Abstract
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 myosin light chain kinase (MLCK) and regulation of non-muscle myosin II. Immunoblotting and immunocytochemistry demonstrated the presence of MLCK, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32P incubation of the inner medulla followed by autoradiography of two-dimensional gels demonstrated increased 32P labeling of both isoforms in response to the V2 receptor agonist [deamino-Cys1,D-Arg8]vasopressin (DDAVP). Time course studies of MLC phosphorylation in IMCD suspensions (using immunoblotting with anti-phospho-MLC antibodies) showed that the increase in phosphorylation could be detected as early as 30 s after exposure to vasopressin. The MLCK inhibitor ML-7 blocked the DDAVP-induced MLC phosphorylation and substantially reduced [Arg8]vasopressin (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.
Highlights
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
These results demonstrate that myosin regulatory light chain (MLC) phosphorylation by myosin light chain kinase (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
Jasplakinolide stabilizes F-actin [32] and induces condensation of actin filaments in the cortical region of cells. 1 M jasplakinolide caused a significant 66% reduction in vasopressin-stimulated water permeability (basal, 199 Ϯ 27 m/s; AVP, 636 Ϯ 67 m/s; and AVP ϩ jasplakinolide, 216 Ϯ 54 m/s (n ϭ 4; p Ͻ 0.02)). These results demonstrate that the vasopressin-dependent water permeability in the IMCD is dependent on the state of organization of actin microfilaments in the collecting duct cells
Summary
Vol 279, No 47, Issue of November 19, pp. 49026 –49035, 2004 Printed in U.S.A. Non-muscle Myosin II and Myosin Light Chain Kinase Are Downstream Targets for Vasopressin Signaling in the Renal Collecting Duct*. 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. Studies by Hays and co-workers have demonstrated that vasopressin and cAMP depolymerize F-actin in the apical region of toad urinary bladder [17,18,19] and IMCD cells [20] They have proposed from these studies that a dense cortical matrix of microfilaments in the subapical region of collecting duct cells constitutes a barrier blocking the access of water channel vesicles to the apical plasma membrane and that vasopressin stimulates fusion of these vesicles with the apical plasma membrane by depolymerizing this matrix and eliminating the barrier. We use immunochemical methods, mass spectrometry-based proteomics methods, phospho-MLC-specific antibodies, and water permeability measurements in isolated perfused tubules to investigate the potential role of myosin II, MLC, and MLCK in the water permeability response to vasopressin in the rat IMCD
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