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1 Department of Surgery, The Toronto General Hospital and University Health Network, Toronto, ON, Canada
2 Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA
* To whom correspondence should be addressed. E-mail: akapus{at}uhnres.utoronto.ca.
Hyperosmotic stress initiates adaptive responses, including the phosphorylation of myosin light chain (MLC), and the concomitant activation of the Na+-K+-Cl- cotransporter (NKCC). Since the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so what are the triggering factors; and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by an affinity pull-down assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration, and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength, but not in individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker, Clostridium Toxin B. The ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions, and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, ML-7 abolished the hypertonic activation of NKCC, while Y-27632 failed to inhibit this response. Thus, hyperosmolarity activates Rho, and the Rho/ROK pathway contributes to the basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK-independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.
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