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Am J Physiol Cell Physiol (February 23, 2005). doi:10.1152/ajpcell.00631.2004
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Submitted on December 22, 2004
Accepted on February 16, 2005

Is Myosin Light Chain Phosphorylation a Regulatory Signal for the Osmotic Activation of the Na+-K+-2Cl- Cotransporter?

Caterina Di Ciano-Oliveira1, Monika Lodyga1, Lingzhi Fan1, Katalin Szaszi1, Hiroshi Hosoya1, Ori D Rotstein1, and Andras Kapus1*

1 Surgery, The Toronto General Hospital and University Health Network, Toronto, Ontario, Canada

* To whom correspondence should be addressed. E-mail: akapus{at}uhnres.utoronto.ca.

Myosin light chain 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 a 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 LLC-PK1 kidney tubular cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independent 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. Further, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a non-phosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC-mutant (DD-MLC), which mimics the diphosphorylated form, neither stimulated the isotonic, nor potentiated the hypertonic NKCC activity. Furthermore, depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolishment 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.




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