Muscarinic Activation Inhibits T-Type Ca²⁺ Current in Hen Granulosa Cells

The effect of the muscarinic agonist carbachol on Ca²⁺ currents in hen granulosa cells isolated from the largest ovarian follicle was studied. The major Ca²⁺ current observed using the perforated patch technique with a recording solution containing 10 m.M Ca²⁺, but no Na⁺ or K⁺, exhibited characteristics typical of T-type Ca²⁺ current: maximal amplitude at -20 mV, rapid inactivation (half-time of 42 ± 3 msec at -20 mV), inhibition by 100 μM Ni²⁺, and insensitivity to the dihydropyridine Ca²⁺ channel antagonist, nifedipine. In all cells studied, carbachol (0.5 mM) caused an inhibition of this current (elicited by depolarizing pulses from -70 to -20 mV) to an average maximal decrease of 90 ± 2% below basal values. In some 50% of the cells, the Ca²⁺ current also partially recovered during the 10-min exposure to the muscarinic agonist. These effects were prevented by the muscarinic antagonist atropine (1 μM). To test whether this inhibition was due to increases in intracellular free Ca²⁺ concentrations ([Ca²⁺]_i), [Ca²⁺]_i was simultaneously measured in fura-2-loaded cells. For cells incubated in normal solution, [Ca²⁺]_i was 0.15 ± 0.02 μM, but increased to 0.25 ± 0.6 μM in cells exposed to the recording solution. Under these conditions, carbachol failed to produce the expected [Ca²⁺]_i transients, but, rather, caused a small decrease (8 ± 2%) in basal [Ca²⁺]_i attributable to its diminution of Ca²⁺ current. Thus, the results demonstrated an important muscarinic inhibition of the T-type Ca²⁺ current not related to [Ca²⁺]; fluctuations. They indicate, on the other hand, that [Ca²⁺]_i can strongly modulate carbachol-induced mobilization of Ca²⁺ from the intracellular stores.