Contractile regulation of the Na⁺-K⁺-2Cl⁻cotransporter in vascular smooth muscle

Abstract

Vasoconstrictors activate the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 in rat aortic smooth muscle, but the mechanism is unknown. Efflux of (86)Rb(+) from rat aorta in response to phenylephrine (PE) was measured in the absence and presence of bumetanide, a specific inhibitor of NKCC1. Removal of extracellular Ca(2+) completely abolished the activation of NKCC1 by PE. This was not due to inhibition of Ca(2+)-dependent K(+) channels since blocking these channels with Ba(2+) in Ca(2+)-replete solution did not prevent activation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited 70% by 75 microM ML-9, 97% by 2 microM wortmannin, and 70% by 2 mM 2,3-butanedione monoxime, each of which inhibited isometric force generation in aortic rings. Bumetanide-insensitive Rb(+) efflux, an indication of Ca(2+)-dependent K(+) channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to 120% of normal almost completely blocked the stimulation of NKCC1 by PE without inhibiting the stimulation by hypertonic shrinkage. We conclude that activation of the Na(+)-K(+)-2Cl(-) cotransporter by PE is the direct result of smooth muscle contraction through Ca(2+)-dependent activation of myosin light chain kinase. This indicates that the Na(+)-K(+)-2Cl(-) cotransporter is regulated by the contractile state of vascular smooth muscle.

Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na+-K+-2Cl−cotransporter

“…Regulation of vascular smooth muscle contractility plays a major role in the maintenance of normal blood pressure. The observations that NKCC1 activity is altered in response to vasoactive compounds (1) and that inhibitors of NKCC1 alter vascular contractility (reviewed in Ref. 13) suggest that eliminating NKCC1 could affect vascular tone.…”

“…Vasoactive agents alter the activity of NKCC1 in vascular smooth muscle (2,39,47) and in endothelial cells (6,27,35). There is evidence that NKCC1 is responsible for the maintenance of intracellular Cl Ϫ concentrations and cell volume during agonist stimulation of endothelial cells (38) and that its activity is regulated in response to changes in vascular smooth muscle contractility (1). The loop diuretic bumetanide, an inhibitor of NKCC1, causes a reduction in the sensitivity of rat aortic rings to phenylephrine-induced contraction (2).…”

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

“…Similar arguments hold for BDM, which is a well-known "chemical phosphatase" (43) and may induce the dephosphorylation of volume-regulatory proteins involved in NKCC activation. In agreement with this possibility, Akar et al (2) observed that inhibition of contraction did not correlate precisely with NKCC inhibition, suggesting that the two processes are not tightly coupled.…”

“…Lysates were subjected to a pressure of 900 lb./in. 2 , followed by the addition of 1% sarcosyl and centrifugation at 12,000 rpm for 10 min (4°C). The supernatant was supplemented with 1% Triton X-100 and incubated with glutathione-Sepharose beads for 1 h. The GST-RBD-covered beads were washed extensively with STE buffer and stored at 4°C.…”

“…The notion that cell contractility, presumably through the phosphorylation state of MLC, regulates NKCC was based on findings that pharmacological agents interfering with MLCK (ML-7, ML-9) or myosin ATPase (2,3-butanedione-2-monoxime, BDM) inhibited the agonist-induced [phenylephrine, forskolin (2,14)] or hyperosmotic (23,34) activation of the cotransporter. Because ML-7 did not prevent the hypertonicityprovoked phosphorylation of NKCC per se (23), the involvement of other targets, predominantly MLC, appeared an attractive mechanism.…”