omega-conotoxin-(conus-magus) 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

Undifferentiated rat pheochromocytoma PC12 cells were voltage clamped using the whole cell technique. After blockade of outward currents, calcium currents were elicited from -40 and -100 mV. A subpopulation of cells displayed only one current component activated at -10 mV and slowly decaying. In other cells this current coexisted with a component activated around -40 mV and decaying with a faster time constant. We conclude that undifferentiated PC12 cells can express two types of calcium channels, L (long-lasting) and N (neuronal)-type channels.

Topics: Adrenal Gland Neoplasms; Animals; Calcium Channel Blockers; Calcium Channels; Cell Line; Electric Conductivity; Membrane Potentials; Mollusk Venoms; Nitrendipine; omega-Conotoxins; Pheochromocytoma; Rats; Tumor Cells, Cultured

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