Astroglial uptake is modulated by extracellular K+.

Primary cultures of rat brain astrocytes were used to examine the uptake of the glucose analogue, 2-deoxy-d-glucose (2-DOG). 2-DOG competes with glucose for uptake, indicating that both are transported by the same carrier system. Extracellular K + at 1 1 . 9 m ~ increased the uptake of 2-DOG at 2-DOG concentrations greater than 100 p ~ . Uptake appears Na+-dependent only at high concentrations of 2-DOG. This suggests that the extracellular concentrations of Na+ and K' may regulate the astrocytic uptake of 2-DOG. THE REGULATION of glucose uptake in the nervous system is poorly understood. Glucose is virtually the sole carbon source for the adult mammalian brain (KETY, 1957; BALAZS, 1970). It is rapidly taken up both in oiuo (KETY, 1957; PARDRIDGE & OLDENDORF, 1975) and in uitro (BACHELARD, 1971; HORTON et al., 1973) and metabolized to amino acids, nucleic acids, and proteins (MAKER et al., 1976). Glucose is transported into brain by either passive or facilitated diffusion (PARDRIDGE & OLDENDORF, 1975), but little is known about the mechanism of, or regulation of glucose uptake in the various cell types in brain. The uptake of glucose may be the rate-limiting step in cerebral glycolytic metabolism (ROLLESTON & NEWSHOLME, 1967; MAKER et al., 1976). It appears to be ion-dependent, requiring both K + and Na+ (SCHULTZ & CURRAN, 1970). This implies that extracellular K + may act as a modulator of glucose uptake in uioo. The close apposition of neuronal and glial membranes (PETERS & PALAY, 1965) gives credence to this possibility. Extracellular K + may reach levels of 9-1 2 mM in the stimulated intact cat cortex, and at the upper limits of [K'l0,,, glial membranes depolarize (SOMJEN, 1975). Increased extracellular K + elevates the O2 consumption of brain slices (MCILWAIN, 1951, 1953; HERTZ & CLAUSEN, 1963), of hand dissected glia (HERTZ, 1966) and of cultured astrocytes (HERTZ, 1973a, h ; HERTZ et a/., 1973). Indeed, HERTZ, (1973a, b) has suggested that astrocytes may be the cerebral cell type most affected by shifts in extracellular K + A technique for the culture of pure populations of non-neoplastic astrocytes (CUMMINS & GLOVER, 1978) has allowed us to explore the control of glucose uptake by K + . We demonstrate here that K + modulates the uptake of the glucose analog 2-deoxy-dglucose (2-DOG) in astrocytes in uitro. MATERIALS AND METHODS The technique for culturing astrocytes from neonatal rat brain has been described elsewhere (CUMMINS & GLOVER. 1978). Briefly, forebrains of 3-4-day-old neonatal rats were dissected under sterile conditions, minced and trypsinized in 0.25% trypsin in Earle's Balanced Salt Solution (BSS). The suspension was centrifuged, and the pellet was washed once in BSS, and diluted to yield 10 60 x 15 mm plates (3002 series, Falcon Plastics) per forebrain. Cells were grown in a medium composed of 10% fetal bovine serum in MEM, with either Earle's or Hank's BSS. The medium was supplemented with 100 U penicillin, 100 pg streptomycin, and lOOU polymyxin per ml medium. Cells were grown in a National incubator, in an atmosphere of 95% air and 5% C02 . Under these conditions, cells grew to confluency in 12-14 days. Glial specific stains were used to aid in identifying and characterizing the confluent cells (CUMMINS & GLOVER, 1978). Radioactive 2-deoxy-d-glucose, C3H(G)], was obtained from New England Nuclear (Boston, MA) and had a specific activity of 10 Ci/mmol. Crystalline 2-DOG was obtained from Sigma Chemical Co., and PL Biochemicals. The purity of both preparations was tested by tlc, and found to contain less than 1.0% glucose. Uptake of 2-DOG was performed on confluent cultures 16-18 days after plating. Sixty millimeter Petri dishes (Falcon Ware, 3002 Series) were used routinely. Dishes were removed from the incubator, media poured off and the edges blotted. The plates were washed with 12-15ml of 0.9% NaCl at room temperature, inverted, and blotted. Two milliliters of warm (37°C) incubating solution containing the desired concentrations of 2-DOG and K ' ions were added and the dishes incubated at 37°C. At the indicated times, the incubation solution was poured off, and plates were rapidly washed 3 times each with approx 20 ml of ice-cold 0.9% NaCI. Two milliliters of 0.4 N-NaOH was immediately added to stop the uptake and digest the cell monolayer (KLETZIEN & PURDUE, 1974). To determine non-specific binding of radioactive 2-DOG, fresh dishes were washed with ice cold 0.9% NaCI.