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 mM 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 microM Ni2+, and insensitivity to the dihydropyridine Ca2+ channel antagonist, nifedipine. In all cell 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 microM). 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 microM, but increased to 0.25 +/- 0.6 microM 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+]i fluctuations. They indicate, on the other hand, that [Ca2+]i can strongly modulate carbachol-induced mobilization of Ca2+ from the intracellular stores.