Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells

摘要

class="enumerated" style="list-style-type:decimal">In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca²⁺ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca²⁺ channel subtypes undergo different patterns of inactivation during cell depolarization.The net Ca²⁺ uptake (measured using ⁴⁵Ca²⁺) into hyperpolarized cells (bathed in a nominally Ca²⁺-free solution containing 1·2 mM K⁺) after application of a Ca²⁺ pulse (5 s exposure to 100 mM K⁺ and 2 mM Ca²⁺), amounted to 0·65 ± 0·02 fmol cell⁻¹; in depolarized cells (bathed in nominally Ca²⁺-free solution containing 100 mM K⁺) the net Ca²⁺ uptake was 0·16 ± 0·01 fmol cell⁻¹.This was paralleled by a dramatic reduction of the increase in the cytosolic Ca²⁺ concentration, [Ca²⁺]i, caused by Ca²⁺ pulses applied to fura-2-loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells.A similar decrease was observed when studying catecholamine release. Secretion was decreased when K⁺ concentration was increased from 1·2 to 100 mM; the Ca²⁺ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells.The inactivation by depolarization of Ca²⁺ entry and secretion occluded the blocking effects of combined ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca²⁺ channels.This was strengthened by two additional findings: (i) nifedipine (3 μM), an L-type Ca²⁺ channel blocker, suppressed the fraction of Ca²⁺ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L-type Ca²⁺ channel ‘activator’, dramatically enhanced the entry of Ca²⁺ and the secretory response in depolarized cells.In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca²⁺ channel current carried by 10 mM Ba²⁺ (IBa). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176.Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.