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Am J Physiol Cell Physiol 281: C579-C584, 2001;
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Vol. 281, Issue 2, C579-C584, August 2001

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

Fatma Akar1, Gengru Jiang1, Richard J. Paul2, and W. Charles O'Neill1,3

1 Renal Division, Department of Medicine, and 3 Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322; and 2 Department of Biophysics and Molecular Physiology, University of Cincinnati, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vasoconstrictors activate the Na+-K+-2Cl- cotransporter NKCC1 in rat aortic smooth muscle, but the mechanism is unknown. Efflux of 86Rb+ 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 Ca2+ completely abolished the activation of NKCC1 by PE. This was not due to inhibition of Ca2+-dependent K+ channels since blocking these channels with Ba2+ in Ca2+-replete solution did not prevent activation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited 70% by 75 µM ML-9, 97% by 2 µM 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 Ca2+-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 Ca2+-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.

sodium-potassium-2-chloride cotransport; myosin light chain kinase; phenylephrine; contraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NKCC1, the secretory or basolateral form of the Na+-K+-2Cl- cotransporter, participates in salt transport in secretory epithelia, but is also present in nonepithelial cells where it functions to regulate cell volume and intracellular [Cl-]. Consistent with this role, NKCC1 is activated by cell shrinkage and inhibited intracellular Cl-. The transporter is also activated by inhibitors of protein phosphatases and is inhibited by kinase inhibitors (9, 22), indicating that regulation occurs through protein phosphorylation. Stimuli that activate NKCC1 also phosphorylate the transporter (3, 13, 14), probably at the same site or sites (12), suggesting that NKCC1 is activated by a specific protein kinase. We have recently identified c-Jun NH2-terminal kinase as a volume-sensitive kinase capable of phosphorylating NKCC1 in vitro (7).

There is also evidence for an additional regulatory pathway that may be independent of NKCC1 phosphorylation. In several cell types, shrinkage increases phosphorylation of myosin light chain (MLC) (8, 28), and inhibition of myosin light chain kinase (MLCK) blocks NKCC1 activation in endothelial (8), Ehrlich ascites (10), and colonic epithelial (5) cells. Activation of the cotransporter in colonic epithelial cells is also affected by alterations in F-actin (5, 15, 16). The mechanism by which the cellular contractile apparatus or cytoskeleton regulates NKCC1 is not known.

To help elucidate this putative contractile regulation of NKCC1, we have turned to a contractile tissue, vascular smooth muscle. We have previously shown that NKCC1 is present in smooth muscle from rat aorta, where it is activated by vasoconstrictors and inhibited by nitrovasodilators (1), consistent with contractile regulation of NKCC1. This regulation of NKCC1 is important in smooth muscle function since inhibition of the cotransporter reduces force generation. We speculate that NKCC1 may promote contraction by maintaining or increasing intracellular [Cl-]. However, it is possible that this regulation of NKCC1 could be mediated by other actions of these compounds, including changes in cell volume. The present studies were undertaken to determine the role of the contractile apparatus in the activation of NKCC1 in rat aorta by the vasoconstrictor phenylephrine.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. The descending aorta was excised from male Sprague-Dawley rats (200-350 g), dissected free of the adventitia, and opened lengthwise. The endothelium was removed with a cotton swab, after which the vessel was cut into 6-12 pieces that were used immediately for assays.

Na+-K+-2Cl- cotransporter activity. Cotransporter activity was measured as unidirectional K+ efflux, using 86Rb as a tracer, as previously described (1). In each assay, the basal rate of efflux was measured over the 6 min before addition of test conditions, and the stimulated rate of efflux was measured between 8 and 10 min after stimulation. Data are presented as means ± SE. Standard isotonic solution was Earle's salts with HEPES substituted for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, containing (in mM) 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 1 NaH2PO4, 5 glucose, and 26 HEPES, pH 7.4. The measured osmolality was 290 mosmol/kgH2O. 86RbCl was obtained from DuPont-NEN. Bumetanide was a gift of Dr. Peter Sorter (Hoffmann-LaRoche, Nutley, NJ) and stored as a stock solution of 100 mM in dimethyl sulfoxide (DMSO). ML-9 was obtained from Biomol (Plymouth Meeting, PA) and stored as a stock solution of 30 mM in DMSO. 2,3-Butanedione monoxime (BDM) was obtained from Sigma (St. Louis, MO) and stored as a stock solution of 1 M in 15% ethanol. Wortmannin was also purchased from Sigma and used as an aqueous solution.

Isometric force measurements. Male Sprague-Dawley rats (178-430 g) were anesthetized in a precharged CO2 chamber and killed by cervical dislocation. The aorta was dissected and cleaned of loose fat and connective tissue, and endothelium was removed. A ring of 5 mm was cut from the thoracic aorta; blot weights were between 4 and 5 mg. Rings were mounted between a fixed stainless steel post and one connected to a Kistler-Morse force transducer whose output was monitored with a computer-based data collection system (BioPac). The length between the wires could be adjusted by a micrometer-driven device. Force measurements were then performed as previously described (17). The rings were mounted in a 15-ml organ bath and allowed to equilibrate at 37°C for 1 h, during which the tension was adjusted to 50 mN. This force was chosen because it places the ring at a length within the range for optimal active force generation. The aorta was then stimulated by addition of KCl (3 M) to bring the bath concentration to 50 mM. The arteries were then relaxed by exchanging the bathing solution; this contraction/relaxation cycle was repeated until reproducible forces were generated.

The protocol for studies with ML-9, wortmannin, and BDM involved a control contraction/relaxation cycle to 10 µM phenylephrine. The aortas were then preincubated with these agents for 10 min, 2 h, and 10 min, respectively, to parallel the design of the K+ efflux experiments. In the continued presence of inhibitor, a second test contraction/relaxation to phenylephrine (10 mM) was conducted. The effects of these agents are reported as a percentage of the initial control force. After each experiment, ring dimensions and blot weight were measured. Aortic wall thickness (t) was estimated using the formula t = blot weight/(1.05 × length × circumference), and the cross-sectional area (CSA) for force normalization was taken as CSA = 2 × t × length. Studies were performed at 37°C in the standard isotonic solution (above), bubbled with 100% O2.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Phenylephrine produced a rapid increase in the efflux of Rb+ from rat aortic smooth muscle (Fig. 1). Much of this was not inhibited by bumetanide, but there was also a significant increase in bumetanide-sensitive efflux as previously reported (1), indicating activation of NKCC1. To test the role of Ca2+ in this activation of NKCC1, Ca2+ was omitted from the medium and EGTA was added to chelate any contaminating Ca2+. In initial experiments, this resulted in a very high basal K+ efflux as previously reported in vascular smooth muscle (27), which could be minimized by increasing the concentration of Mg2+ to 11 mM isosmotically (27). Ca2+-free conditions were initiated at the beginning of the flux measurements, 10 min before adding phenylephrine. As shown in Fig. 2, the increase in both bumetanide-sensitive and bumetanide-insensitive efflux of Rb+ by phenylephrine was completely blocked by removal of Ca2+. The inhibition of the bumetanide-insensitive efflux is indicative of Ca2+-dependent K+ channels. We have previously shown that these channels are responsible for activation of NKCC1 by Ca2+-mobilizing agonists in vascular endothelial cells, probably through loss of cell chloride and cell shrinkage (19). To determine whether a similar mechanism was operant in vascular smooth muscle, efflux was measured in the presence of 3 mM Ba2+. As shown in Fig. 3, Ba2+ completely blocked the increase in bumetanide-insensitive Rb+ efflux by phenylephrine, providing further evidence that this flux is mediated by K+ channels, but did not prevent activation of NKCC1. Our previous demonstration that NKCC1 in rat aorta is also activated by KCl at concentrations that would actually increase cell volume and [Cl-] (up to 80 mM isosmotically substituted for Na+) also indicates that shrinkage cannot explain the activation of NKCC1 by agonists (1). These results indicate that vasoconstrictors directly activate NKCC1 in aortic smooth muscle via a Ca2+-dependent pathway.


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  		Fig. 1.   Effect of phenylephrine (PE) on the efflux of Rb+ from rat aortic smooth muscle. Aortic segments were loaded for 2 h with 86Rb+ after which efflux was measured before and after addition of 10 µM PE, in the absence (Total) and presence (+bumetanide) of 50 µM bumetanide (A). B: bumetanide-sensitive efflux. Results are means of 4 separate experiments, each performed in triplicate. Error bars, SEs.



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  		Fig. 2.   Effect of Ca2+-free medium on the efflux of Rb+ from rat aortic smooth muscle. Aortic segments were loaded for 2 h with 86Rb+ after which efflux was measured before and after addition of 10 µM PE, in the absence (Total) and presence (+bumetanide) of 50 µM bumetanide in Ca2+-free medium containing 1 mM EGTA. Results are means of 4 separate experiments, each performed in triplicate. Error bars, SEs.



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  		Fig. 3.   Effect of Ba2+ on the activation of NKCC1 by PE. A: efflux in the absence (Total) and presence (+bumetanide) of 50 µM bumetanide. B: bumetanide-sensitive efflux. Data are means of 3 independent experiments. Error bars, SEs.

We have previously shown in endothelial cells (8) that activation of NKCC1 by shrinkage is associated with phosphorylation of myosin light chain and blocked by inhibition of myosin light chain kinase (MLCK). To test the role of MLCK in rat aorta, two unrelated inhibitors of this kinase were employed: ML-9 and wortmannin. ML-9 is a relatively specific inhibitor (6, 25) that is very closely related to ML-7, the inhibitor used in previous studies in endothelial cells (8). Wortmannin is also a potent inhibitor of MLCK but has other actions including inhibition of phosphatidylinositol-3 kinase (2). ML-9 produced a dose-dependent inhibition of NKCC1 activation by phenylephrine without altering basal efflux (Fig. 4). The inhibition by 75 µM ML-9 was 70%. Wortmannin at 2 µM almost completely blocked (97%) stimulation of NKCC1 by phenylephrine (Fig. 5). There was a small increase in basal NKCC1 activity that was not statistically significant. Activation of NKCC1 was also substantially inhibited (70% at 2 mM) by BDM (Fig. 6), a compound that inhibits actin-myosin interaction in skeletal muscle and also inhibits vascular smooth muscle contraction (21). ML-9, wortmannin, and BDM each inhibited isometric force generation by phenylephrine in aortic rings (Fig. 7). In the case of ML-9 and wortmannin, inhibition of force generation correlated closely with inhibition of NKCC1 activation, while higher concentrations of BDM were required to inhibit force generation.


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  		Fig. 4.   Effect of ML-9 on the activation of NKCC1 by PE. ML-9 was added at the start of the efflux measurements. Each set of bars represents the means of 5, 3, and 5 independent experiments for 0, 37.5, and 75 µM ML-9, respectively. Error bars, SEs. * P < 0.02 vs. control (0 µM ML-9) for stimulation by PE.



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  		Fig. 5.   Effect of wortmannin on the activation of NKCC1 by PE. Wortmannin (2 µM) was added 2 h before the flux measurements. Data are means of 5 independent experiments. Error bars, SEs. * P < 0.01 vs. control for stimulation for PE.



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  		Fig. 6.   Effect of 2,3-butanedione monoxime (BDM) on the activation of NKCC1 by PE. BDM was added at the start of the efflux measurements. Each set of bars represents the means of 5 independent experiments except for 10 mM, which is a single experiment performed in triplicate. Error bars, SEs. * P < 0.02, ** P < 0.001 vs. control (0 mM BDM) for stimulation by PE.



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  		Fig. 7.   Inhibition of isometric force generation in rat aorta. Aortic rings were treated with 10 µM PE to determine maximum force generation. The PE was removed and the rings were then treated with ML-9, wortmannin (WORT), and BDM as described for efflux measurements before reexposure to PE. Each bar is the mean of triplicate measurements. Error bars, SEs.

BDM and ML-9 have also been reported to decrease Ca2+ influx (4, 29) and could inhibit contraction by reducing intracellular [Ca2+]. The activity of Ca2+-dependent K+ channels, which is measurable as bumetanide-insensitive efflux, can serve as an indirect measure of intracellular [Ca2+]. As shown in Fig. 8, both basal and stimulated bumetanide-insensitive efflux was reduced by 75 µM ML-9, consistent with a reduction in intracellular [Ca2+]. However, stimulated efflux was unaffected by 2 mM BDM or 2 µM wortmannin, indicating that sizable reductions in Ca2+ influx or intracellular Ca2+ release were probably not occurring with these compounds at these concentrations. Wortmannin actually increased basal bumetanide-insensitive efflux (P < 0.01), the mechanism of which is unknown.


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  		Fig. 8.   Bumetanide-insensitive K+ efflux. Efflux in the presence of 50 µM bumetanide was measured before and after addition of 10 µM PE. ML-9 (75 µM) and BDM (2 mM) were added at the start of the assay while wortmannin (2 µM) was added 2 h before assay. Results are the means of 5 individual experiments.

The link between smooth muscle contraction and activation of NKCC1 was investigated further by mounting aortic rings on tubing of different sizes before measurement of efflux. Aortic rings (without endothelium) were everted and placed over the tubing before loading with 86Rb+, with the luminal surface facing outward. Tubing with the following compositions and outer diameters was used: 1.91 mm and 2.08 mm polyethylene (Becton Dickinson, Parsippany, NJ), 1.96 mm Silastic (Dow Corning, Midland, MI), and 2.00 mm Nalgene PVC and 2.16 mm Nalgene silicone (Nalge, Rochester, NY). The measured lumen diameter of the rat aortic segments used in this study was ~1.80 mm. As shown in Fig. 9, stimulation of NKCC1 by phenylephrine was maintained up to a lumen diameter of 1.96 mm (approximately a 10% increase), after which there was progressive inhibition. By comparison, the stimulation of NKCC1 by hypertonic shrinkage remained intact and actually increased. This indicates that stretching of aorta is not producing a nonspecific inhibitory effect on NKCC1. Bumetanide-insensitive efflux was not affected by rigid tubing (data not shown).


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  		Fig. 9.   Effect of lumen diameter on the activation of NKCC1. Aortic rings (without endothelium) were everted and placed over tubing, as described in RESULTS, with the luminal surface facing outward. Efflux of 86Rb+ was then measured as described in METHODS, and the increase in bumetanide-sensitive efflux with either PE (10 µM) or hypertonic medium (150 mM sucrose) is plotted as a function of lumen diameter. Results are the means of at least 3 experiments. Error bars, SEs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that inhibition of smooth muscle contraction at multiple steps can block activation of the Na+-K+-2Cl- cotransporter by phenylephrine. The Ca2+ dependence of the stimulation of NKCC1 by phenylephrine is consistent with the Ca2+ dependence of alpha 1-adrenergic contraction (18). Although contraction by phenylephrine can be mediated by release of Ca2+ from internal stores in the absence of external Ca2+, force is reduced about 75% and is not sustained (17). It is unlikely that Ca2+ is having a direct effect on NKCC1 because we were not able to demonstrate any stimulation of NKCC1 by Ca2+ in endothelial cells provided that cell volume was held constant (19). Ca2+ could activate NKCC1 through opening of Ca2+-dependent K+ channels with resulting decreases in cell volume and intracellular [Cl-], as we have previously shown in endothelial cells (19). However, this is not the case in vascular smooth muscle since blockade of the channels by Ba2+ did not prevent activation of NKCC1 by phenylephrine. Ba2+ would be expected to increase aortic contraction by inhibiting Ca2+-dependent K+ channels and depolarizing the membrane, and through direct activation of the contractile mechanism (24). Furthermore, NKCC1 is also activated by isosmotic KCl, which raises intracellular [Ca2+] and also increases cell volume and intracellular [Cl-]. It is clear then that vasoconstrictors can activate NKCC1 in vascular smooth muscle in a Ca2+-dependent manner that does not involve activation of K+ channels and decreases in cell volume or intracellular [Cl-].

This Ca2+-dependent, volume-independent activation of NKCC1 by phenylephrine suggests involvement of MLCK, consistent with our previous results showing that activation of NKCC1 in cultured endothelial cells is dependent on myosin phosphorylation (8). This was confirmed by showing that two different inhibitors of MLCK reduced the stimulation of NKCC1 in aorta. Furthermore, activation of NKCC1 was also blocked by BDM, an agent that interferes with the interaction between actin and myosin in skeletal muscle and inhibits contraction of smooth muscle (21). However, inhibition of NKCC1 did not correlate precisely with inhibition of contraction, with NKCC1 stimulation being more sensitive to BDM, suggesting that contraction and inhibition of NKCC1 are not tightly coupled or may occur through different mechanisms.

It is important to note that the mechanism by which BDM inhibits smooth muscle contraction may be distinctly different from that in skeletal muscle. There is no inhibition of contraction in permeabilized smooth muscle, suggesting that the major effect of BDM is not on the contractile proteins themselves, but rather on Ca2+ delivery during excitation (11, 26). BDM can inhibit Ca2+ influx (4), but this may only occur at high concentrations. Although we did not directly measure intracellular [Ca2+], the activity of Ca2+-dependent K+ channels provided an indirect measure. The fact that 2 mM BDM did not lower bumetanide-insensitive efflux indicates that Ca2+-dependent K+ channel activity was not reduced and that BDM probably did not substantially affect intracellular [Ca2+] at a concentration that blocked stimulation of NKCC1 and reduced force generation. Our results do not rule out an effect of BDM on local Ca2+ concentrations or Ca2+ "sparks" that might not affect Ca2+-dependent K+ channels. A higher concentration of BDM (10 mM) completely abrogated the stimulation of bumetanide-insensitive K+ efflux by phenylephrine (data not shown), indicating that effects on Ca2+ influx or intracellular release can occur at higher concentrations.

The evidence that stimulation of NKCC1 is dependent on the contractile apparatus was bolstered by studies showing a length dependence for this stimulation. Increasing lumen diameter beyond 10% produced a progressive decrease in the stimulation of NKCC1 by phenylephrine. The basis for this length dependence is unknown but could be due to prevention of contraction. Although there was no measurable contraction of aorta on even the narrowest tubing, cell shortening could still be occurring, counterbalanced by stretching of the extracellular elastic component of the muscle. Larger tubing may stretch this elastic component to the point where it cannot stretch further to accommodate cell shortening, thus creating a state of true isometric contraction. It is of interest that stimulation of NKCC1 by hypertonic shrinkage was not only maintained in stretched aortas but was actually enhanced. Thus the volume sensitivity of NKCC1 is also length dependent, but in the opposite direction from agonist sensitivity.

On the basis of our data, we propose a novel pathway for regulation of NKCC1 in vascular smooth muscle involving the contractile apparatus. Agonists increase intracellular [Ca2+], activating MLCK and resulting in MLC phosphorylation, stimulation of myosin ATPase, cell contraction, and activation of NKCC1. The mechanism by which isotonic contraction activates NKCC1 is unclear. One possibility is an interaction between the cotransporter and the contractile apparatus or cytoskeleton. This is consistent with the observation that agents that perturb the actin cytoskeleton can alter NKCC1 activity in other cells (5, 15, 16). Another possibility is that contraction of vascular smooth muscle initiates a kinase signaling cascade that results in phosphorylation of NKCC1. This is suggested by our previous demonstration that phenylephrine and KCl increase phosphorylation of NKCC1 in rat aorta (1) and is consistent with the ability of mechanical strain to activate protein kinases in cultured vascular smooth muscle cells (23). Further studies in vascular smooth muscle should help elucidate the contractile regulation of the Na+-K+-2Cl- cotransporter


    ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health Grants HL-47449 and DK-07656 (to W. C. O'Neill) and HL-54829 and HL-61974 (to R. J. Paul). F. Akar was supported by the North Atlantic Treaty Organization Scientific Fellowship Program of the Scientific and Technical Research Council of Turkey (TUBITAK).


    FOOTNOTES

Address for reprint requests and other correspondence: W. C. O'Neill, Emory Univ. School of Medicine, Renal Division, WMB 338, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: woneill{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 August 2000; accepted in final form 8 March 2001.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(2):C579-C584
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