CALCIUM SIGNALING IN ISOLATED SINGLE CHROMAFFIN CELLS OF THE RAINBOW-TROUT (ONCORHYNCHUS-MYKISS)

Chromaffin cells were isolated from the posterior cardinal vein of rai nbow trout (Oncorhynchus mykiss) to assess their suitability as a mode l system for studying mechanisms of catecholamine secretion in fish an d to evaluate intracellular calcium changes associated with cholinorec eptor stimulation. Immunocytochemistry in concert with fluorescence mi croscopy was employed to identify characteristic chromaffin cell prote ins and thus to confirm the presence of these specific cells in suspen sions and cultures. Dopamine-beta-hydroxylase, an enzyme of the catech olamine-synthesising ng Blaschko pathway, was identified in cytoplasmi c vesicles of the isolated chromaffin cells. The actin filament-severi ng protein, scinderin, was co-localized with actin in the sub-plasmale mmal membrane of these chromaffin cells. Intracellular calcium [Ca2+]( i) was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca-?(2+)]( i) were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K+ or by the stimulation o f the cell by the cholinergic receptor agonists, nicotine, acetylcholi ne or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexam ethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible choline rgic agonist, carbachol. These results demonstrate the presence of fun ctional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these c ells as a model system for studying aspects of catecholamine secretion in fish.