Plasma membrane calcium pump-mediated calcium efflux and bulk cytosolic free calcium in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto normotensives rats

Abstract

To compare the efficacy of the calcium pump-mediated calcium efflux pathway in spontaneously hypertensive rats (SHR) with that in Wistar-Kyoto normotensive rats (WKY), at rest and after angiotensin II stimulation. The intracellular free calcium concentration and calcium-45 efflux were measured in parallel, in cultured aortic smooth muscle cells isolated from 10-week-old male SHR and WKY rats. The intracellular free calcium concentration and calcium-45 efflux were studied in confluent vascular smooth muscle cells in culture. Experiments were performed in the absence of added extracellular calcium and sodium. Fura-2 was used to measure basal and angiotensin II-stimulated intracellular free calcium concentration. Effluxed calcium-45 was measured over 5s intervals to determine basal and angiotensin II-stimulated calcium efflux rates in SHR and in WKY rats. No significant difference between SHR and WKY rats was observed in basal intracellular free calcium concentration or 100nmol/l angiotensin II-stimulated peak intracellular free calcium concentration. However, significantly elevated basal and 100 nmol/l angiotensin II-stimulated calcium-45 efflux rates were found in SHR. The calcium-45 efflux rates in SHR were elevated when the efflux was normalized with respect to the bulk intracellular free calcium concentration. The time taken to reach the maximum calcium-45 efflux rate after angiotensin II stimulation was reduced in SHR compared with that in WKY rats and was dose-dependent in both rat strains. The calcium-pump mediated calcium efflux pathway appears to be more efficient in SHR. This may be the result of post-translational modification, enhanced calcium pump sites in a critical region of the membrane, or the presence of a pool of calcium near the plasma membrane that is not readily detected by cytosolic Fura-2 but is higher in SHR both before and after angiotensin II stimulation.