Non-muscle Myosin II and Myosin Light Chain Kinase Are Downstream Targets for Vasopressin Signaling in the Renal Collecting Duct

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 timeof-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32 P incubation of the inner medulla followed by autoradiography of twodimensional gels demonstrated increased 32 Vasopressin regulates the water permeability of the renal collecting duct epithelium in part by inducing translocation of aquaporin-2-containing intracellular vesicles to the apical region of the principal cells, where they fuse with the apical plasma membrane (1). The resulting increase in water permeability accelerates water reabsorption from the tubule lumen to the blood. Regulation of the water permeability of the collecting duct is a key element of the homeostatic process whereby vasopressin regulates body water balance.Vasopressin acts in collecting duct principal cells by binding to the V 2 receptor located in the basolateral membrane. Ligand binding activates adenylyl cyclase VI (2) via the heterotrimeric G-protein G s , resulting in an increase in cAMP production. In addition, V 2 receptor occupation by vasopressin elicits an increase in intracellular calcium (3-7). The increase in intracellular calcium is seen at physiological concentrations of vasopressin and can be elicited in response to exposure of the cells to cell-permeant forms of cAMP (7), suggesting that vasopressin-induced calcium mobilization is triggered by cAMP, presumably via protein kinase A activation. Recent confocal imaging studies of the inner medullary collecting duct (IMCD) 1 have shown that the vasopressin-induced increase in intracellular calcium is oscillatory in nature (8). The calcium release is mediated by ryanodine-sensitive intracellular calcium channels in the collecting duct cells (7).Isolated perfused tubule studies have demonstrated that the ability of vasopressin to increase water permeability in the IMCD is blocked by intracellular Ca 2ϩ chelation with BAPTA or by blockade of ryanodine-sensitive Ca 2ϩ channels by ryanodine, indicating that vasopressin-induced Ca 2ϩ mobilization is important for translocation of aquaporin-2 and the water permeability response in the IMCD (7). Furthermore, inhibitors of calmodulin, viz. W-7 and trifluoroperazine, cause a reversible inhibition of vasopressin-d...

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

“…7F) and so was forskolininduced water permeability increase ( Fig. S5 and Table S3), consistent with the prior finding that latrunculin B inhibits vasopressin induced osmotic water permeability increases in isolated perfused rat collecting ducts (31). The vehicle control (DMSO) did not affect dDAVP-induced apical AQP2 localization (Fig.…”

“…As the actomyosin tension relaxes in the cell center, the remaining peripheral tension can be expected to pull F-actin toward the cell periphery. An increase in the peripheral actomyosin tension is also a possibility because vasopressin has been shown to activate myosin light chain kinase (31), which, in turn, activates myosin light chain and hence nonmuscle myosin II activity (55). In the ear's organ of Corti, the nonmuscle myosin IIB and IIC, not IIA, are organized as a sarcomeric network surrounding the apical perimeter of the cells (56).…”

“…The P values were obtained using Fisher's exact test against all transcripts expressed in mpkCCD cells (4). (31,(36)(37)(38)(39)(40)(41)(42)(43). Furthermore, inhibition of Rho, a small GTPase protein involved in actin polymerization (44), results in AQP2 membrane localization (36,38,42,45).…”

Calmodulin Is Required for Vasopressin-stimulated Increase in Cyclic AMP Production in Inner Medullary Collecting Duct

“…Preincubation of isolated perfused rat inner medullary collecting duct (IMCD) with the CaM inhibitors W-7 and trifluoperazine (TFP) blocked AVP-stimulated water permeability. Further investigation revealed that CaM activates myosin light chain kinase and subsequent nonmuscle myosin II-dependent vesicle trafficking of AQP2 (3).…”

Quantitative phosphoproteomics of vasopressin-sensitive renal cells: Regulation of aquaporin-2 phosphorylation at two sites

“…Water and urea transport across this epithelium is regulated by the peptide hormone vasopressin via V2-type receptors. Ligand binding triggers an incompletely understood signaling response that includes activation of at least two adenylyl cyclase isoforms via the heterotrimeric G protein G s (7)(8)(9), an increase in intracellular cAMP (10), an increase in cytosolic Ca 2ϩ concentration (11), calmodulin-mediated activation of myosin light-chain kinase (12), and phosphorylation of the water channel aquaporin-2 (AQP2) (13) as well as the urea transporter isoforms A1 and A3 (UT-A1͞3) (14). The work presented here identifies a large number of previously unknown phosphorylation sites in IMCD proteins, including sites in AQP2 and UT-A1͞3.…”