Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct.
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.
Chou
Chung-Lin
CL
Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1603, USA.
Christensen
Birgitte M
BM
Frische
Sebastian
S
Vorum
Henrik
H
Desai
Ravi A
RA
Hoffert
Jason D
JD
de Lanerolle
Primal
P
Nielsen
Soren
S
Knepper
Mark A
MA
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D020346:Aquaporins / Q000378:metabolism; D001381:Azepines / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D047630:Depsipeptides / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D000070559:Gizzard, Non-avian / Q000378:metabolism; D015151:Immunoblotting; D007120:Immunochemistry; D007150:Immunohistochemistry; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D024462:Myosin Type II / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D009281:Naphthalenes / Q000494:pharmacology; D009995:Osmosis; D010455:Peptides / Q000737:chemistry; D010477:Perfusion; D010766:Phosphorylation; D020033:Protein Isoforms; D020543:Proteome; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D013997:Time Factors; D014421:Turkey; D014667:Vasopressins / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D000199:Actins; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D020346:Aquaporins; D001381:Azepines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D009281:Naphthalenes; D010455:Peptides; D020033:Protein Isoforms; D020543:Proteome; D013844:Thiazoles; D053778:Thiazolidines; D014867:Water; C057531:jasplakinolide; C070571:ML 7; D014667:Vasopressins; D009219:Myosin-Light-Chain Kinase; D024462:Myosin Type II; D003894:Deamino Arginine Vasopressin; C037068:latrunculin B