title: Evidence for the release of newly acquired ascorbate and alpha-aminoisobutyric acid from the cytosol of adrenomedullary chromaffin cells through specific transporter mechanisms.
abstract: 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)
authors: Department of Medicinal Biochemistry, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
authors: J
authors: J
authors: Knoth
authors: O H
authors: OH
authors: Viveros
authors: E J
authors: EJ
authors: Diliberto
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism; D000313:Adrenal Medulla / Q000378:metabolism*; D000621:Aminoisobutyric Acids / Q000378:metabolism*; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001692:Biological Transport; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin; D007700:Kinetics
chemical_list: D000621:Aminoisobutyric Acids; D002395:Catecholamines; C100049:2-aminoisobutyric acid; D000110:Acetylcholinesterase; D004072:Digitonin; D001205:Ascorbic Acid