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Primary cultures of bovine adrenomedullary cells actively take up ascorbic acid and alpha-aminoisobutyric acid (AIB). Following a brief incubation with L-[14C] ascorbic acid and alpha-[methyl-3H]aminoisobutyric acid, cells stimulated with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide or by membrane depolarization with high [K+] or veratridine released newly acquired ascorbic acid (NA-ascorbate) and AIB. NA-ascorbate and endogenous catecholamines are differentially released under a variety of conditions suggesting that release of both substances cannot originate from the same subcellular compartment. In contrast, the release profile for NA-ascorbate and AIB, a putative cytosolic marker, suggest that both of these molecules are released from a cytosolic compartment. Cells permeabilized with the detergent digitonin release catecholamines only in the presence of external Ca2+, whereas release of NA-ascorbate and AIB is Ca2+-independent and time- and detergent concentration-dependent. If the osmolality of the external medium is made either hyper- or hypoosmotic, 1,1-dimethyl-4-phenylpiperazinium iodide-induced release of endogenous catecholamines is inhibited. Release of NA-ascorbate and AIB, however, is progressively inhibited with increasing osmolality and enhanced with decreasing osmolality. Furthermore, differential release of NA-ascorbate and AIB as compared to soluble acetylcholinesterase, which is apparently released form the cisternae of the endoplasmic reticulum, was also observed. To determine the mechanism by which NA-ascorbate and AIB are released from the cell, the requirements for their maximal release were investigated. Release of NA-ascorbate and AIB was sensitive to inhibitors (both metabolic and transport) and to changes in the external ionic environment. The metabolic inhibitors carbonyl cyanide p-trifluoromethoxyphenylhydrazone and KCN (when incubated simultaneously with 2-deoxyglucose) inhibited NA-ascorbate and AIB release by greater than 75%. In contrast, the Na+-K+-ATPase inhibitor ouabain enhanced veratridine-induced release of NA-ascorbate by nearly 100% and had an even greater effect on AIB release. Changes in the external ionic environment (i.e. Na+ and/or Cl- substitution) inhibited both NA-ascorbate and AIB release to varying degrees. Substitution of Cl- by various anions inhibited NA-ascorbate and AIB release to a much greater degree than endogenous catecholamine release. Complete substitution of NaCl with sucrose inhibited release of NA-ascorbate and AIB release by greater than 80%, while Na+ substituted with Li+ inhibited release of all three molecules by about 50%.(ABSTRACT TRUNCATED AT 400 WORDS) |
