OSMOTIC SWELLING STIMULATES ASCORBATE EFFLUX FROM CEREBRAL ASTROCYTES

Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromo dulator, and antioxidant that is stored at millimolar concentrations i n the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effe cts of osmotic stress on astrocytic transport of ascorbate, Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 m in) increased by incubation in hypotonic medium, Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium, Swelling-induced ascorbat e efflux was inhibited by the anion-transport inhibitors 4,4'-diisothi ocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2',7'-bis(carboxyeth yl)-5-(or -6)-carboxyfluorescein (BCECF), was retained in the swollen astrocytes, Na+-dependent ascorbate uptake into astrocytes was inhibit ed slightly during the first minute of hypotonic stress, indicating th at the sodium ascorbate cotransporter does not mediate swelling-induce d efflux, Cell concentration of authentic ascorbate was measured by HP LC with electrochemical detection. When astrocytes were incubated in a scorbate-free medium, hypotonicity decreased cell ascorbate concentrat ion by 50% within 3 min. When astrocytes were incubated in ascorbate-s upplemented hypotonic medium, intracellular ascorbate concentration wa s restored within 10 min because uptake remained effective, Many patho logical conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may cont ribute to cellular osmoregulation in the short-term and the scavenging of reactive oxygen species.