dihydropyridines and Pheochromocytoma

In this study, we report the effect of pertussis toxin pretreatment on dihydropyridine modulation of voltage-sensitive calcium channels in PC12 cells. The rise in intracellular calcium concentration caused by potassium depolarization is not affected significantly by pertussis toxin pretreatment. Nicardipine, a dihydropyridine derivative, added either before or after potassium-induced depolarization, reduces the resultant elevation in cytosolic calcium level both in control and in pertussis toxin-treated cells. The dihydropyridine agonist Bay K 8644, when added before potassium, is able to enhance the potassium-induced spike of cytosolic calcium levels, an effect significantly reduced by pertussis toxin pretreatment. Moreover, the addition of Bay K 8644 after potassium holds the intracellular calcium concentration at a cytosolic sustained level during the slow inactivating phase of depolarization. This effect of Bay K 8644 is inhibited by nicardipine. Pertussis toxin pretreatment slightly weakens the effect of Bay K 8644 when added after potassium-induced depolarization, whereas it significantly reduces the nicardipine inhibition of cytosolic calcium rise stimulated by potassium and Bay K 8644, but not by potassium alone. In conclusion, our findings suggest that a pertussis toxin-sensitive guanine nucleotide regulatory protein could be involved in the interaction between dihydropyridine derivatives and voltage-dependent calcium channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenal Gland Neoplasms; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Electrophysiology; Nicardipine; Pertussis Toxin; Pheochromocytoma; Potassium; Tumor Cells, Cultured; Virulence Factors, Bordetella

Topics: Adrenal Gland Neoplasms; Adult; Blood Pressure; Calcium Channel Blockers; Catecholamines; Dihydropyridines; Dose-Response Relationship, Drug; Female; Humans; Hypertension; Isradipine; Male; Middle Aged; Pheochromocytoma

Chronic treatment of PC 12 cells with the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine [5 x 10-8M/5 days] and the activator S Bay K 8644 [5 x 10-7 M/5 days] resulted in up- and down-regulation of 1,4-dihydropyridine binding site density by 29 and 24%, respectively, without change in affinity. These changes in binding site density represent functional changes as indicated by the corresponding changes in K+ depolarization-induced 45Ca2+ uptake and in whole cell currents carried by Ba2+ ions. This homologous regulation of voltage-dependent Ca2+ channels [VDCC] by potent and specific ligands parallels that observed for other classes of membrane receptors.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Barium; Calcium; Calcium Channels; Cell Line; Dihydropyridines; Kinetics; Membrane Potentials; Pheochromocytoma; Radioligand Assay; Rats; Receptors, Nicotinic

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.

Topics: Acetylcholine; Animals; Calcium; Calcium Channels; Dihydropyridines; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Ion Channel Gating; Mollusk Venoms; Neurons; omega-Conotoxins; Pheochromocytoma; Rats; Thionucleotides; Tumor Cells, Cultured

Binding of omega-conotoxin, a peptide toxin specific for some subtypes of voltage-operated calcium channels (VOCCs), was investigated in IMR32 neuroblastoma and PC12 pheochromocytoma cell lines. In both cell types, binding was specific, saturable and of high affinity. Association was rapid and dissociation almost non-existent. Dihydropyridines and verapamil failed to affect toxin binding, while high concentrations of CaCl2 completely antagonized it. Depolarization with high K+ induced a [Ca2+]i rise (revealed by the fura-2 fluorimetric technique) that consisted of an initial (0.5-1 min) peak followed by a prolonged (several minutes) plateau phase. omega-Conotoxin blocked mainly the first phase, while the dihydropyridine Ca2+ channel blocker, nitrendipine, primarily affected the plateau. This result suggests that in the two cell lines investigated, omega-conotoxin acts mainly on a subgroup of VOCCs that is resistant to dihydropyridines.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Calcium Channel Blockers; Calcium Chloride; Dihydropyridines; Humans; Ion Channels; Mollusk Venoms; Neuroblastoma; Nitrendipine; omega-Conotoxins; Pheochromocytoma; Potassium; Rats; Tumor Cells, Cultured

The release of catecholamines from adrenal chromaffin cells is known to be blocked by dihydropyridines, such as nitrendipine, and enhanced by others, such as BAY K8644. On the other hand, release from sympathetic neurons is predominantly insensitive to these agents. Release of [3H]norepinephrine from undifferentiated PC-12 pheochromocytoma cells resembles that from chromaffin cells in that it is extremely sensitive to dihydropyridines. Following differentiation, however, release of catecholamine becomes predominantly insensitive to both nitrendipine and BAY K8644. Under both growth conditions, release remains completely blocked by 3 mM Co2+ or by removal of Ca2+ from the release media. Dose-response curves to K+ show that following differentiation, cells become more sensitive, releasing transmitter at lower K+ concentrations. In contrast, depolarization-induced uptake of 45Ca2+ remains sensitive to dihydropyridines and shows similar sensitivity to K+ stimulation in both growth conditions. These results can be explained by invoking a model involving dihydropyridine-sensitive and -insensitive types of voltage-sensitive calcium channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Bucladesine; Calcium; Cell Differentiation; Dihydropyridines; Nerve Growth Factors; Nifedipine; Nitrendipine; Norepinephrine; Pheochromocytoma; Potassium; Pyridines; Rats; Secretory Rate

Methyl 1,4-dihydro-2, 6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated 45Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 nM BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of 45Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K+, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenal Gland Neoplasms; Animals; Calcium; Cell Line; Dihydropyridines; Dose-Response Relationship, Drug; Nifedipine; Nitrendipine; Pheochromocytoma; Pyridines; Rats