Effects of tyramine and calcium on the kinetics of secretion in intact and electroporated chromaffin cells superfused at high speed

Abstract

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K⁺ glutamate-based solution containing 50 nM free Ca²⁺ and 2 mM adenosine 5′-triphosphate, dipotassium salt (K₂ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca²⁺ concentrations ([Ca²⁺]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca²⁺] (3–100 μM), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200–400 vesicles per cell. Pretreatment of cells with tyramine (10 μM for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca²⁺] in the range of 0.1–3 μM, the activation rate of secretion increased from 2.3 to 35.3 nA · s⁻¹, reached a plateau between 3–30 μM and rose again from 100 to 1000 μM [Ca²⁺] to a maximum of 91.9 nA · s⁻¹. In contrast, total secretion first increased (0.1–1 μM Ca²⁺), then plateaud (1–100 μM Ca²⁺) and subsequently decreased (100–1000 μM Ca²⁺). At 30 and 1000 μM extracellular [Ca²⁺] or [Ca²⁺]_o, the activation rates of secretion from intact cells depolarised with 70 mM K⁺were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 μM Ca²⁺) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 μA · s at 1000 μM Ca²⁺) than in electroporated cells (42.4 μA,s at 1 μM Ca²⁺). Separation of the time-courses of changes in intracellular [Ca²⁺] or [Ca²⁺]_i and secretion in intact chromaffin cells depolarised with 70 mM K⁺was demonstrated at different [Ca²⁺]_o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca²⁺]_i. In contrast, the decline of secretion was faster than the decline of the peak [Ca²⁺]_i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca²⁺ by the rates of opening and closing of voltage-dependent Ca²⁺ channels of the L-and Q-subtypes, which control the local [Ca²⁺]_i near to exocytotic sites.