The export of glutamine from astrocytes, and the uptake of glutamine by neurons, are integral steps in the glutamateglutamine cycle, a major pathway for the replenishment of neuronal glutamate. We review here the functional and molecular identi®cation of the transporters that mediate this transfer. The emerging picture of glutamine transfer in adult brain is of a dominant pathway mediated by system N transport (SN1) in astrocytes and system A transport (SAT/ATA) in neurons. The participating glutamine transporters are functionally and structurally related, sharing the following properties: (a) unlike many neutral amino acid transporters which have proven to be obligate exchangers, these glutamine transporters mediate net substrate transfer energized by coupling to ionic gradients; (b) they are sensitive to small pH changes in the physiological range; (c) they are susceptible to adaptive and humoral regulation; (d) they are related structurally to the AAAP (amino acid and auxin permeases) family of transporters. A key difference between SN1 and the SAT/ATA transporters is the ready reversibility of glutamine¯uxes via SN1 under physiological conditions, which allows SN1 both to sustain a glutamine concentration gradient in astrocytes and to mediate the net outward¯ux of glutamine. It is likely that the ASCT2 transporter, an obligate exchanger of neutral amino acids, displaces the SN1 transporter as the main carrier of glutamine export in proliferating astrocytes.

L-Kynurenine (KYN), an intermediary product in the kynurenine pathway of tryptophan metabolism, is the common precursor from which are formed both quinolinic acid, a potent endogenous "excitotoxin," and kynurenic acid, a nonselective antagonist of excitotoxins. The present work examines 3H-KYN transport in primary astrocyte cultures derived from the cerebra of newborn mice. Influx and efflux of 3H-KYN were attributable almost entirely to carrier-mediated transport. The tritium recovered in uptake experiments was identifiable as 3H-KYN, indicating a low rate of KYN metabolism during incubations up to 30 min. KYN uptake decreased in the presence of extracellular Na+, at least in part because KYN efflux was accelerated. Marked trans stimulation of KYN efflux by extracellular KYN provided evidence of the exchanging nature of the carrier. Saturation curves for the initial velocity of KYN uptake conformed to a 1-component saturable system with Km of 32 microM and Vmax of 2.1 nmol mg-1 protein min-1. KYN was notably concentrated by the astrocytes, with an estimated steady-state distribution ratio of 180-fold for 1 microM KYN. Analog inhibition studies showed that the KYN transporter exhibited a clear preference for large neutral amino acids; leucine, tryptophan, and phenylalanine were recognized with relatively higher affinity than KYN. In summary, KYN is concentratively transported into astrocytes by a Na+-independent exchanger with high affinity for branched-chain and aromatic neutral amino acids. The substrate specificity and high affinity of this transport system resemble the properties of neutral amino acid transport across the blood-brain barrier in the rat and human.

Raising extracellular K+ concentration ([K+]o) induces an alkaline shift of intracellular pH (pHi) in astrocytes. The mechanism of this effect was examined using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in primary cultures of mouse cerebral astrocytes. Raising [K+]o from 3 to 12 mM increased pHi by 0.28 pH units in 26 mM HCO(3-)-buffered solution. In nominally HCO(3-)-free solution (containing approximately 95 microM HCO3-), the alkalinization fell to 0.21 pH units and further to 0.08 pH units on removal of atmospheric CO2, suggesting a process with high affinity for HCO3-. This effect was Na+ dependent, Cl- independent, and inhibited by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, indicating the involvement of Na(+)-HCO3- cotransport. The relationship between pHi and log[K+]o was found to be linear and to predict a stoichiometry of at least two HCO3- transported with each Na+. After removal of exogenous CO2/HCO3-, the direction of changes in pHi elicited by adding 1 mM HCO3- showed that net flux of HCO3- via the Na(+)-HCO3- cotransporter was outward at rest and was reversed by depolarization.

We describe an unconventional response of intracellular pH to NH4Cl in mouse cerebral astrocytes. Rapid alkalinization reversed abruptly to be replaced by an intense sustained acidification in the continued presence of NH4Cl. We hypothesize that high-velocity [Formula: see text] influx persisted after the distribution of ammonia attained steady state. From the initial rate of acidification elicited by 1 mM NH4Cl in bicarbonate-buffered solution, we estimate that [Formula: see text] entered at a velocity of at least 31.5 nmol ⋅ min−1 ⋅ mg protein−1. This rate increased with NH4Cl concentration, not saturating at up to 20 mM NH4Cl. Acidification was attenuated by raising or lowering extracellular K+ concentration. Ba2+ (50 μM) inhibited the acidification rate by 80.6%, suggesting inwardly rectifying K+ channels as the primary[Formula: see text] entry pathway. Acidification was 10-fold slower in rat hippocampal astrocytes, consistent with the difference reported for K+ flux in vitro. The combination of Ba2+ and bumetanide prevented net acidification by 1 mM NH4Cl, identifying the Na+-K+-2Cl−cotransporter as a second [Formula: see text] entry route.[Formula: see text] entry via K+ transport pathways could impact “buffering” of ammonia by astrocytes and could initiate the elevation of extracellular K+concentration and astrocyte swelling observed in acute hyperammonemia.

Glucose utilization in primary cell cultures of mouse cerebral astrocytes was studied by measuring uptake of tracer concentrations of [3H]2-deoxyglucose ([3H]2-DG). The resting rate of glucose utilization, estimated at an extracellular K+ concentration ([K+]o) of 5.4 mM, was high (7.5 nmol glucose/mg protein/min) and was similar in morphologically undifferentiated and "differentiated" (dibutyryl cyclic AMP-pretreated) cultures. Resting uptake of [3H]2-DG was depressed by ouabain, by reducing [K+]o, and by cooling. These observations suggest that resting glucose utilization in astrocytes was dependent on sodium pump activity. Sodium pump-dependent uptake in 2-3-week-old cultures was about 50% of total [3H]2-DG uptake but this fraction declined with culture age from 1 to 5 weeks. Uptake was not affected by changes in extracellular bicarbonate concentration ([HCO3-]o) in the range of 5-50 mM but was significantly reduced in bicarbonate-free solution. At high [HCO3-]o (50 mM) uptake was insensitive to pH (pH 6-8), whereas at low [HCO3-]o (less than 5 mM) uptake was markedly pH-dependent. Elevation of [K+]o from 2.3 mM to 14.2-20 mM (corresponding to extremes of the physiological range of [K+]o) resulted in a 35-43% increase in [3H]2-DG uptake that was not affected by culture age or by morphological differentiation. Our results indicate a high apparent rate of glucose utilization in astrocytes. This rate is dynamically responsive to changes in extracellular K+ concentration in the physiological range and is partially dependent on sodium pump activity.