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We compared Ca2+ signaling and inositol polyphosphate metabolism in NIH-3T3 cells stably transfected with cDNA encoding either the wild-type G protein G16 alpha subunit or a GTPase-deficient alpha 16 subunit (Q212L-alpha 16). Constitutive activation of phosphoinositidase C (PIC) in cells expressing Q212L-alpha 16 was demonstrated by 1) an increased basal level of [3H]inositol polyphosphates, 2) an enhanced rate of [3H]inositol polyphosphate accumulation in cells treated with 10 mM LiCl, and 3) an increased rate of incorporation of [3H]inositol into cell lipids. Q212L-alpha 16 cells had a diminished cell growth rate. Basal intracellular Ca2+ concentration was equivalent in Fura-2 acetoxymethyl ester-loaded Q212L-alpha 16 cells compared with controls; however, calcium release in Q212L-alpha 16 cells exposed to ionomycin, ATP (a G protein-linked agonist), or platelet-derived growth factor (a tyrosine kinase-linked agonist) was decreased. Permeabilized, 45Ca-loaded Q212L-alpha 16 cells released less 45Ca at each concentration of inositol-1,4,5-trisphosphate than did control cells. Accordingly, the total amount of inositol trisphosphate (IP3) receptor protein was decreased in Q212L-alpha 16 cells relative to controls. These data demonstrate that Q212L-alpha 16 cells maintain physiological levels of cytoplasmic calcium and partially loaded Ca2+ stores in the face of constitutively active PIC. This is accomplished, at least in part, by down-regulation of IP3 receptor number. Thus, diminution in cell growth rate in Q212L-alpha 16 cells seems to be attributable to a combination of at least two effects: a direct effect of PIC activation leading to partial depletion of Ca2+ stores and an indirect, adaptive response resulting in a decreased IP3 receptor number.

The possible existence of multiple [3H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [3H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s-1 [corrected]. Maximal specific [3H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [3H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [3H]ouabain and 125I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [3H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells.

Myocardial cell swelling occurs in ischemia and in reperfusion injury before the onset of irreversible injury. Swelling has been attributed to failure of the Na+/K+ pump and the accumulation of intracellular Na+. To evaluate the role of the pump-leak model of cell volume maintenance, short term changes in cell volume in response to Na+/K+ pump inhibition were studied in aggregates of cultured embryonic chick cardiac myocytes using optical and biochemical methods. Exposure to 100 µM ouabain over 20 min induced cell shrinkage of approximately 10%. Cell water was also decreased by Na+/K+ pump inhibition; incubation for 1 hr either in the presence of 100 µM ouaain or in K+-free solution reduced cell water by 18.4% and 28.4% respectively. When exposed to ouabain in the absence of extracellular Ca2+, the aggregates swelled by approximately 15%, indicating that extracellular Ca2+ was required for the ouabain-induced shrinkage to occur. Ouabain still caused shrinkage, however, in the presence of the Ca2+ channel blockers verapamil (10 µM) and nifedipine (10 µM), suggesting that Na+/Ca2+ exchange, rather than Ca2+ channels, is the route for Ca2+ influx during Na+/K+ pump inhibition. Efflux of amino acids (taurine, aspartate, glutamate, glycine and alanine) from confluent monolayers of chick heart cells exposed to ouabain for 20 min was nearly double that observed in control solution. These results suggest that, during Na+/K+ pump inhibition, chick heart cells can limit accumulation of intracellular sodium by means of Na+/Ca2+ exchange, and that a rise in intracellular [Ca2+], also mediated by Na+/Ca2+ exchange, promotes the loss of amino acids and ions to cause cell shrinkage. Therefore, swelling during ischemic injury may not result from Na+/K+ pump failure alone, but may reflect the exhaustion of alternative volume regulatory transport mechanisms.