Taurine transport in skate hepatocytes. II. Volume activation, energy, and sulfhydryl dependence

Abstract

Isolated skate (Raja erinacea) hepatocytes swollen in hypotonic media exhibited a regulatory volume decrease (RVD) that was associated with only a small increase in K+ or 86Rb+ efflux but a substantial increase in the release of taurine, an amino acid found in high concentrations in skate hepatocytes. Taurine efflux was stimulated in media made hypotonic by addition of H2O or removal of NaCl, as well as in cells swollen in isotonic media containing rapidly penetrating solutes (202 mM ethylene glycol or 202 mM additional urea substituted for 101 mM NaCl), suggesting that cell swelling rather than hyposmolarity is the stimulus for the activation of taurine release. In contrast, release of glutathione, L-[14C]alanine and other alpha-amino acids (e.g., threonine, serine, glutamate, glutamine, glycine, or valine) was unaffected by dilution with 40% H2O. Taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ and was only slightly diminished by replacing Cl- with NO3-. Addition of 50 mM taurine or hypotaurine to the incubation media also had no effect on volume-stimulated [14C]taurine efflux, suggesting that the taurine concentration gradient across the plasma membrane is not the driving force. Volume-stimulated taurine transport was temperature sensitive, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibitable (0.5 mM), and nearly completely blocked by metabolic inhibitors (2,4-dinitrophenol, KCN, sodium azide, oligomycin, carbonyl cyanide m-chlorophenylhydrazone, and antimycin A), suggesting an active energy-dependent process. Sulfhydryl-reactive reagents (N-ethylmaleimide, diamide, iodoacetate, tert-butyl hydroperoxide, and mercury) also blocked volume-stimulated taurine efflux, whereas efflux was unaffected by Ca2+ ionophore, phorbol ester, dibutyryl-adenosine 3',5'-cyclic monophosphate, vasopressin, or pretreatment with ouabain or furosemide. N-ethylmaleimide, diamide, 2,4-dinitrophenol, and iodoacetate plus KCN also inhibited the RVD. These findings suggest that, in contrast to hepatocytes from most vertebrate species, RVD in skate hepatocytes is associated with the release of only a small fraction of intracellular K+ but a substantial fraction of intracellular taurine and perhaps other organic osmolytes. This volume-activated taurine transport mechanism is energy and sulfhydryl group dependent and is not related to the taurine concentration gradient across the skate hepatocyte plasma membrane.