We have previously demonstrated that rat type-1 cerebellar astrocytes express a very active Na(+)/Ca(2+) exchanger which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of Ca (i) (2+) induced by physiological agonist. In this chapter, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signalling in rat cerebellar astrocytes. Laser-scanning confocal microscopy experiments using immunofluorescence labelling of Na(+)/Ca(2+) exchanger and RyRs demonstrated that they are highly co-localized. The most important finding presented in this chapter is that L-glutamate activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing a Na(+) entry through the electrogenic Na(+)-glutamate co-transporter and not through the ionophoric L-glutamate receptors as confirmed by pharmacological experiments with specific blockers of ionophoric L-glutamate receptors, electrogenic glutamate transporters and the Na/Ca exchange.

SUMMARY1. The efflux of Na in dialysed axons of the squid has been used to monitor the sidedness of the interactions of the Na pump with Na+ ions, K+ ions and ATP. The axons were under conditions such that most of the Na efflux went through the Na pump by means of a complete cycle of ATP hydrolysis.2. With 310 mM-Kj, 70 mM-Nat and 10 mM-K+ artificial sea water (ASW) more than 97 % of the Na efflux was abolished by removal of ATP. The efflux of Na was stimulated by ATP with a Kj of about 200 /1M. This is similar to the KI of 150 /SM found for the ATP dependence of a ouabain-sensitive Na,K-ATPase activity in membrane fragments isolated from squid optical nerves.3. A 100-fold reduction in the ATP concentration (from 3-5 mm to 30-50 /M) increased the apparent affinity of the Na pump for K+ about 8-fold. In addition, the maximal rate of K+-stimulated Na efflux was reduced by a similar factor. Analogous results were seen in axons dialysed with 310 mM-Kt or without Kt.4. The relative effectiveness of external monovalent cations as activators of the Na efflux was a function of the ATP concentration inside the axon. With 3-5 mM-ATP the order of effectiveness was K+ > NH+ > Rb+. With 30-50 ,uM-ATP the sequence was NH4+ > K+ > Rb+. These results were not affected by the removal of Kt.5. When the ATP concentration was 3 mm and the Nat concentration 70 mm, the removal of Kt produced a slight and reversible increase in the total efflux ofNa (15 %) and no change in the ATP-dependent Na efflux. When the ATP concentration was reduced to 30-50 /,tM, or the Nat concentration lowered to 5-10 mm, the removal of Kt reversibly increased the total and the ATP-dependent efflux of Na. The largest increase in Na efflux was seen when both ATP and Nat were simultaneously reduced. The ATP-dependent extra Na efflux resulting from the exclusion of Kt was abolished by 10-4 M-ouabain in the sea waters.6. The increase in the ATP-dependent Na efflux observed in axons dialysed with 0 Kt+ 10 mM-K+ ASW was not seen in axons perfused with 310 mM-Kt+450 mr-K+ ASW. However, both experimental conditions gave rise to a similar (and small) ATP-independent and ouabain-insensitive efflux of Na. This indicates that the effects 0022-3751/81/4720-1160 $07.50 (© 1981 The Physiology Society 458 L. BEA UGE AND R. Di POLO on the Na pump of removing K+ are not due to the simultaneous membrane depolarization. In addition, it suggests that Kt has an inhibitory effect on the Na pump, and that that effect is antagonized by Naj+ and ATP.7. The present results are consistent with the idea that the same conformation of the Na pump (and Na,K-ATPase) can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. This enzyme conformation produces an enzyme-K complex from which K+ ions are not easily released unless high concentrations of ATP are present. This also stresses a non-phosphorylating regulatory role of ATP.

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