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In earlier studies we demonstrated that cyclical mechanical strain on vascular smooth muscle cells increases intracellular Na+ and upregulates the alpha-1 and alpha-2 isoform expression of Na+,K+-ATPase, and that the increase of intracellular Na+ and upregulation of the alpha-2 isoform expression are blocked by Gd3+, which blocks entry of ions (including Na+) through stretch-activated channels. The present study was designed to investigate the role of intracellular Na+ in Na+,K+-ATPase regulation by increasing intracellular Na+ with chronic ouabain treatment. In parallel experiments, we measured Na+,K+-ATPase alpha isoform expression, Na+-pump activity and intracellular Na+ in cultured aortic smooth muscle cells after treatment with two concentrations of ouabain for various time periods. Treatment with 100 nM ouabain resulted in a significant elevation in intracellular Na+ after 1 (21%) and 2 h (12%), but the value returned to baseline after 12 h. Both alpha-1 and alpha-2 subunits of Na+,K+-ATPase were significantly upregulated after 1 through 4 days. Na+-pump activity was also stimulated, and the time course of this effect closely followed protein expression. At 200 microM of ouabain, the effects on intracellular Na+, isoform expression and Na+-pump activity at earlier time points (1 h through 1 day) were similar to those with 100 nM treatment, but prolonged treatment (2 and 4 days) resulted in an accumulation of intracellular Na+ and inhibition of the isoform expression and Na+-pump activity, possibly due to general dysfunction of the cells as a result of chronic exposure to high concentrations of ouabain. We conclude that elevated intracellular Na+ can serve as a signal to mediate the alpha isoform upregulation and the regulatory process requires less than one day.

This study was designed to test the role of Na+ and Ca2+ entry in the stretch-induced Na(+)-K(+)-ATPase alpha 1- and alpha 2-isoform upregulation observed in our previous studies. We measured intracellular Na+ in cyclically stretched rat aortic smooth muscle cells, with or without gadolinium treatment, for various durations and performed Western blotting to analyze the effects of stretch and the calcium channel blocker isradipine on the expression of alpha-isoforms. Intracellular Na+ was elevated significantly after 1- and 2-h stretch, but returned to baseline after 1-, 2-, and 4-day stretch. This increase in intracellular Na+ was blocked by gadolinium. Both alpha 1- and alpha 2-isoforms were upregulated after either 2 or 4 days of cyclical stretch. Isradipine had no apparent effect on stretch-induced upregulation on either alpha-isoform, thus suggesting that Ca2+ entry through L-type channels is not involved in the stretch-induced upregulation. We therefore conclude that a transient intracellular Na+ elevation during stretch may serve as a signal to mediate the alpha 1- and alpha 2-isoform upregulation.

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.

We have previously demonstrated that vascular sodium pump activity is stimulated in several rat models of hypertension. In addition, others have reported an upregulation of mRNA for the Na+,K+-ATPase alpha1-subunit in hypertension. To test the effect of sustained, cyclic, stretch-relaxation stimuli on the expression of alpha1- and alpha2-subunits of Na+,K+-ATPase in vascular smooth muscle cells, we used the Flexercell strain unit to stretch rat aortic smooth muscle cells for several days on a collagen-coated silicone elastomer substratum. Six-second cycles of stretch-relaxation were applied to obtain 10% average surface elongation (22% maximum) for 4 days. Control cells were not stretched but were grown on a similar surface. The effect of Gd3+, a blocker of stretch-activated channels, was also investigated. At the end of 4 days, protein expression of alpha1- and alpha2-subunits was determined by Western blot analysis. Intensity of the bands for alpha1- and alpha2-subunits was quantified with the use of a computerized image analyzer. In the stretched cells, both the alpha1- and the alpha2-subunit protein-band intensities were significantly increased compared with those of the non-stretched cells. Treatment with 50 micromol/L Gd3+ during the application of stretch prevented the upregulation of alpha2-expression but not that of alpha1-expression. Sodium pump activity, the functional counterpart of Na+,K+-ATPase, was inhibited as a result of stretch; Gd3+ had no effect on this variable. Our results suggest that in vascular smooth muscle, stretch may be a signal for the upregulation of both the alpha1- and alpha2-isoforms. However, a differential response of the two isoforms to the blocker of stretch-activated channels implies involvement of different mechanisms. This alteration in protein expression is not reflected in the function of the enzyme.

The catalytic alpha- and smaller glycosylated beta-subunits of the membrane transport enzyme Na(+)-K(+)-adenosinetriphosphatase (ATPase) occur in different molecular forms, alpha 1, alpha 2, alpha 3, beta 1, and beta 2. The catalytic alpha 1-, alpha 2-, and alpha 3-subunits of the enzyme have varying affinities for digitalis and exist in different tissues with unique distribution patterns. In this report we document for the first time the existence of alpha 1-, alpha 2-, alpha 3-subunit proteins (all approximately 97.5 kDa) in cultured rat aortic smooth muscle cells and rat tail arteries. In addition to the three molecular forms of the alpha-protein we detected a minor band at approximately 68-kDa position in aortic smooth muscle cells, which may correspond to the truncated alpha 1-protein reported earlier.