oxadiazoles and Pheochromocytoma

Biochemical studies have suggested a voltage-dependent dihydropyridine-sensitive catecholamine release in adrenal chromaffin cells. This release is inhibited by activation of alpha 2-adrenergic and muscarinic receptors; the underlying molecular mechanism is not known. We used undifferentiated PC-12 cells to study the effect of epinephrine and carbachol on transmembranous currents. Applying the patch-clamp technique in the whole cell configuration and using Ba2+ as charge carrier, we identified a high voltage-activated Ca2+ channel current. Both epinephrine (10 microM, in the presence of 1 microM propranolol) and carbachol (10 microM) reversibly inhibited the Ca2+ channel current by 30-40%. Yohimbine abolished and clonidine mimicked the effect of epinephrine. Phenylephrine failed to inhibit the Ca2+ channel current. The effect of carbachol was abolished by atropine. Epinephrine and carbachol did not affect the Ca2+ channel current reduced by the dihydropyridine, PN 200-110 (1 microM), suggesting a selective inhibition of dihydropyridine-sensitive Ca2+ channels. The Ca2+ channel current and its inhibition by receptor agonists were not influenced by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM). Pretreatment of cells with pertussis toxin or intracellular infusion of the GDP analogue guanosine-5'-O-(2-thiodiphosphate) was without effects on the control Ca2+ channel current but abolished its hormonal inhibition. Four pertussis toxin-sensitive G proteins were identified in membranes of PC-12 cells: two members of the Gi family, Gi1 and Gi2, and two members of the Go family, Go2 and another Go subtype (possibly Go1). The present data indicate that activated alpha 2-adrenergic and muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ channels via pertussis toxin-sensitive G proteins without the involvement of a cAMP-dependent intermediate step.

Topics: Adrenal Gland Neoplasms; Amino Acid Sequence; Animals; Atropine; Barium; Calcium Channel Blockers; Calcium Channels; Carbachol; Cell Line; Cell Membrane; Enkephalin, Leucine-2-Alanine; Epinephrine; Evoked Potentials; GTP-Binding Proteins; Immune Sera; Isradipine; Membrane Potentials; Molecular Sequence Data; omega-Conotoxins; Oxadiazoles; Peptides; Peptides, Cyclic; Pertussis Toxin; Pheochromocytoma; Propranolol; Rats; Receptors, Adrenergic, beta; Receptors, Muscarinic; Virulence Factors, Bordetella; Yohimbine

Exposure to ethanol for several days increases the expression of dihydropyridine-sensitive, voltage-dependent Ca2+ channels in brain and in the neural cell line PC12. Since protein phosphorylation is a major mechanism by which ion channels are regulated, we used protein kinase inhibitors to investigate whether ethanol-induced up-regulation of Ca2+ channels involves activation of a protein kinase. Sphingosine and polymixin B, which inhibit protein kinase C and calmodulin-dependent kinases, prevented the enhancement of 45Ca2+ uptake induced by exposure of PC12 cells to ethanol for 4 days. In addition, sphingosine blocked the ability of ethanol to increase the number of [3H]dihydropyridine binding sites in PC12 cell membranes. Sphingosine's effect was prevented by simultaneous exposure to phorbol 12,13-dibutyrate, a potent activator of protein kinase C. Therefore, protein kinase C appears to be involved in the up-regulation of dihydropyridine-sensitive Ca2+ channels during prolonged exposure to ethanol.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Cell Membrane; Ethanol; Isradipine; Kinetics; Oxadiazoles; Pheochromocytoma; Phorbol 12,13-Dibutyrate; Potassium; Protein Kinase C; Rats; Sphingosine

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenal Gland Neoplasms; Animals; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Ethanol; Isradipine; Nerve Tissue; Oxadiazoles; Pheochromocytoma; Rats; Receptors, Nicotinic; Stereoisomerism; Time Factors; Tumor Cells, Cultured

We correlated the binding of the dihydropyridines, nitrendipine and PN200-110, with their pharmacological actions on voltage-dependent membrane calcium channels. Binding was studied in clonal rat adrenal medullary cells (PC12) and in plasma membranes prepared from them. Calcium currents were studied using whole cell and single channel patch clamp methods. For both [3H]-(+/-)-nitrendipine and [3H]-(+)-PN200-110, high affinity binding sites with dissociation constants of 0.6 and 0.04 nM, respectively, were identified both in membrane fragments and in intact cells. In crude membrane preparations a low affinity nitrendipine-binding site was also found. The dissociation constant for binding at this site was affected by ionic strength and the presence of divalent cations. In 500 mM KCI, 0.1 mM CaCl2, 50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), the KD is about 70 nM. The number of high affinity binding sites for dihydropyridines was between 30 and 100 fmol/mg of protein while the number of low affinity sites was between 30 and 70 pmol/mg of protein. In whole cells the measured number of high affinity sites was between 2000 and 4000/cell and, by extrapolation from the membrane preparation, the low affinity sites correspond to several million sites per cell. The electrophysiological effects of both of the dihydropyridines on Ca2+ currents were voltage dependent. When nitrendipine was applied, a small increase in calcium current occurred and this was followed by a decrease. The inhibitory effect was more pronounced at depolarized membrane holding potentials and was relieved by hyperpolarizing the membrane, whereas the stimulatory effect was pronounced at negative membrane holding potentials. In 10 nM nitrendipine these effects were also observed in single channels; they were not due to changes in channel conductance or dwell times in the open state but, rather, were due to changes in the probability of opening. The half-maximal inhibitory concentration (IC50) for nitrendipine was 67 nM and the IC50 for the effect of (+)-PN200-110 was 9 nM using protocols which favored the depolarized state of the channel. No excitatory effect was seen. The IC50 from electrophysiological estimates is higher than the KD for the high affinity binding site for both compounds. Using a simple model of voltage-dependent binding, we could not account for the difference. The number of functional channels calculated from the relation between whole cell and single channel calcium cur

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Cell Membrane; Clone Cells; Electrophysiology; Ion Channels; Isradipine; Mathematics; Membrane Potentials; Nitrendipine; Oxadiazoles; Pheochromocytoma; Rats

In view of existing evidence that Ca2+ may be important for tumor cell growth and metastasis, we investigated the effects of three antineoplastic drugs on K+-stimulated 45Ca2+ uptake through voltage-dependent Ca2+ channels of the PC12 neurosecretory cell line. The agents chosen for study (vinblastine, doxorubicin, and tamoxifen) were those previously shown to inhibit Ca2+/calmodulin- or Ca2+/phospholipid-activated protein kinases. Neither vinblastine nor doxorubicin altered 45Ca2+ uptake at concentrations that inhibit these Ca2+-dependent enzymes. However, tamoxifen reduced uptake [50% inhibitory dose, 8.6 +/- 0.9 (SE) microM] and competed for Ca2+ channel antagonist binding sites labeled by [3H]-(+)PN200-110 (ki = 2.2 +/- 0.3 microM). Ca2+ channel antagonist properties may contribute to the effects of antineoplastic agents such as tamoxifen.

Topics: Animals; Calcium; Calcium Channel Blockers; Cell Division; Cell Line; Cell Survival; Isradipine; Oxadiazoles; Pheochromocytoma; Rats; Receptors, Estrogen; Tamoxifen; Tritium