Nonmuscle Myosin II and Myosin Light Chain Kinase are Downstream Targets for Vasopressin Signaling in the Renal Collecting Duct

Title: Nonmuscle Myosin II and Myosin Light Chain Kinase are Downstream Targets for Vasopressin Signaling in the Renal Collecting Duct
Authors: Chou, Chung-Lin; Christensen, Birgitte Monster; Frische, PhD, Sebastian; Vorum, MD, PhD, Henrik; Desai, Ravi A.; Hoffert, Jason; de Lanerolle, Primal; Nielsen, Soren; Knepper, Mark
Publisher: Journal of Biological Chemistry
Date Published: August 30, 2004
Reference Number: 665
We have previously demonstrated that vasopressin increases the water permeability of the inner medullary collecting duct (IMCD) by inducing trafficking of aquaporin-2 to the apical plasma membrane, and that this response is dependent on intracellular calcium mobilization and calmodulin activation. Here, we address the hypothesis that this water permeability response is mediated in part through activation of the calcium/calmodulin-dependent kinase, myosin light chain kinase (MLCK), and regulation of nonmuscle myosin II. Immunoblotting and immunocytochemistry demonstrated MLCK, myosin regulatory light chain (MLC) and the IIA and IIB isoforms of non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32P incubation of inner medulla followed by autoradiography of 2-D gels demonstrated increased 32P labeling of both isoforms in response to the V2 receptor agonist dDAVP. Time course studies of MLC phosphorylation in IMCD suspensions (using immunoblotting with anti-phospho-MLC antibodies) showed that the increase in phosphorylation can be detected as early as 30 seconds after exposure to vasopressin. The MLCK inhibitor ML-7 blocked the dDAVP-induced MLC phosphorylation and substantially reduced the AVP-stimulated water permeability. AVP-induced MLC phosphorylation was associated with a rearrangement of actin filaments (Alexa Fluor 568-phalloidin) in primary cultures of IMCD cells. These results demonstrate that MLC phosphorylation by MLCK represents a downstream effect of AVP-activated calcium/calmodulin signaling in IMCD cells and point to a role for non-muscle myosin II in regulation of water permeability by vasopressin.
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