Functional AT1-, and α-adrenoceptors in cultured smooth muscle cells from WKY and SHR rats

Altered regulation of intracellular Ca²⁺ is associated with the development of hypertension. We analysed the effects of angiotensin II (Ang II 2×10^-7 M) and norepinephrine (NE, 1×10^-6M) treatment on the intracellular Ca²⁺ concentrations (|Ca ²⁺|i) in cultured aortic smooth muscle cells (SMC) from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at three stages of hypertension development: at 3-4-, 10-12-, and 28-30-weeks. In the 10-12 week-old group we also evaluated the effect of α1- adrenoceptors activation on the intracellular distribution of the Ca ²⁺-dependent isoforms of the enzyme protein kinase C (PKC). |Ca ²⁺|i was measured using the fluorescent Ca²⁺ indicator Fluo-3, and monitoring the excitation and emission wavelength at 490 and 530 nm with a SPEX Fluorolog 112. Intracellular distribution of PKC was determined in cytosolic and membrane fractions of cultured SMC from 10-12-week-old WKY and SHR, by SDS-PAGE electro-Dhoresis and Western immunoblotting, using a monoclonal antibody against the α, β, and γ-isoforms of PKC |Ca²⁺|i under basal, unstimulated conditions was similar in SMC from both strains of rats at all three age groups measured. Treatment of SMC with Ang II led to a significant increase in |Ca ²⁺|i in the 3-4-week-old WKY and in both strains of SMC of the two older age groups, confirming the expression of functional AT, receptors in these cells. AT, receptor function, however was similar in WKY and SHR SMC at all three ages. NE treatment of cultured SMC induced elevations in |Ca ²⁺|i in SMC from both strains at 10-12 weeks and 28-30 weeks, but not at 3-4 weeks, thus affirming the oresence of functional α¹-adrenoceptors in the two older age groups, α1-adrenoceptor function did not differ between WKY and SHR cells. In the 10-12-week-old WKY SMC treatment with NE or PE induced a translocation of PKC from the cytoplasmic to the membrane fraction, demonstratingenzyme activation consequent to α¹-adrenoceptor simulation. No such translocation of PKC was detected in the 10-12-week-old SHR SMC, indicating that the Ca²⁺-dependent PKC isoforms are not expressed in the hypertensive cells. Our results are consistent with the expression of functional AT1 receptors in cultured SMC from the older WKY and SHR. They further support the expression of functional α1-adrenoceptors in SMC from both strains of rats in the older age groups but not in the 3-4-week-old group.