Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation.
Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because 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 the triggering factors are, 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 affinity pulldown 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 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. 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, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to 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.
Department of Surgery, Toronto General Hospital and University Health Network, 200 Elizabeth St., Toronto, ON, Canada M5G 2C4.
Caterina
C
Di Ciano-Oliveira
Gábor
G
Sirokmány
Katalin
K
Szászi
William T
WT
Arthur
András
A
Masszi
Mark
M
Peterson
Ori D
OD
Rotstein
András
A
Kapus
D000818:Animals; D006982:Hypertonic Solutions / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D018374:LLC-PK1 Cells; D018994:Myosin Light Chains / Q000378:metabolism*; D009997:Osmotic Pressure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013552:Swine; D014882:Water-Electrolyte Balance / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
D006982:Hypertonic Solutions; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins