Endothelin-1 gene suppression by shear stress: Pharmacological evaluation of the role of tyrosine kinase, intracellular calcium, cytoskeleton, and mechanosensitive channels

Physiological fluid shear stress regulates endothelin-1 (ET-1) gene express ion in endothelial cells by inducing an early transient upregulation follow ed by a sustained suppression, at times greater than 2 h in duration. We ev aluated the mechanism of ET-1 mRNA downregulation in confluent monolayers o f bovine aortic endothelial (BAE) cells by applying a 6 h steady laminar sh ear stress of magnitude 20 dyn/cm(2). Inhibition of tyrosine kinases using herbimycin A (875 nM) abolished the shear-induced decrease in ET-1 mRNA exp ression. Similarly, chelation of intracellular calcium ([Ca2+](i)) with qui n2-AM (10 mu M) blocked the suppression of ET-1 mRNA by shear. To examine t he role of the endothelial cytoskeleton in the response to flow, cytochalas in D was used to disrupt F-actin microfilaments. This treatment induced cel l retraction and detachment under flow, whereas stabilization of F-actin wi th phalloidin (1 mu M) did not affect shear-induced ET-1 downregulation. In contrast, disruption of the microtubule network. with nocodazole (10 mu g/ ml) completely prevented, while microtubule stabilization with taxol (10 mu M) did not affect the suppression of ET-1 mRNA by flow. To determine the p ossible contributions of mechanosensitive channels, barium (1 mM BaCl2), wa s added to confluent BAE monolayers in a low-sulfate/low-phosphate: modifie d medium and was noted to abrogate the downregulation of ET-1 gene expressi on and to attenuate the shear-induced increase in cytoplasmic free calcium concentration. Tetraethylammonium (3 mM TEA) partially inhibited the suppre ssion of ET-1 mRNA by shear: in contrast, gadolinium (10 mu M GdCl3), an in hibitor of the stretch-activated cation channel I-SA had no effect. Membran e depolarization by elevated extracellular potassium ([K+](o)) also attenua ted the suppression of ET-1 mRNA by flow at [K+](o) = 70 mM and completely inhibited it at [K+](o)=135 mM. In summary, the steady-state downregulation of ET-1 mRNA by physiological levels of fluid shear stress shares signalin g features with the morphological and cytoskeletal response to shear stress . These include requirement for intracellular calcium, tyrosine kinase acti vity, an intact microtubule network, and independence from a Gd3+-sensitive I-SA. Unlike shear-induced changes in cell morphology and the actin cytosk eleton, the shear-induced decrease in ET-1 mRNA level is blocked by cell de polarization and by Ba2+, a blocker of the shear-activated I-KS which also decreases shear-induced cytoplasmic calcium increase. (C) 1999 Academic Pre ss.