Search

The ability of adrenal chromaffin cells to fast-release catecholamines relies on their capacity to fire action potentials (APs). However, little attention has been paid to the requirements needed to evoke the controlled firing of APs. Few data are available in rodents and none on the bovine chromaffin cell, a model extensively used by researchers. The aim of this work was to clarify this issue. Short puffs of acetylcholine (ACh) were fast perifused to current-clamped chromaffin cells and produced the firing of single APs. Based on the currents generated by such ACh applications and previous literature, current waveforms that efficiently elicited APs at frequencies up to 20 Hz were generated. Complex waveforms were also generated by adding simple waveforms with different delays; these waveforms aimed at modeling the stimulation patterns that a chromaffin cell would conceivably undergo upon strong synaptic stimulation. Cholinergic innervation was assessed using the acetylcholinesterase staining technique on the supposition that the innervation pattern is a determinant of the kind of stimuli chromaffin cells can receive. It is concluded that 1) a reliable method to produce frequency-controlled APs by applying defined current injection waveforms is achieved; 2) the APs thus generated have essentially the same features as those spontaneously emitted by the cell and those elicited by fast-ACh perifusion; 3) the higher frequencies attainable peak at around 30 Hz; and 4) the bovine adrenal medulla shows abundant cholinergic innervation, and chromaffin cells show strong acetylcholinesterase staining, consistent with a tight cholinergic presynaptic control of firing frequency.

The view that Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC) and through nicotinic receptors for acetylcholine (nAChRs) causes equal catecholamine release responses in chromaffin cells, was reinvestigated here using new protocols. We have made two-step experiments consisting in an ACh prepulse followed by a depolarizing pulse (DP). In voltage-clamped bovine chromaffin cells an ACh prepulse caused a slow-rate release but augmented 4.5-fold the much faster exocytotic response triggered by a subsequent depolarizing pulse (measured with capacitance and amperometry). If the ACh prepulse was given with mecamylamine or in low external Ca(2+), the secretion increase disappeared. This suggests a two-step model for the effects of ACh: (1) meager Ca(2+) entry through nAChRs mostly serves to keep loaded with vesicles the secretory machine; and (2) in this manner, the cell is prepared to respond with an explosive secretion of catecholamine upon depolarization and fast high Ca(2+) entry through VDCC.

Depending on experimental conditions, cell model, and pattern and type of depolarizing stimuli, the relationship between calcium entry ([Ca2+]c) and the release of neurotransmitters and hormones varies from exponential (power of 3-4) to near linear (power of 1.5) or linear function. Here, we present a study using the more physiological stimulation pattern based on acetylcholine (ACh)-like action potentials, in voltage-clamped bovine chromaffin cells, with the perforated-patch configuration of the patch-clamp technique and 2 mM extracellular calcium. Trains of ACh-like action potentials or square depolarizing pulses of increasing length were applied, and calcium currents (ICa), total calcium entry (QCa), and exocytosis (DeltaCm) measured.