ALTERED BETA-ADRENERGIC REGULATION OF NA-K-CL COTRANSPORT IN CULTURED SMOOTH-MUSCLE CELLS FROM THE AORTA OF SPONTANEOUSLY HYPERTENSIVE RATS - ROLE OF THE CYTOSKELETON NETWORK

To verify the hypothesis of Na-K-Cl cotransport (COTR) involvement in ion transport abnormalities as revealed in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR), we compared the rate of ouabain-insensitive, bumetanide-inhibited Rb-86 influx in quiescent and growing cultures of VSMC from the aorta of SHR and normotensive (BN.lx) rats and its regulation by the cAMP signaling system. Basal COTR was not altered in quiescent cells from SHR but was decreased by 30% to 40% (P <.02) in growing SHR VSMC as compared to BN.lx rats. In quiescent BN.lx VSMC, isoproterenol inhibited COTR by 50% and induced cell shape transition of >90% of cells, resulting in the appearance of rounded VSMC with arborized cytoplasms. In contrast, isoproterenol elicited cell shape transition in only 50% of quiescent SHR VSMC and did not modify COTR. In growing cells, it decreased COTR by 85% to 95% and altered cell morphology in >95% of VSMC without differences between SHR and BN.lx rats. Neither inhibitors of protein kinase A (H-89 and KT-5720) nor an inhibitor of phosphoprotein phosphatase (okadaic acid) affected cell shape transition and COTR suppression in isoproterenol-treated VSMC. The COTR suppression and cytoplasm arborization was also demonstrated by the addition of cytochalasin B, a disintegrator of microfilament bundles, and staurosporine, an inhibitor of protein kinase C. The effects of these compounds on COTR and SHR and BN.lx VSMC morphology were not different. The calmodulin antagonist R24571 decreased COTR by 60% to 70% in quiescent BN.lx VSMC and did not modify this carrier in SHR VSMC. Other inhibitors of protein kinase C (calphostin C) and calmodulin (W-13) did not alter COTR and VSMC shape. Unlike cytochalasin B, inhibitors of microtubule polymerization (vinblastine, colchicine) did not arborize VSMC and increased COTR by 50% to 80% in SHR and BN.lx rats. These compounds prevented isoproterenol-induced cell shape transition and decreased the effectiveness of beta-adrenergic COTR regulation in VSMC. Thus, data obtained in this study demonstrated a blunted regulation of COTR and cytoskeleton organization by cAMP in quiescent SHR VSMC.