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

Abstract

Physiological fluid shear stress regulates endothelin-1 (ET-1) gene expression in endothelial cells by inducing an early transient upregulation followed by a sustained suppression, at times greater than 2 h in duration. We evaluated the mechanism of ET-1 mRNA downregulation in confluent monolayers of bovine aortic endothelial (BAE) cells by applying a 6 h steady laminar shear stress of magnitude 20 dyn/cm2. Inhibition of tyrosine kinases using herbimycin A (875 nM) abolished the shear-induced decrease in ET-1 mRNA expression. Similarly, chelation of intracellular calcium ([Ca2+]i) with quin 2-AM (10 microM) blocked the suppression of ET-1 mRNA by shear. To examine the role of the endothelial cytoskeleton in the response to flow, cytochalasin D was used to disrupt F-actin microfilaments. This treatment induced cell retraction and detachment under flow, whereas stabilization of F-actin with phalloidin (1 microM) did not affect shear-induced ET-1 downregulation. In contrast, disruption of the microtubule network with nocodazole (10 micrograms/ml) completely prevented, while microtubule stabilization with taxol (10 microM) did not affect the suppression of ET-1 mRNA by flow. To determine the possible contributions of mechanosensitive channels, barium (1 mM BaCl2), was added to confluent BAE monolayers in a low-sulfate/low-phosphate modified medium and was noted to abrogate the downregulation of ET-1 gene expression and to attenuate the shear-induced increase in cytoplasmic free calcium concentration. Tetraethylammonium (3 mM TEA) partially inhibited the suppression of ET-1 mRNA by shear; in contrast, gadolinium (10 microM GdCl3), an inhibitor of the stretch-activated cation channel ISA, had no effect. Membrane depolarization by elevated extracellular potassium ([K+]o) also attenuated 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 signaling features with the morphological and cytoskeletal response to shear stress. These include requirement for intracellular calcium, tyrosine kinase activity, an intact microtubule network, and independence from a Gd(3+)-sensitive ISA. Unlike shear-induced changes in cell morphology and the actin cytoskeleton, the shear-induced decrease in ET-1 mRNA level is blocked by cell depolarization and by Ba2+, a blocker of the shear-activated IKS which also decreases shear-induced cytoplasmic calcium increase.