Different effects of low and high dose cardiotonic steroids on cytosolic calcium in spontaneously active hippocampal neurons and in co-cultured glia.

The Na+ pump is crucial for the regulation of [Na+]i (the intracellular Na+ concentration) in all cells. Three Na+ pump alpha subunit isoforms, alpha1, alpha2 and alpha3, are expressed in rat hippocampal neurons, and alpha1 and alpha2 are expressed in glia, but the significance of these isoforms is not understood. We exploited the different ouabain affinities of the Na+ pump alpha subunit isoforms in rat (alpha1, low ouabain affinity; alpha2 and alpha3, high ouabain affinity) to probe their possible physiological roles. Low and intermediate doses (1-10 microM) of ouabain and its readily reversible analog, dihydroouabain, altered the spontaneous elevations of [Ca2+]i (the intracellular Ca2+ concentration) in neurons and induced [Ca2+]i transients in glia. Complete inhibition of all Na+ pump isoforms (>/=100 microM ouabain) caused sustained increases in global neuronal [Ca2+]i in rat neuronal/glial hippocampal co-cultures and transient [Ca2+]i increases in surrounding glia. High dose ouabain was also associated with increased [Na+]i and [H+]i in neurons and glia. In contrast, 1 microM ouabain (a concentration that completely inhibits only alpha2 and alpha3) was not associated with sustained increases in global neuronal [Ca2+]i or the sustained derangements in [Na+]i and [H+]i observed with high dose ouabain. Reduction of [K+]o to 1 mM suppressed the spontaneous [Ca2+]i oscillations in neurons and induced Ca2+ transients in some glia; removal of external K+ induced sustained elevation of neuronal [Ca2+]i. These studies indicate that the alpha1 isoform is the 'housekeeper' required for maintenance of the global Na+ gradient. As suggested by their restricted plasmalemmal distribution, the high ouabain-affinity Na+ pump isoforms may have more specific roles in neurons and glia.