Experiments were conducted to test the effect of 1 microM ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 microM ouabain is sufficient to inhibit the alpha2 Na,K-ATPase, but not the alpha1. Immortalized astrocytes derived from Na,K-ATPase alpha2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 microM ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 microM ouabain, a significantly greater calcium response to ATP was observed. 1 microM ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase alpha2 and alpha1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase alpha1 (rat kidney). In intact astrocytes, 1 microM ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase alpha2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 microM ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase alpha2 isoform has the potential to change calcium signaling and CCE.
In the lens, different cells appear to be specialized such that some have a high capacity for energy-dependent ion transport while others do not. This short review describes the distribution of functional Na,K-ATPase activity and Ca-ATPase activity in the lens. Movement of ions in the extracellular space between lens fibers, a topic studied by David Maurice 25 years ago, is discussed together with cell-to-cell movement of ions which is facilitated by extensive coupling in the lens cell mass. The expression of different Na,K-ATPase and Ca-ATPase isoforms in lens epithelium and fiber cells is considered along with mechanisms that potentially regulate the activity of these transport proteins.
Lens 45Ca uptake was shown to increase when the external calcium concentration was less than 1 mM, suggesting that calcium ions themselves are able to interact with lens membranes in a way that influences the mechanism of calcium permeability. Strontium ions, added to a calcium-deficient bathing solution, were shown to attenuate the increased 45Ca uptake by the lens. There was no evidence for extracellular binding of calcium. Lens membrane permeability to calcium appeared not to be voltage-dependent since 45Ca uptake was not affected when the lens was depolarized by high potassium solutions. Calcium channel blockers were shown not to reduce the increased calcium leak resulting from exposure of the lens to a calcium-deficient medium.
Chlorpromazine (CPZ) concentrations of 5 x 10(-5)M or greater were found to disrupt lens electrolyte and water balance significantly. Lens sodium and calcium levels increased while potassium decreased. These changes were accompanied by water accumulation. Such effects of CPZ were not dependent upon exposure to ultraviolet light. Electrical measurements revealed that CPZ induced depolarization of the lens potential but little change of overall lens conductance. 86Rb efflux from the lens was unaltered by a 1 hr exposure to CPZ. The active sodium pump in the lens was inhibited by CPZ, as evidenced by a reduction in Na- K-ATPase activity.
In the rabbit, cryoextraction of the lens and subsequent storage in Tyrode's solution did not alter the Na,K-ATPase activity from that determined in immediately excised rabbit lenses. Similarly, the procedures employed with the rabbit eye to simulate collection and storage of normal human eyes (eye banking) had no effect upon the Na,K-ATPase activity of the lens. These results permitted the investigation of human lenses with the knowledge that measured Na,K-ATPase activity had not been altered grossly by any manipulation procedures. Analysis of Na,K-ATPase activity in 44 eye bank lenses, 14 primary nuclear cataracts, 11 primary cortical cataracts, 18 primary posterior subcapsular cataracts, and 31 mixed cataracts revealed no significant difference in the enzyme activity between these groups. Similarly, there was no correlation between electrolyte levels and Na,K-ATPase in a further 18 mixed cataracts. It is concluded that, despite an often pronounced electrolyte imbalance, human cataract can develop without significant alteration in Na,K-ATPase activity.
