title: Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?
abstract: Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na+-K+-2Cl- cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl- depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl- depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC.
authors.affiliation: Department of Surgery, The Toronto General Hospital and University Health Network, Ontario, Canada.
authors.forename: Caterina
authors.initials: C
authors.lastname: Di Ciano-Oliveira
authors.forename: Monika
authors.initials: M
authors.lastname: Lodyga
authors.forename: Lingzhi
authors.initials: L
authors.lastname: Fan
authors.forename: Katalin
authors.initials: K
authors.lastname: Szászi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hosoya
authors.forename: Ori D
authors.initials: OD
authors.lastname: Rotstein
authors.forename: András
authors.initials: A
authors.lastname: Kapus
mesh_terms: D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006982:Hypertonic Solutions / Q000494:pharmacology; D026121:Indole Alkaloids; D007672:Kidney Cortex / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D018374:LLC-PK1 Cells; D008722:Methods; D009154:Mutation; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009218:Myosins / Q000502:physiology; D009281:Naphthalenes / Q000494:pharmacology; D009995:Osmosis; D009997:Osmotic Pressure; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D013552:Swine
chemical_list: D001381:Azepines; D002227:Carbazoles; D002712:Chlorides; D004791:Enzyme Inhibitors; D006982:Hypertonic Solutions; D026121:Indole Alkaloids; D018994:Myosin Light Chains; D009281:Naphthalenes; D012333:RNA, Messenger; D028021:Sodium-Potassium-Chloride Symporters; C070571:ML 7; C049985:staurosporine aglycone; D009218:Myosins