Ca(2+)-activated K+ channels modulate muscarinic secretion in cat chromaffin cells

Abstract

1. This study was aimed at testing the hypothesis that Ca(2+)-dependent K+ channels regulate the release of catecholamines mediated by muscarinic stimulation of cat adrenal chromaffin cells. Two parameters were measured: the secretory response to brief pulses of methacholine (100 microM for 10 s) in intact cat adrenal glands perfused at a high rate with oxygenated Krebs solution; and the changes in cytosolic Ca2+ concentrations, [Ca2+]i, produced by puff applications of methacholine pulses (also 100 microM for 10 s) in isolated single cat adrenal chromaffin cells loaded with Fura-2. 2. A pulse of methacholine released 805 +/- 164 ng of catecholamines (mean of thirty-two pulses). d-Tubocurarine (DTC) increased the secretory response in a concentration-dependent manner. The maximum increase (around 1000 ng catecholamines over control values) was reached at 100 microM-DTC and the EC50 was around 10 microM. 3. The secretory responses to methacholine alone, or to the combination of methacholine plus DTC, were strongly dependent on the extracellular Ca2+ concentration, [Ca2+]o. Thus Ca2+o removal from the perfusing solution for 5-10 min abolished catecholamine release. 4. At 0.1 microM, isradipine (an L-type Ca2+ channel blocker) inhibited by 71% the secretory response to DTC plus methacholine. At 1 microM, Bay K 8644 (an L-type Ca2+ channel activator) increased 2-fold the secretory response to DTC plus methacholine (2746 ng of catecholamines). 5. Apamin (1 microM) increased 3.5-fold the secretory response to methacholine pulses (from 500 to 1800 ng of catecholamines). 6. Methacholine pulses enhanced [Ca2+]i from the resting level of 100 nM to a peak of 1000 nM which quickly declined to basal level. DTC (100 microM) enhanced by 20% the [Ca2+]i peak and substantially prolonged its duration. 7. Apamin (1 microM) increased by 60% the [Ca2+]i peak evoked by methacholine, and delayed the initiation of decline of the [Ca2+]i peak. 8. These results are compatible with the idea that muscarinic stimulation depolarizes the cat adrenal chromaffin cell through an unidentified mechanism. Depolarization is probably counteracted by activation of Ca2+i-dependent K+ channels. Therefore, inhibition of these channels enhances depolarization and firing of action potentials which activate voltage-dependent L-type Ca2+ channels to increase further the Ca2+i signal and the secretory response. Thus Ca2+i-dependent K+ channels, probably of the small-conductance type (SK), seem to be involved in the modulation of muscarinic-evoked catecholamine release responses in cat adrenal chromaffin cells.