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LIVER AND BILIARY TRACT

[Ca²⁺]_i-independent contractile force generation by rat hepatic stellate cells in response to endothelin-1

¹Liver Center and Department of Medicine, University of California San Francisco, San Francisco; and ²Department of Physiology, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California

Submitted 20 July 2005 ; accepted in final form 22 August 2005

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca²⁺]_i, myosin activation, and migration were quantified using standard techniques. [Ca²⁺]_i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca²⁺. Removal of extracellular Ca²⁺ did not alter endothelin-1-stimulated force development or [Ca²⁺]_i. Ionomycin, a Ca²⁺ ionophore, triggered an increase in [Ca²⁺]_i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca²⁺]_i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca²⁺ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca²⁺]_i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca²⁺]_i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.

myosin light chain phosphorylation; myosin light chain kinase; rho-associated kinase; cytosolic calcium concentration

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