Channel Blocker D600 Research Articles

Calcium entry blockers inhibit contractures induced by the molluscicide Frescon in Lymnaea stagnalis smooth and cross-striated muscles

The molluscicide Frescon induces irreversible contracture in smooth and cross-striated muscles of the freshwater snail Lymnaea stagnalis. This contracture can be inhibited or reversed by elevated Mg 2+ and reduced Ca 2+ concentrations, by the heavy metals Ni 2+, Co 2+, and Mn 2+, by the rare earth La 3+, and by the calcium channel blocker D-600. The removal of extracellular Ca 2+ also prevents the expression of Frescon action. These results are consistent with the hypothesis that Frescon causes an increase in the Ca 2+ permeability of the sarcolemma.

Veratridine and ouabain stimulate calcium-dependent prolactin release.

We have investigated the effects of veratridine, a Na+ channel activator, and ouabain, an inhibitor of Na+-K+-ATPase, on short term (1-h) PRL release from primary cultures of rat anterior pituitary cells and from the rat anterior pituitary cell line GH4C1 in culture. Both compounds should increase intracellular Na+. Veratridine (20-500 microM) and ouabain (0.1-3 mM) stimulated PRL release from normal cells. The stimulation was inhibited by the omission of Ca++ from the release buffer or by preincubation with the calcium channel blocker D600 (20-500 microM), suggesting a role for Ca++ in the action of these compounds. Ouabain (1 mM), but not veratridine (200 microM), stimulated PRL release from GH4C1 cells, an effect that was also inhibited by calcium channel blockers. In the presence of the dopaminergic agonist bromocriptine (30 nM), the amount of stimulated release by veratridine (200 microM) and ouabain (1 mM) was reduced by 50%. The veratridine effect was only partially inhibited by preincubation of the cells with the Na+ channel blocker tetrodotoxin (1 and 10 microM), but the effect was inhibited completely when Na+ in the buffer was replaced by choline, suggesting that the action of veratridine requires extracellular Na+. The results of this study indicate that 1) ouabain- and veratridine-stimulated PRL release are largely dependent on Ca++; 2) veratridine appears to act through a tetrodotoxin-insensitive mechanism; and 3) stimulation of PRL release by these compounds is similar to that by 50 mM KCl and cAMP in its sensitivity to bromocriptine.

Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose.

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINm5F) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101 +/- 5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7 +/- 0.8 min. In contrast to the action of K+, exposure of the RINm5F cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity. This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in beta-cells. Unlike the case in normal beta-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINm5F cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINm5F cells exposed to glucose.

Paralysis of frog skeletal muscle fibres by the calcium antagonist D-600.

The Ca2+ channel blocker D-600 (methoxyverapamil) paralyses single muscle fibres of the frog: fibres exposed to the drug at 7 degrees C give a single K+ contracture after which they are paralysed, unable to contract in response to electrical stimulation or further applications of K+. Paralysed fibres contract in response to caffeine and have normal resting potentials and action potentials. Fibres treated with D-600 at 22 degrees C are not paralysed. Paralysed fibres warmed to 22 degrees C recover contractile properties: they twitch and give K+ contractures. Other workers have shown that D-600 blocks a Ca2+ channel at room temperature; thus, the paralytic action of D-600 is probably mediated by a different membrane protein, perhaps a different Ca2+ channel from that blocked at room temperature. These results suggest that the binding of D-600 can disrupt the mechanism coupling electrical potential changes across the T membrane to Ca2+ release from the sarcoplasmic reticulum.

Calcium-dependent protein phosphorylation and dephosphorylation in intact brain neurons in culture

Preincubation of intact fetal rat brain neurons in culture with 32P i results in the incorporation of 32P i into about 20 specific proteins. Upon stimulation by electrical field stimulation or by K +-induced depolarization, highly significant calcium-dependent increase in phosphorylation of a protein of app. M r 43 000 and decrease in phosphorylation of an app. M r 55 000 protein occur. These changes can be attributed to the entry of Ca 2+ into the cellular cytoplasm since they can occur upon selective permeabilization of the cell membrane to Ca 2+ by the Ca 2+-ionophore A23187 and are not observed upon stimulation of the cells in the presence of the Ca 2+ channel blocker D-600. These data suggest that these phosphoproteins may be involved in the regulation of processes underlying neurotransmitter release.

Interaction of verapamil and other calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors.

To determine the specificity of the previously demonstrated competition of verapamil with radioligand binding to alpha-adrenergic receptors, we examined the interaction of calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors on several tissues. Verapamil competed for [3H] prazosin binding to alpha 1-adrenergic receptors and for [3H]yohimbine binding to alpha 2-adrenergic receptors in several tissues (human platelets, rat kidney and heart, and cultured muscle cells) with dissociation constants of 0.6-6 microM. The calcium channel blockers D600, D591, fendiline, and prenylamine--which are structural analogues of verapamil--also competed for [3H]yohimbine binding to human platelets. Two other calcium channel blockers, diltiazem and nifedipine, did not compete for [3H] yohimbine binding to human platelets or [3H]prazosin binding to membranes prepared from rat ventricles. We used [3H]nitrendipine binding to identify putative calcium channels on rat myocardial membranes. Nifedipine and verapamil blocked these [3H]nitrendipine-binding sites on ventricular membranes, but epinephrine and prazosin did not, indicating that the ventricular alpha 1 receptors and calcium channels are distinct. We found no specific [3H]nitrendipine binding to human platelets. We conclude that the interaction of verapamil with alpha-adrenergic receptors is not receptor subtype or tissue specific, that interaction with alpha-adrenergic receptors is not a property of all calcium channel blockers, and that the interaction of verapamil with alpha-adrenergic receptors and its interaction with calcium channels occur at at least two distinct sites.

Parathyroid hormone secretion in the absence of extracellular free Ca 2+ and transmembrane Ca 2+ influx

The involvement of extracellular Ca 2+ and Ca 2+ influx across the plasma membrane in parathyroid hormone (PTH) secretion was investigated in vitro using a new preparation of bovine parathyroid cells. Incubation of these cells in the presence of 25 μM or 2.5 μM free ambient Ca 2+ induced a maximal rate of PTH secretion. Low free Ca 2+ secretion is not associated with changes in membrane permeability, requires metabolic energy, and is reversible. The Ca 2+ channel blocker D600 had no effect on either 45Ca-influx or PTH secretion in these cells. These results, showing that extracellular Ca 2+ and Ca 2+ influx across the plasma membrane are not required for PTH secretion by parathyroid cells, emphasize the differences in the cellular mechanisms underlying the secretion of PTH vs that of other secretory cells.

Conductance of the slow inward channel in the rabbit sinoatrial node.

The elementary conductance of the slow inward current channel in the rabbit sinoatrial node was measured by analysis of the current fluctuations. The preparations were voltage-clamped to -30 mV, where d infinity and f infinity of the slow inward current (Isi) intersect. In the presence of Ba, which increases Isi and decreases outward currents, a small steady-state component of Isi could be detected. The fluctuations of the current in 10 mM Ba were smaller due to the block of the outward channels. They were further reduced after the inhibition of Isi by the Ca channel blocker D600. The spectral power density distribution of the current fluctuations originating from Isi could be fitted at frequencies less than 30 Hz with a single Lorentzian which was attributed to the inactivation process. The corner frequency was 5.28 +/- 1.16 Hz (n = 10), corresponding to an average open time of the single channel of about 30 msec at -30 mV. The single channel conductance was determined to 6.50 +/- 3.15 pS (S.D., n = 10).

Does the organic calcium channel blocker D600 act from inside or outside on the cardiac cell membrane?

The effects of extra- and intracellularly applied D600 (methoxyverapamil) and D890 (a quarternary derivative) on the action potentials of isolated guinea pig myocytes were compared. We also studied the extracellular myocytes were compared. We also studied the extracellular effects of these drugs on the calcium current (hybride sucrose gap) and contractile force of right ventricular trabeculae of the cat heart. The following results were obtained: 1. In ventricular trabeculae D600 suppressed the calcium current, tension and the plateau of the action potential. In contrast, D890 even in a 50 times higher concentration did not display any effect on these parameters. 2. In single isolated cells external application of D890 did not alter the configuration of the action potential. In contrast, external application of D600 suppressed the plateau and shortened the action potential in a dose-dependent way. 3. Intracellular injection of D600 or D890 strongly lowered the height of the plateau and abbreviated the action potential. The onset of the effects of both drugs was more rapid on intracellular application than that of external D600. Whereas the effect of an intracellular injection of D600 was reversible, that of D890 was not. These results support the hypothesis that the organic calcium channel blocker D600 enters the cell in the uncharged lipid soluble form and reaches its receptor associated with the calcium channel from inside. Because of its inability to pass the hydrophobic cell membrane, D890 is ineffective from outside but displays blocking effects on intracellular application.

Pharmacological study of the contractile activity of leukotriene C4 and D4 on isolated human airway smooth muscle.

Synthetic leukotriene C4(LTC4) and leukotriene D4(LTD4) were examined on isolated human tracheal and bronchial smooth muscle. Both LTC4 and LTD4 produced slowly developing contraction of trachealis and bronchus which were submaximal (70-85% of the carbachol maximum) and were not blocked by 1.0 microM atropine, 5.0 microM mepyramine, or 5.6 microM indomethacin. The EC50's for LTC4 and LTD4 were 10.3 and 1.8 nM on trachealis and 1.8 and 1.7 nM on bronchus, respectively. LTD4 was in excess of 20 000 times more potent than acetylcholine on some bronchial and tracheal tissues. Similar differences in potency were obtained in some tissues when LTC4 and LTD4 were compared with histamine. Tissues pretreated for 30 min with 2.0-4.0 microM FPL-55712 were less reactive to LTC4 and LTD4 than untreated control tissues. Established contractions to LTC4 and LTD4 persisted despite washing with fresh Krebs solution, were partially reversed by FPL-55712 (0.5-10 microM) and the Ca2+ channel blocker D-600 (5-10 microM), but were completely reversed by the catecholamine adrenaline (1.0 microM) and by high concentrations of FPL-55712 (greater than 20 microM). The marked potency of the leukotrienes on human tracheal and bronchial smooth muscle suggests an important role for these lipoxygenase products in respiratory disease.

Biological activities of cystic fibrosis serum. IV. Stimulation of the calcium mediated K+ efflux from rat submandibular gland fragments.

Cystic fibrosis (CF) and heterozygote sera stimulate a significant K+ efflux from rat submandibular gland fragments in the presence of 1 mM ouabain. This sensitive parameter can be maximally stimulated by as little as 0.5% CF serum and is inhibited by the calcium channel blocker D600 and EGTA. Specific receptor blockers propranolol, phenoxybenzamine or atropine do not inhibit the CF serum-stimulated K+ efflux and agonists do not supramaximally stimulate K+ efflux when added with serum. CF serum-induced K+ efflux did not result in the leakage of lactic dehydrogenase into the bathing media nor did it mimic the action of the calcium ionophore A23187 when added in the presence of D600. In addition, ultrafiltrates of CF serum (less than 10,000 daltons) also stimulated K+ efflux from rat submandibular gland tissue fragments.

Fusion of chick embryo skeletal myoblasts: Role of calcium influx preceding membrane union

Extracellular calcium is required for normal progression through the initial recognition-alignment phase of myogenesis. We have found that a net calcium influx into fusion-competent myoblasts is a requisite step in membrane fusion, in addition to the previously demonstrated requirement for extracellular calcium. This conclusion is based on the following evidence: (1) a measurable increase in net calcium influx occurs just prior to fusion; (2) ethylene glycol bis(β-aminoethyl ether) N,N′-teraacetic acid can block the fusion process at a stage after alignment but prior to membrane union; (3) the calcium ionophore A23187 induces precocious fusion; and (4) the calcium channel blocker D600 inhibits cell fusion but not alignment. Based on ultrastructural analyses of other investigators, we suggest a mechanism whereby the increase in intracellular calcium could trigger membrane union between two aligned, aposed, fusion-competent myoblasts.

Effect of calcium on differentiation of Friend leukemia cells

Induction of hemoglobin synthesis of Friend leukemia cells is inhibited by changing the ratio between internal and external Ca 2+ concentrations. The concentration ratio can be successfully manipulated by the addition of the growth medium of (1) Ca 2+ channel blocker D600 (90 n M-4 × 10 2 n M), (2) Ca 2+ ionophore A23187 (1 × 10 2–2 × 10 2 n M), and (3) EGTA at molar concentrations comparable to the Ca 2+ concentration of the medium formulation (3 × 10 2 μ M). The observations suggest that a specific ratio between intra- and extracellular Ca 2+ is required for erythroid differentiation to proceed.

Biological activities of cystic fibrosis serum III. CF serum induced uptake of 45Ca ++ by rabbit tracheal explants

Cystic Fibrosis (CF) serum and its isolated component IgG fraction produce an increased uptake of 45Ca ++ in rabbit tracheal explants when compared to control serum and its isolated IgG fraction. Heterozygote serum also produced an increased uptake of 45Ca ++ but not to the same extent as CF serum. The calcium channel blocker D600 inhibited the CF serum induced uptake of 45Ca ++ indicating that CF serum may be acting on the plasma membrane to produce changes in calcium permeability in rabbit tracheal explants.

Voltage-sensitive calcium channels regulate guanosine 3',5'-cyclic monophosphate levels in neuroblastoma cells.

Veratridine or high potassium concentration increased guanosine 3',5'-cyclic monophosphate (cGMP) levels in neuroblastoma cells of clone N1E-115 without affecting levels of adenosine 3',5'-cyclic monophosphate (cAMP). The increases in cGMP appear to be a direct result of the depolarizing action of these agents and not due to the action of substances released from the cells upon depolarization. The increase in cGMP produced by depolarization was dependent upon extracellular calcium and could be prevented by the calcium channel blockers D600 and cobalt. Carbachol, acting on muscarinic acetylcholine receptors, also caused a calcium-dependent increase in cGMP in these cells. The carbachol and potassium effects were additive from 5 to 100 mM potassium and from 1 to 3 mM calcium. The carbachol response was nearly as sensitive as the potassium response to inhibition by D600 but was much less sensitive to inhibition by cobalt. The results suggest that depolarization increases cGMP levels in these cells by opening voltage-sensitive calcium channels and that activation of muscarinic receptors opens separate, voltage-insensitive calcium channels.