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.
Highlights
THE NAϩ-Kϩ-2CLϪ COTRANSPORTER (NKCC) is a loop diureticsensitive electroneutral ion translocator that plays vital roles in salt and water secretion and reabsorbtion, thereby regulating the fluid homeostasis of the whole organism
In the presence of DD-myosin light chain (MLC), the hyperosmotic NKCC activity reached the same level as that of control cells (DD-MLC, Fig. 5). These results show that while AA-MLC can suppress hyperosmotically induced MLC phosphorylation, it has no significant effect on hyperosmotic NKCC activation
MLC kinase (MLCK) inhibition using K252a, which abolished basal MLC phosphorylation, showed the same effect as blebbistatin in that both compounds and to a similar extent, inhibited the hyperosmolarity-induced NKCC activity. These findings suggest that myosin ATPase activity is a significant factor in the osmotic regulation of NKCC, and they are consistent with the possibility that MLCK may regulate NKCC activity by directly regulating myosin ATPase activity
Summary
THE NAϩ-Kϩ-2CLϪ COTRANSPORTER (NKCC) is a loop diureticsensitive electroneutral ion translocator that plays vital roles in salt and water secretion and reabsorbtion, thereby regulating the fluid homeostasis of the whole organism. While in certain cell types ML-7 potently inhibited the shrinkage-induced activation of NKCC (with Ki Ͻ 0.5 M corresponding to its effect on MLCK) [27, 43], the required inhibitor concentration was two orders of magnitude higher in other cells [9, 26], in which the drug may not be sufficiently specific and could affect NKCC activation independently of MLCK [3, 25, 49] It remained to be tested whether ML-7 selectively abrogates the hypertonic regulation of NKCC or interferes with the contractilityindependent (e.g., ClϪ depletion induced) activation of the cotransporter. Basal myosin ATPase activity is a prerequisite for the complete osmotic activation of NKCC, suggesting that it exerts a permissive effect
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