INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IN ADDITION TO ION channels, vascular smooth muscle cells (VSMC) possess a variety of ion transporters, the activities of which are influenced by vasoactive substances and growth factors, indicating potential roles in vasoconstriction and smooth muscle hypertrophy. Particular attention has been focused on monovalent cation transporters including the Na⁺-K⁺ pump, Na⁺/H⁺ antiporter NHE1, and Na⁺-K⁺-2Cl cotransporter NKCC1, in part because they transport Na⁺ (37). The importance of these transporters may lie in their ability to regulate not only intracellular Na⁺ concentration ([Na⁺]) but also intracellular Cl concentration ([Cl]), cell volume, and membrane potential. Although various abnormalities in the function of these transporters in essential hypertension and in hypertension models have been described, their significance remains unclear because the findings have generally been limited to nonvascular cells or to VSMC in culture. Because VSMC rapidly lose their contractile phenotype in culture and convert to a proliferative and synthetic phenotype (46), findings for cultured cells may not be indicative of the function and regulation of ion transporters in vivo.

NKCC1 is a prominent transporter in VSMC in culture (29, 31, 32, 42). It is activated by cell shrinkage (29, 30) and is responsible for volume recovery (30), and it is also activated by ANG II both acutely and chronically (2, 32, 48), suggesting a role in smooth muscle contraction and hypertrophy. NKCC1 activity is reduced in cells cultured from aortas of spontaneously hypertensive rats (37, 41). Because of the proliferative phenotype in culture, and particularly because NKCC1 is activated by growth factors and may participate in cell growth, these findings are of questionable relevance to smooth muscle in vivo (33, 34). However, few studies of NKCC1 in intact vascular smooth muscle have been performed. Deth et al. (8) observed a Na⁺-dependent, Cl-dependent Rb⁺ influx in rat and rabbit aortas that was inhibited by furosemide, thereby establishing the presence of NKCC1 in intact smooth muscle. Furosemide reduced both the Ca²⁺ and contractile responses to phenylephrine in this study and has been shown to inhibit smooth muscle contraction in other studies (9, 47), consistent with its vasodilatory action in vivo. However, furosemide is not a specific inhibitor of NKCC1 and can block Cl transport through other pathways. Davis et al. (6) provided additional evidence for the cotransporter in the rat femoral artery by showing that bumetanide lowered intracellular [Cl]. This decrease in intracellular [Cl] was augmented in rats made hypertensive by the administration of deoxycorticosterone and a high-salt diet, suggesting upregulation of NKCC1. Norepinephrine produces an increase in intracellular [Cl] that is partly blocked by bumetanide (7), indicating activation of NKCC1, but the response of the cotransporter in intact smooth muscle to other vasoconstrictors or to vasodilators has not been examined.

Studies of ion transport in vascular tissue have been limited by technical considerations. NKCC1 activity is usually measured as the influx of ⁸⁶Rb⁺, a tracer for K⁺, but measurements of influx are hampered by problems with trapped extracellular tracer, with normalization (various proportions of cells vs. matrix), and with the large amount of tissue required. NKCC1 activity has also been assayed as changes in intracellular [Cl] measured with intracellular electrodes (6), but this is technically very demanding and time consuming. Because NKCC1 mediates bidirectional transport, NKCC1 activity can also be measured as the efflux of ⁸⁶Rb⁺ (15). Although net transport is inward under physiological conditions, there is a sizable efflux representing Rb⁺/K⁺ exchange. The measurement of efflux avoids the problems associated with influx measurements, particularly sample heterogeneity, because results can be normalized to intracellular tracer content. The validity of measuring NKCC1 activity as ⁸⁶Rb⁺ efflux was established by previous studies of vascular endothelial cells (15, 28). The efflux of Rb⁺ in rat aorta has previously been measured and, although somewhat lower than the efflux of K⁺, showed qualitatively similar responses to norepinephrine and KCl depolarization (43). We have now employed this technique to measure NKCC1 and its regulation by vasoconstrictors and vasodilators in intact smooth muscle from rat aorta. Coupled with the measurement of force generation, this enabled us to demonstrate that the cotransporter has a functional role in vascular smooth muscle.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tissue preparation. Descending aortas were 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.

Cell culture. VSMC were cultured from aortas of Sprague-Dawley rats and were grown in DMEM (high glucose) containing 10% fetal bovine serum. Once the cells reached ~70% confluency, they were serum starved for 72 h in serum-free medium containing insulin, transferrin, and ascorbate (19), with daily medium changes. All studies were performed with monolayers in plastic multiwell plates.

NKCC1 activity. Cotransporter activity was measured as unidirectional K⁺ efflux as previously described (35), with ⁸⁶Rb⁺ as a tracer. The standard isotonic solution was Earle's salts, with HEPES substituted for HCO₃, 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. The solution was made hypertonic by adding 150 mM sucrose. ⁸⁶RbCl was obtained from DuPont NEN. Bumetanide was a gift from Dr. Peter Sorter (Hoffmann-LaRoche, Nutley, NJ) and was stored as a 100 mM stock solution in DMSO.

Cotransporter phosphorylation. Aortas were prepared as described above and divided lengthwise, and each half was incubated in 1-2 ml of phosphate-free Dulbecco's MEM containing 0.1 mCi/ml of [³²P]orthophosphate for 3 h at 37°C in a CO₂ incubator. Test substances were added at the end of this incubation. Each half-aorta was homogenized in a ground-glass homogenizer in 250 µl of 250 mM sucrose, 10 mM triethanolamine, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), pH 7.6. The homogenizer was rinsed with an additional 250 µl, and the two aliquots were combined. SDS was added to a final concentration of 1%, and the mixture was then incubated for 10 min at room temperature. The mixture was passed through an insulin syringe several times and then added to 5 ml of a solution of 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in extraction buffer containing (in mM) 25 Tris · HCl (pH 7.9), 100 sodium pyrophosphate, 100 NaF, 250 NaCl, 10 EGTA, 5 EDTA, 0.2 PMSF, and 0.5 benzamidine, with 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml soybean trypsin inhibitor. After incubation on ice for 1 h, particulate matter was removed by centrifugation and the supernatant was incubated at 4°C for 12 h with a monoclonal antibody prepared against the Na⁺-K⁺-2Cl cotransporter from human colonic carcinoma cells (T4 antibody; kindly provided by Dr. C. Lytle). Protein G-Sepharose (GIBCO, Grand Island, NY), washed and equilibrated in the 2% CHAPS extraction buffer, was added (50 µl of a 40% suspension per sample) with gentle agitation for 3 h at 4°C. The Sepharose was washed six times with 1% Triton X-100 in extraction buffer and once with K⁺-free PBS. Bound material was removed by heating the Sepharose in SDS electrophoresis sample buffer and was separated on 7.5% polyacrylamide gels according to the method of Laemmli (17).

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. A ring of 5 mm was cut from the thoracic aorta; blot weights were 4-5 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 (24). 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. Then the aorta was stimulated by the 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. Then a cumulative concentration-force relation for phenylephrine was generated. Bumetanide (10 µM) was added to the bath, and after 20 min the concentration-force relation was again measured. This experimental protocol for KCl stimulation was repeated in a separate study. After each experiment, ring dimensions and blot weight were measured. Aortic wall thickness (t) was estimated from 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 with a physiological saline solution consisting of (in mM) 118 NaCl, 4.73 KCl, 1.2 MgCl₂, 0.026 EDTA, 1.2 KH₂PO₄, 2.5 CaCl₂, 5.5 glucose, and 25 NaH₂CO₃, which was bubbled with 95% O₂-5% CO₂ at pH 7.4 and 37°C.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although normal physiological conditions dictate a net influx of ions through NKCC1, the transporter is bidirectional and exhibits a considerable rate of K⁺/K⁺ (Rb⁺) exchange. Thus cotransporter activity can be measured as Rb⁺ efflux, which has distinct advantages for intact smooth muscle. Multiple time points for the same segment of aorta can be obtained, substantially reducing the amount of tissue required, and measurement of dry weight or protein or DNA content is not required because the data are normalized to Rb⁺ content (rate coefficients). A typical assay is shown in Fig. 1. Data are presented as fractions of tracer remaining in the cells (top) and as rate coefficients (bottom). A stable basal efflux was obtained, after which the addition of 50 µM bumetanide, a specific inhibitor of NKCC1, reduced efflux to a new, stable level. Subtraction of the two rate coefficients yields the bumetanide-sensitive flux, which accounts for approximately one-third of the total efflux. The remaining bumetanide-insensitive efflux represents fluxes through K⁺ channels and K⁺ leak pathways. This procedure permits the calculation of bumetanide-sensitive efflux for each aortic segment. It has previously been shown that bumetanide-sensitive Rb⁺ influx in aorta is dependent on extracellular Na⁺ and Cl (8), consistent with NKCC1 activity.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Efflux of 86Rb+ from rat aortic smooth muscle. Efflux was measured at 2-min intervals, before and after addition of 50 µM bumetanide (arrows), from rat aorta prepared and loaded with 86Rb+ as described in MATERIALS AND METHODS. Top: fraction of 86Rb+ remaining after each time point. Bottom: fraction of 86Rb+ recovered in medium at each time point expressed as rate coefficient. Results are means ± SE of a representative experiment performed in triplicate.

To measure acute changes in cotransport, parallel assays were performed in the presence and absence of bumetanide (Fig. 2). In this experiment, the addition of 150 mM sucrose to produce hypertonic shrinkage increased total efflux and reduced bumetanide-insensitive efflux, indicating a rapid doubling of cotransporter activity. Cotransporter activity returned to baseline when isotonicity was restored (data not shown). This is consistent with the volume sensitivity of NKCC1 in virtually all cells, including cultured VSMC (29), and indicates that Rb⁺ efflux provides a reliable assay of NKCC1 activation in intact vascular smooth muscle.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of hypertonic medium on Rb+ efflux in rat aorta. 86Rb+ efflux was measured in isotonic and hypertonic medium (arrows indicate transition). Top: efflux in absence or presence of bumetanide (50 µM). Bottom: bumetanide-sensitive efflux. Results are means ± SE from 3 aortas, each assayed in triplicate.

ANG II (100 nM) significantly increased the efflux of Rb⁺ (Fig. 3). A portion of the increase was inhibited by 50 µM bumetanide, revealing a transient increase in NKCC1 activity that peaked at 152 ± 11% of control (n = 7, P < 0.001). The increase in bumetanide-insensitive efflux presumably represents the opening of K⁺ channels. More prolonged incubation with ANG II (performed during loading with ⁸⁶Rb⁺) revealed a second, delayed activation of NKCC1 (Fig. 4). There was no stimulation after 1 h of incubation with 100 nM ANG II, but significant stimulation was apparent after 2 h (82 ± 20%; n = 9, P < 0.02) and 4 h (22 ± 3%; n = 3, P < 0.02). Losartan, an antagonist of type 1 ANG II receptors, completely blocked both the acute and delayed stimulation of NKCC1 at a concentration of 10 µM (Fig. 5). There was a small but statistically significant reduction in basal NKCC1 activity (22%) with losartan. However, neither 1 µM saralasin (a peptide antagonist of ANG II) nor 4 µM lisinopril, an inhibitor of ANG II-converting enzyme, reduced basal NKCC1 activity (data not shown). For comparison, efflux in VSMC cultured from rat aorta was also measured. Bumetanide-insensitive efflux was much higher in cells (0.0189 ± 0.0012 min¹) than in aorta, rendering bumetanide-sensitive efflux difficult to measure. However, values for bumetanide-sensitive efflux under basal conditions (0.00228 ± 0.00067¹), after acute ANG II stimulation (0.00423 ± 0.00089¹), and after 2 h of ANG II (0.00544 ± 0.00080¹) were similar to those observed in intact vascular smooth muscle.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of ANG II on Rb+ efflux in rat aorta. Top: efflux of 86Rb+ was measured in absence or presence of bumetanide (50 µM). Bottom: bumetanide-sensitive efflux derived from top data. ANG II (100 nM) was added at time indicated (arrows). Results are means ± SE from 7 aortas, each assayed in triplicate.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Delayed stimulation of Na+-K+-2Cl cotransporter NKCC1 by ANG II. Aortas were preincubated with (ANG II) or without (control) ANG II (100 nM) for times indicated, after which efflux of 86Rb+ was measured in absence or presence of bumetanide (50 µM). Results are means ± SE from 3 aortas, each assayed in triplicate. * P < 0.02 vs. control.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of losartan on bumetanide-sensitive Rb+ efflux in rat aorta. Aortic segments were preincubated for 2 h with (ANG II) or without (basal) ANG II (100 nM) and with (losartan) or without (control) losartan (10 µM), and efflux of 86Rb+ was measured in presence and absence of bumetanide (50 µM). Results are means ± SE of 7 aortas for basal flux and 4 aortas for flux in presence of ANG II, each measurement performed in triplicate. * P < 0.01 vs. basal control; ** P < 0.001 vs. basal control; *** P < 0.001 vs. ANG II control.

NKCC1 activity in rat aorta was also stimulated acutely by 10 µM phenylephrine (50 ± 13%; P < 0.01, n = 8), 50 nM endothelin (53 ± 17%; P < 0.02, n = 6), and 30 mM KCl (54 ± 6%; n = 5, P <0.01) as shown in Fig. 6. For the KCl experiments, KCl was isosmotically substituted for NaCl to avoid any hypertonic stimulation of NKCC1. However, some cell swelling would be expected in this medium, and this swelling would reduce NKCC1 activity. Thus the stimulation of NKCC1 by membrane depolarization may be greater than that achieved in these experiments. The stimulation of Rb⁺ efflux by extracellular K⁺ is unlikely to be due to direct enhancement of K⁺/Rb⁺ exchange (trans stimulation) by NKCC1 because stimulation was quantitatively similar at 80 mM (not shown) and because raising extracellular K⁺ concentration ([K⁺]) does not increase bumetanide-sensitive Rb⁺ efflux in endothelial cells (28), which lack voltage-sensitive Ca²⁺ channels (1). As in the case of ANG II, there was a significant increase in bumetanide-insensitive efflux. Unlike the case of ANG II, there was no delayed stimulation by phenylephrine or endothelin (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of vasoconstrictors on bumetanide-sensitive Rb+ efflux in rat aorta. Phenylephrine (10 µM), endothelin (50 nM), and KCl were added at time indicated (arrows). Results are means ± SE from 8 (phenylephrine), 6 (endothelin), and 3 aortas (KCl), each assayed in triplicate.

Nitrovasodilators had an opposite effect on NKCC1. Nitric oxide was tested by using 10 µM 2-(N,N-diethylamino)diazenolate-2-oxide (DEA-NO) as a donor. As shown in Fig. 7, bumetanide-sensitive efflux was inhibited 34%. A similar degree of inhibition was observed with 8 µM nitroprusside, with no additional inhibition at higher concentrations of nitroprusside. S-nitroso-N-acetyl-D,L-penicillamine also inhibited NKCC1, but this did not reach statistical significance. The inhibition of NKCC1 was more apparent when aortas were pretreated with phenylephrine, a process akin to relaxation assays of preconstricted vessels. As shown in Fig. 8, nitroprusside (8 µM) completely reversed the stimulation of NKCC1 by phenylephrine (10 µM).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of nitrovasodilators on bumetanide-sensitive Rb+ efflux in rat aorta. Efflux was measured before and after addition (arrow) of 10 µM 2-(N,N-diethylamino)diazenolate-2-oxide (DEA-NO). Inset: mean bumetanide-sensitive efflux before (control) and after (vasodilator) addition of nitrovasodilators from 6 aortic segments. SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetyl-D,L-penicillamine. * P < 0.01 vs. control. Error bars, SE.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effect of SNP on stimulation of Rb+ efflux in rat aorta by phenylephrine (PE). PE (10 µM) and SNP (8 µM) were added at times indicated (arrows). Results are from 1 experiment performed in triplicate and are representative of 3 additional experiments. Inset: composite data from 4 experiments. Solid bar, mean basal efflux (2-6 min); open bar, mean peak PE efflux (10 min); cross-hatched bar, mean efflux at 16 min. Error bars, SE.

In other cells, stimulation of NKCC1 is associated with phosphorylation of the transporter (10, 14, 20). To confirm this relationship in smooth muscle and to provide definitive evidence for NKCC1 in this tissue, NKCC1 was immunoprecipitated from aortic smooth muscle preincubated with [³²P]orthophosphate. As shown in Fig. 9, changes in NKCC1 phosphorylation paralleled changes in bumetanide-sensitive Rb⁺ efflux, with significant stimulation by phenylephrine, KCl, and hypertonicity and inhibition by nitroprusside. These data confirm that the efflux results accurately represent changes in NKCC1 activity.

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Phosphorylation of NKCC1 in vascular smooth muscle. Rat aortas were labeled with [32P]orthophosphate and treated for 5 min with 30 mM KCl, 10 µM PE, or 10 µM SNP or for 15 min in hypertonic medium (150 mM sucrose; HYPER). This was followed by immunoprecipitation of NKCC1 as described in MATERIALS AND METHODS. A: representative autoradiogram. kD, kilodaltons. B: change in phosphorylation, expressed as percentages of concurrent control (isotonic) samples and measured by densitometry. Bars are means ± SE of 3-5 separate experiments.

To determine the functional significance of cotransporter activation by vasoconstrictors, force generation in aortic rings was measured before and after a 20-min incubation with 10 µM bumetanide (Fig. 10). Bumetanide significantly reduced the contractile response to submaximal concentrations of phenylephrine (63 and 26% at 10 and 30 nM, respectively) without altering maximal contraction. This resulted in approximately a doubling of the half-maximal phenylephrine concentration. In contrast, there was no inhibition of contraction by KCl, under which condition bumetanide would not be expected to lower cell volume or intracellular [Cl].

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Effect of bumetanide on aortic contraction. Isometric force in aortic rings was measured as described in MATERIALS AND METHODS before () and after () 20 min in 10 µM bumetanide. * P < 0.05, ** P < 0.02 vs. force at same concentration of PE in absence of bumetanide. Error bars, SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Rb⁺ efflux from rat aortic smooth muscle was inhibited 30-40% by bumetanide, a highly specific inhibitor of Na⁺-K⁺-2Cl cotransport, indicating the presence of NKCC1 in vascular smooth muscle. Definitive evidence was provided by immunoprecipitation of the phosphorylated cotransporter from the aorta. This extends previous findings by Deth et al. (8) of furosemide-sensitive, Na⁺- and Cl-dependent Rb⁺ influx in aortas from rats and rabbits and the findings by Davis et al. (5) that bumetanide decreases intracellular [Cl] in vascular smooth muscle from the rat femoral artery. The significant stimulation of bumetanide-sensitive efflux in hypertonic medium is further evidence for the Na⁺-K⁺-2Cl cotransporter, which is universally stimulated by cell shrinkage. The present findings and previous studies contradict the recent contention that NKCC1 is present only in cultured smooth muscle cells and not in intact vascular smooth muscle (38). There are two forms of this transporter, encoded by separate genes (11). Because one form (NKCC2) is found only in the apical membrane of the thick ascending limb of Henle in the kidney, it is assumed that NKCC1 is the form present in vascular smooth muscle, and this is consistent with the presence of NKCC1 mRNA in cultured rat aortic smooth muscle cells (38). The inability of Raat et al. (38) to detect NKCC1 mRNA in freshly isolated rat aortic smooth muscle cells is difficult to explain because we observed similar rate coefficients for bumetanide-sensitive efflux in rat aortas and in rat aortic smooth muscle cells in culture.

All vasoconstrictors tested produced a rapid increase in total Rb⁺ efflux, consistent with previous studies (3, 21-23, 44). Although this had been ascribed to the opening of K⁺ channels, we find that NKCC1 contributes substantially to this increase, a contribution ranging from 30% (ANG II) to 36% (phenylephrine). That portion of the KCl-induced K⁺ efflux previously described as Ca²⁺ independent and swelling independent (21) may in fact be mediated by NKCC1. The stimulation of NKCC1 by KCl, which acts by depolarizing smooth muscle and activating voltage-sensitive Ca²⁺ channels to raise intracellular [Ca²⁺], indicates that the stimulation by other vasoconstrictors is a direct effect of increased intracellular [Ca²⁺] and not an effect of other receptor-mediated actions. In addition to immediate stimulation of NKCC1, ANG II also demonstrated a second, delayed stimulation between 2 and 4 h that was not observed with phenylephrine. This suggests at least two modes of activation of NKCC1 in vascular smooth muscle and is consistent with both immediate and delayed signaling by ANG II in VSMC (40).

NKCC1 is known to be activated by cell shrinkage or a decrease in intracellular [Cl]. We have previously shown that activation of NKCC1 by Ca²⁺-mobilizing agonists in endothelial cells is the result of cell shrinkage or loss of Cl due to the opening of Ca²⁺-dependent K⁺ channels and possibly Cl channels as well (28). This does not appear to be the case in vascular smooth muscle because equivalent stimulation of NKCC1 occurred with KCl (up to 80 mM), which would prevent cell shrinkage and loss of Cl through K⁺ and Cl channels (28). Furthermore, intracellular [Cl] in rat arterial smooth muscle does not decrease and actually increases in the presence of phenylephrine (7). Thus stimulation of NKCC1 by vasoconstrictors appears to be a direct effect of raising intracellular [Ca²⁺] rather than a secondary response to decreased intracellular [Cl] or cell volume. One possible mechanism may be the recently described link between myosin light chain phosphorylation and NKCC1 activity in endothelial cells (14).

Nitrovasodilators had the opposite effect on NKCC1, inhibiting its basal activity and reversing its stimulation by phenylephrine. The inhibition of NKCC1 by nitroprusside or nitric oxide has not previously been described. cGMP, a mediator of nitrovasodilator effects, stimulates NKCC1 in VSMC in culture (27), which is the opposite of the effect of nitroprusside in rat aorta. However, cGMP is reported to inhibit NKCC1 in cultured endothelial cells (26). Thus the effect of cGMP may vary between cells and with culture conditions. Nitroprusside is reported to lower intracellular [Ca²⁺] in vascular smooth muscle (22), which would be consistent with the role of intracellular Ca²⁺ in the stimulation of NKCC1 by vasoconstrictors. Whether nitroprusside has effects on cell volume or intracellular [Cl] that could inhibit NKCC1 is unknown.

Activation of NKCC1 in other cells depends on phosphorylation (20, 36) and is associated with the phosphorylation of the transporter (10, 14, 20), although it is not known whether the phosphorylation of NKCC1 is required for activation. Consistent with this, we observed changes in the phosphorylation of NKCC1 that paralleled changes in activity. This suggests that vasoconstrictors and nitrovasodilators affect NKCC1 through a common pathway that alters its phosphorylation. The results also indicate that the changes in NKCC1 activity observed in smooth muscle are not due to kinetic effects related to changes in intracellular ion activities.

The opposing effects of vasoconstrictors and vasodilators on NKCC1 suggest that the cotransporter has an important role in smooth muscle contraction, which was confirmed in direct measurements of contraction by phenylephrine. The inhibition by bumetanide was most apparent at low agonist concentrations likely to be physiologically relevant. Bumetanide was recently shown to inhibit the contraction of rat aorta induced by norepinephrine (18), but the response to different doses of agonist and the effect on maximal contraction were not studied. The effect of bumetanide is consistent with the known vasodilatory effect of furosemide in vitro and in vivo (8, 9). However, furosemide is not specific for NKCC1 and can inhibit other Cl transport pathways. Therefore the effect of bumetanide provides definitive proof that inhibition of NKCC1 can produce vasodilation and indicates that this is the likely mechanism for the vasodilatory effect of furosemide. Vasodilation is not a prominent feature of "loop" diuretics because extensive protein binding limits systemic inhibition of NKCC1. Assuming that 90% of bumetanide is protein bound (4) and that the volume of distribution is 0.068 l/kg (39), a standard dose of 0.015 mg/kg in humans would produce a free plasma concentration of ~60 nM. This is well below the half-inhibitory concentration of ~200 nM (13), but a maximal dose could approach this concentration.

The lack of inhibition of vasoconstriction by KCl indicates possible mechanisms by which NKCC1 promotes contraction. Because the cotransporter would be expected to mediate a net influx under physiological conditions, bumetanide could have several effects in smooth muscle, specifically, a decrease in intracellular [Na⁺], intracellular [Cl], or cell volume. The last two would be prevented or significantly reduced by raising external [K⁺], but the decrease in intracellular [Na⁺] would not. The fact that the vasodilatory effect of bumetanide is not observed after KCl contraction therefore implicates intracellular [Cl] or cell volume. It also eliminates the possibility that bumetanide is acting nonspecifically through an effect independent of NKCC1. Bumetanide has been shown to decrease intracellular [Cl] in vascular smooth muscle (6), indicating a role for NKCC1 in regulating this ion. The activation of NKCC1 by vasoconstrictors may therefore serve to raise intracellular [Cl], thereby maintaining or increasing the Cl current that contributes to membrane depolarization and subsequent Ca²⁺ influx (16, 18, 45). This raises the possibility that alterations in NKCC1 could influence vascular smooth muscle function and blood pressure in vivo.