45Ca2+ and the efflux of glutamate or aspartate induced by veratrine or high K+ was blocked (54-76%) by AP7 (IC50 46-250 microM). AP5 and AP4 were less effective. (-)-D888 (10 microM) caused 100% block of evoked 45Ca2+ uptake. Uptake of 45Ca2+ induced by exogenous glutamate, aspartate, and N-methyl-D-aspartate (NMDA) was also inhibited by AP7, whereas GAMS completely blocked the action of kainate and partially blocked that of glutamate. The action of NMDA in stimulating 45Ca2+ uptake was Mg2+-sensitive, low Mg2+ levels in the incubation medium selectively enhancing the response. It is concluded that Ca2+ uptake evoked by excitatory amino acids is receptor-mediated, and that released excitatory amino acids are responsible for a large part of the action of veratrine and high K+ in stimulating 45Ca2+ uptake.     

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.     

[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

Potassium-mediated stimulation of hepatic glycogenolysis

Increased extracellular potassium concentrations ([K+]o) stimulated transient increases in glucose release and 45Ca2+ washout in the perfused rat liver. Stimulated glucose release had a K0.5 of about 26 mM for [K+]o, was not desensitized by successive infusion intervals of increased [K+]o, was not affected by altering the direction of perfusion, was absolutely dependent on the presence of [Ca2+]o, and was blocked by 2 mM cobalt or 10 microM verapamil. The increase in 45Ca2+ washout resulting from increased [K+]o also was blocked by 2 mM cobalt or 10 microM verapamil. Inhibitors of vascular tone (nitroprusside, atriopeptin II), arachidonic acid metabolism (indomethacin, nordihydroguaiaretic acid), and alpha- or beta-adrenergic or muscarinic nerve stimulation/secretion (phentolamine, propranolol, atropine) were unable to inhibit the [K+]o-stimulated glucose release. ATP, ADP, and AMP concentrations in tissue freeze-clamped 2 min after the onset of infusion of 50 mM K+ were not significantly different from control tissue. Glucose release from freshly isolated suspensions or primary cultured monolayers of hepatocytes or from liver slices, all of which responded to glucagon or phenylephrine, did not respond to increased [K+]o. The results indicate that glycogenolysis stimulated by depolarizing gradients of K+ is dependent on an intact perfused vasculature and may be mediated by potential-sensitive Ca2+ channels present in the vascular endothelium of the liver.     

Cadmium uptake and toxicity via voltage-sensitive calcium channels

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.     

The effect of Lambert-Eaton myasthenic syndrome antibody on slow action potentials in mouse cardiac ventricle

Immunoglobulin G (IgG) from Lambert-Eaton myasthenic syndrome (LEMS) patients acts at motor nerve terminal Ca2+ channels. It was injected into mice to investigate effects on cardiac Ca2+ channels. Intracellular recordings were made of slow action potentials in right ventricular muscle cells in the presence of high K+ concentrations and isoprenaline (1 microM). Reduction in Ca2+ concentration reduced the rate of rise and amplitude, but not the duration, of slow action potentials whereas verapamil (1 microM) blocked them. They were not blocked by tetrodotoxin (10 microM), and 4-aminopyridine (1 mM) prolonged the decay phase without affecting the rate of rise and amplitude. The rate of rise, amplitude and duration of slow action potentials were not affected by LEMS IgG. These results show that LEMS IgG does not act on Ca2+ channel currents that underlie slow action potentials in mouse ventricles, suggesting antigenic differences between Ca2+ channels at motor nerve terminals and heart.     

The actions of diazepam and diphenylhydantoin on fast and slow Ca2+ uptake processes in guinea pig cerebral cortex synaptosomes

The activities of diazepam and diphenylhydantoin as inhibitors of the fast and slow phases of 45Ca2+ uptake in response to K+ depolarization and of [3H]nitrendipine binding were examined in guinea pig cerebral cortex synaptosomes. The slow phase of 45Ca2+ uptake was abolished in Na+-free media (choline substitution) and was more sensitive to inhibition by 3,4-dichlorobenzamil and represents a Na+-dependent Ca2+ uptake process. The fast component of uptake represents activation of voltage-dependent Ca2+ channels. Diazepam (to 300 microM) was selectively active against the fast component of 45Ca2+ uptake. The benzodiazepines Ro 11-3624 and Ro 11-3128 were similarly selective with a modest stereoselectivity against the fast component of 45Ca2+ uptake. Diphenylhydantoin (100 and 200 microM) blocked nonselectively both fast and slow phases of Ca2+ uptake. Diazepam (60 microM) and diphenylhydantoin (200 microM) blocked [3H]nitrendipine binding in a competitive manner. Diazepam and diphenylhydantoin probably exert at least part of their anticonvulsant activity by inhibition of voltage-dependent Ca2+ channels.     

Novel tacrine derivatives that block neuronal calcium channels

A new series of tacrine (9-amino-1,2,3,4-tetrahydroacridine) derivatives were synthesized and their effects on 45Ca(2+) entry into bovine adrenal chromaffin cells stimulated with dimethylphenylpiperazinium (DMPP) or K(+), studied. At 3 microM, compound 1 did not affect (45)Ca(2+) uptake evoked by DMPP. Compounds 14, 15 and 17 inhibited the effects of DMPP by 30%. Compounds 3, 9 and tacrine blocked the DMPP signal by about 50%. Compounds 5 and 12 were the most potent blockers of DMPP-stimulated 45Ca(2+) entry (90%); the rest of the compounds inhibited the effects of DMPP by 70-80%. Compounds 1, 3, 4, 8, 10, 11, 13, 16, 17 and tacrine inhibited 45Ca(2+) uptake induced by K(+) about 20%. Compounds 6, 14 and 15 inhibited the K(+) effects by 10% or less. Compounds 7, 9, 12 and 18 blocked the K(+) signal by 30% and, finally, compounds 2 and 5 inhibited the K(+)-induced 45Ca(2+) entry by 50%. None of the new compounds was as effective as diltiazem (IC(50)=0.03 microM) in causing relaxation of the rat aorta precontracted with 35 mM K(+); the most potent was compound 7 (IC(50)=0.3 microM). Compounds 5, 6, 8, 9, 10 and 13 had IC(50)s around 10 microM and compounds 3, 4, 11 and 12 around 20 microM. Blockade of Ca(2+) entry through neuronal voltage-dependent Ca(2+) channels, without concomitant blockade of vascular Ca(2+) channels, suggests that some of these compounds might exhibit neuroprotectant effects but not undesirable hemodynamic effects.     

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 influx mediated by nicotinic receptors and voltage sensitive calcium channels in SK-N-SH human neuroblastoma cells

When SK-N-SH human neuroblastoma cells were exposed to nicotine (NIC) or KCl they showed a dose-dependent transient increase (2- to 4-fold) in intracellular Ca2+ concentration ([Ca2+])i as detected by quin-2 fluorescence, with half maximal effects (EC50) observed at 13 microM and 26 mM, respectively. Tubocurarine and 1-isodihydrohistrionicotoxin potently blocked the NIC-evoked (IC50 congruent to 1 microM and 0.3 microM, respectively), but not the high [K+]o-evoked [Ca2+]i accumulation. The KCl-induced response was inhibited by verapamil and diltiazem (IC50 = 1.4 and 10.9 microM, respectively). Tetrodotoxin (3 microM) and tetraethylammonium (10 microM) had no effect on [Ca2+]i accumulation induced by either agent. Increases in [Ca2+]i could be evoked sequentially by NIC and KCl in the same cells suggesting independent mechanisms of Ca2+ entry. In a Ca2+-free medium, no response to either KCl or NIC was observed. However, when Ca2+ ions were restored, [Ca2+]i accumulation was enhanced to the same extent as cells suspended in a Ca2+-containing buffer. Long-term (18 hr) pretreatment of SK-N-SH cells with pertussis (100 ng/ml) or cholera toxins (10 nM) had no effect on NIC or KCl-induced [Ca2+]i accumulation. Together, these data demonstrate the presence of NIC receptors and voltage-sensitive Ca2+ channels on SK-N-SH neuroblastoma cells, through which [Ca2+]i may be modulated.     

Direct binding of verapamil to the ryanodine receptor channel of sarcoplasmic reticulum.

Radioligand binding experiments and single channel recordings demonstrate that verapamil interacts with the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum of rabbit skeletal muscle. In isolated triads, verapamil decreased binding of [3H]Ryanodine with an IC50 of approximately 8 microM at an optimal pH 8.5 and pCa 4.3. Nitrendipine and d-cis-diltiazem did not interfere with binding of [3H]Ryanodine to triads, suggesting that the action of verapamil does not involve the dihydropyridine receptor. Single channel recordings showed that verapamil blocked Ca2+ release channels by decreasing open probability, duration of open events, and number of events per unit time. A direct interaction of verapamil with the ryanodine receptor peptide was demonstrated after purification of the approximately 400 kDa receptor protein from Chaps-solubilized triads. The purified receptor displayed high affinity for [3H]Ryanodine with a Kd of approximately 5 nM and a Bmax of approximately 400 pmol/mg. Verapamil and D600 decreased [3H]Ryanodine binding noncompetitively by reducing the Bmax. Thus the presence of binding sites for phenylalkylamines in the Ca2+ release channel was confirmed. Verapamil blockade of Ca2+ release channels may explain some of the paralyzing effects of phenylalkylamines observed during excitation-contraction coupling of skeletal muscle.     

Ca2+ channel blockers inhibit secretory C1-channels in intestinal epithelial cells.

Outwardly rectifying Cl- channels are present in the human colonic cell line (HT29D4). The classical Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoate inhibits Cl- channel activity with a K0.5 value of 20 microM. Epithelial Cl- channel activity is inhibited by Ca2+ channel blockers. Phenylalkylamines are the most effective inhibitors. (+/-)Verapamil and (-)desmethoxyverapamil induce flickering and then the complete blockade of Cl- channels recorded from outside-out patches. K0.5 values are 60 microM and 100 microM for (-)desmethoxyverapamil and (+/-)verapamil, respectively. Other classes of L-type Ca2+ channel blockers have also been studied but they are less active.     

Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules

The purified calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubule membrane consists of three subunits: alpha with Mr 135 000, beta with Mr 50 000, and gamma with Mr 33 000. Purified receptor preparations were incorporated into phosphatidylcholine (PC) vesicles by addition of PC in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and removal of detergent by molecular sieve chromatography. Forty-five percent of the alpha, beta, and gamma polypeptides and the [3H]dihydropyridine/receptor complex were recovered in association with PC vesicles. The rate of dissociation of the purified and reconstituted dihydropyridine/receptor complex was identical with that in T-tubule membranes, and allosteric modulation by verapamil and diltiazem was retained. The reconstituted calcium antagonist receptor, when occupied by the calcium channel activator BAY K 8644, mediated specific 45Ca2+ and 133Ba2+ transport into the reconstituted vesicles. 45Ca2+ influx was blocked by the organic calcium antagonists PN200-110 (K0.5 = 0.2 microM), D600 (K0.5 = 1.0 microM), and verapamil (K0.5 = 1.5 microM) and by inorganic calcium channel antagonists (La3+ greater than Cd2+ greater than Ni2+ greater than Mg2+) as in intact T-tubules. A close quantitative correlation was observed between the presence of the alpha, beta, and gamma subunits of the calcium antagonist receptor and the ability to mediate 45Ca2+ or 133Ba2+ flux into reconstituted vesicles. Comparison of the number of reconstituted calcium antagonist receptors and functional channels supports the conclusion that only a few percent of the purified calcium antagonist receptor polypeptides are capable of mediating calcium transport as previously demonstrated for calcium antagonist receptors in intact T-tubules.     

Depressant action of Ca-antagonists on slow action potentials in guinea pig ventricular muscles

The depressant action of four Ca antagonists, including a novel drug, tiapamil, on Ca channels was investigated using a conventional microelectrode technique. "All or none" slow action potentials were recorded in K+-depolarized guinea-pig papillary muscles. Verapamil and diltiazem decreased the amplitude and maximum rate of rise (Vmax) of the slow action potentials at concentrations up to 2 microM. The depressant effect of a novel Ca-antagonist, tiapamil, on the slow action potentials was as marked as that of verapamil and diltiazem. However, prenylamine was less potent than the other 3 drugs. In addition, the action of all drugs on the slow action potentials was enhanced as the frequency of stimulation was increased between 0.0083 and 1 Hz. It was concluded that tiapamil, as verapamil and diltiazem, produced a frequency-dependent blockade of the slow Ca channel.