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

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.