K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation

Norma C. Adragna1, Richard E. White2, Sergei N. Orlov3, and Peter K. Lauf2

1 Departments of Pharmacology and Toxicology and 2 Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio 45435; and 3 Laboratory of Molecular Medicine, Centre de Recherche de L'Université de Montreal, Quebec H2W 1T8, Canada


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INTRODUCTION
MATERIALS AND METHODS
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K-Cl cotransport, the electroneutral-coupled movement of K and Cl ions, plays an important role in regulatory volume decrease. We recently reported that nitrite, a nitric oxide derivative possessing potent vasodilation properties, stimulates K-Cl cotransport in low-K sheep red blood cells (LK SRBCs). We hypothesized that activation of vascular smooth muscle (VSM) K-Cl cotransport by vasodilators decreases VSM tension. Here we tested this hypothesis by comparing the effects of commonly used vasodilators, hydralazine (HYZ), sodium nitroprusside, isosorbide mononitrate, and pentaerythritol, on K-Cl cotransport in LK SRBCs and in primary cultures of rat VSM cells (VSMCs) and of HYZ-induced K-Cl cotransport activation on relaxation of isolated porcine coronary rings. K-Cl cotransport was measured as the Cl-dependent K efflux or Rb influx in the presence and absence of inhibitors for other K/Rb transport pathways. All vasodilators activated K-Cl cotransport in LK SRBCs and HYZ in VSMCs, and this activation was inhibited by calyculin and genistein, two inhibitors of K-Cl cotransport. KT-5823, a specific inhibitor of protein kinase G, abolished the sodium nitroprusside-stimulated K-Cl cotransport in LK SRBCs, suggesting involvement of the cGMP pathway in K-Cl cotransport activation. Hydralazine, in a dose-dependent manner, and sodium nitroprusside relaxed (independently of the endothelium) precontracted arteries when only K-Cl cotransport was operating and other pathways for K/Rb transport, including the Ca-activated K channel, were inhibited. Our findings suggest that K-Cl cotransport may be involved in vasodilation.

potassium-chloride cotransport; vasodilators; nitric oxide


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INTRODUCTION
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ORGANIC NITRATES ARE PRODRUGS that are converted into nitric oxide (NO) via the cGMP pathway in vascular smooth muscle (VSM) (6, 9, 19, 29). Nitrates are therapeutic drugs widely used for the treatment of angina pectoris and are thought to affect the process of atherosclerosis (29), probably by inducing apoptosis by a cGMP-independent mechanism in atherosclerotic plaques (26). These drugs are not used for long-term therapy, due to the development of tolerance. The mechanism by which tolerance develops is still unclear (19, 29). In contrast, hydralazine (HYZ) is one of the common drugs used in the long-term therapy for the treatment of hypertension, yet its mechanism of action is not well understood, although it is thought to involve NO release. Recent findings suggest that HYZ leads to endothelium-dependent vasorelaxation via a slow accumulation of cGMP in porcine coronary arteries without release of NO or PGI2 (33).

Activation of K channels mediates NO-induced relaxation of canine middle cerebral arteries (25). However, differences in NO production (35) and nitrovasodilator-induced cGMP levels suggest participation of different channels in different vascular beds (6). For instance, the Ca-activated K (BK) channel is expressed at high density and dominates channel activity in porcine coronary arteries during agonist-induced vasorelaxation (10, 34).

Electroneutral K-Cl cotransport (21) mediates efflux of K and Cl driven by their respective chemical gradients in red blood cells (RBCs) of a wide variety of species. At least two isoforms of K-Cl cotransport have been cloned: KCC1 and KCC2, both members of the superfamily of cation-chloride cotransporters (13, 30). The physiological role of KCC1 is volume and ion homeostasis. Among the pharmacological inhibitors of K-Cl cotransport are the loop diuretics, furosemide and bumetanide (22), in concentrations higher than those affecting Na-K-2Cl cotransport, whereas [(dihydroindenyl)oxy]alkanoic acid (DIOA), earlier reported as the most specific inhibitor of K-Cl cotransport (22), inhibits Na-K-Cl cotransport at similar concentrations (14). Several interventions stimulate K-Cl cotransport, such as cell swelling, cytoskeleton breakdown, thiol group modification, decrease of intracellular Ca and Mg concentrations, and pH (between 6.5 and 7) (22, 27). In nucleated cells and immature erythrocytes, K-Cl cotransport plays an important role in regulatory volume decrease (22). Mature low K (LK), but not high K (HK) sheep red blood cells (SRBCs) exhibit K-Cl cotransport and thus serve as models to characterize its physiological properties and to understand its pathophysiological abnormalities in humans. Evidence from our laboratory indicates that, in LK SRBCs, nitrite (NO-2), an NO derivative, stimulates K-Cl cotransport (3-5).

We hypothesized that activation of VSM cell (VSMC) K-Cl cotransport by vasodilators decreases VSM tension. We tested this working hypothesis by cellular and functional studies with commonly used vasodilators: HYZ, sodium nitroprusside (SNP), isosorbide mononitrate (ISSB), and pentaerythritol (PE), in LK SRBCs and in primary cultures of VSMCs from rat aortas. We also tested the effect of K-Cl cotransport activation by HYZ and SNP on VSMC relaxation of isolated porcine coronary rings. Our findings uncover a new mechanism of regulation of K-Cl cotransport involving vasodilators and suggest a potential role for the transporter in vasodilation.


    MATERIALS AND METHODS
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Chemicals. All chemicals were of analytical grade: Na2HPO4, NaH2PO4, and NaNO2 were from J. T. Baker Chemical (Phillisburg, NJ); BaCl2, NaCl, NaNO3, MgCl2, HCl, NaOH, glucose, sucrose, DMSO, HYZ, ISSB, PE, and GdCl3 were from Fisher Scientific (Fairlawn, NJ); N-ethylmaleimide (NEM) was from Mallinckrodt Chemical Works (St. Louis, MO); bumetanide, dithiotreitol (DTT), histamine, tetraethyl ammonium (TEA), and SNP were from Fluka (Milwaukee, WI); ouabain, BSA (catalog no. A7030), Tris, HEPES, and PGF2alpha were from Sigma (St. Louis, MO); and ultrapure RbNO3 and RbCl were from Johnson Mathew Chemicals (Royston, UK). Cell culture media supplements, trypsin, penicillin, streptomycin, amphotericin B, EDTA, and DMEM were from GIBCO (Grand Island, NY); fetal bovine serum was from Hyclone Laboratories (Logan, UT); A-23187 and KT-5823 were from Calbiochem (San Diego, CA); and DIOA was from RBI/Sigma (Natick, MA).

Unless otherwise specified, all stock and working solutions were prepared with double-distilled and deionized water. All solutions containing vasodilators were prepared on the same day of the experiment.

K efflux measurements in LK SRBCs. K efflux rate constants were determined as detailed elsewhere (5, 20). Briefly, an aliquot of whole blood was obtained from LK sheep by staff from the Laboratory of Animal Resources. RBCs were separated from white cells and plasma by centrifugation and were washed and equilibrated in either isotonic or hypotonic buffer (5). After equilibration, the packed cells were resuspended in flux media. Aliquots of cell suspensions were taken to determine the initial internal K concentrations and the K concentrations of cell-free supernatants, taken at five time points, by atomic absorption spectrophotometry (Perkin-Elmer 5000; Perkin-Elmer, Norwalk, CT). Rate constants were calculated as described elsewhere (20).

Rb influx in cultured VSMCs. Rat aortic smooth muscle cells (RASMCs) were kindly provided by Dr. Robert Putnam, Department of Physiology and Biophysics, Wright State University. RASMCs were obtained and cultured according to a published procedure (32). Cells were grown for 3-4 days (80-100% confluence) on 12-well plates in DMEM growth media and assayed for Rb influx in balanced salt solution (BSS) Cl and sulfamate media containing (in mM) 130 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 20 HEPES/Tris buffered to pH 7.4 at 37°C. Cl-free solutions contained Na and K sulfamate and Ca and Mg gluconate salts. In addition, 10-4 M ouabain, 10-5 M bumetanide, and 10-5 M GdCl3 were added as inhibitors of the Na-K pump, Na-K-2Cl cotransport, and K/Rb channel fluxes (36), respectively. Ba, a known K channel inhibitor, and 1 mM TEA were applied to block the BK channel and to eliminate Rb fluxes through other channels (16). K-Cl cotransport was measured as the difference between the (ouabain + bumetanide + barium + TEA + gadolinium)-insensitive Rb influx in Cl and sulfamate. Details of the Rb influx assay and calculations are given below.

Flux procedure. A previously published flux protocol (1, 2) was modified for the experiments with VSMCs. Cells were washed in BSS Cl or sulfamate, equilibrated in BSS Cl or sulfamate plus or minus test drugs and 1% BSA at 37°C for 15 min, unless otherwise specified. Cells were fluxed for a period of time within which initial rates were maintained (5-10 min) in BSS-BSA Cl or sulfamate containing 10 mM Rb [a K congener as published for RBCs (20)], 10-4 M ouabain, 10-5 M bumetanide, and 10-5 M GdCl3. Flux was stopped by adding a cold washing solution containing (in mM) 120 MgCl2, 10 MOPS/Tris, and 5 glucose (pH 7.4 at 4°C, 300 mosM). Cells were subsequently washed and extracted for Rb measurement, and the proteins were determined by the Lowry method (as described in Refs. 1 and 2). Fluxes were calculated by established computer programs using the equations listed below. Rb uptake was measured by flame emission spectrophotometry in a Perkin-Elmer 5000 atomic absorption spectrophotometer. Rb influx, in nanomoles per milligram protein per minute was calculated from the initial velocity of cellular Rb uptake as a function of time using linear regression programs according to Eq. 1: Rb influx = ([Rb]t - [Rb]0)/t, where zero and t refer to zero and experimental time, respectively. The Cl-dependent Rb influx (or K-Cl influx or cotransport) is the calculated difference between Rb influx in Cl and in sulfamate.

Tension studies of coronary arteries. The general protocol was as described elsewhere (10, 34). Briefly, arteries were placed under a dissecting microscope, and fat and connective tissue were removed in ice-cold buffer solution. Two 4- to 5-mm rings were obtained from each left anterior descending coronary artery and were prepared for isometric contractile force recordings as described previously (10, 34). After the initial equilibration in a modified Krebs-Henseleit buffer of the following composition (in mM): 122 NaCl, 4.7 KCl, 15.5 NaHCO3, 1.2 KH2PO4, 1.2 MCl2, 1.8 CaCl2, and 11.5 glucose (pH 7.2, 37°C), oxygenated continuously with 97% O2-3% CO2, arteries were exposed to histamine or PGF2alpha (10 µM) to stabilize the preparations. After washout and a 30-min reequilibration, these vasoconstrictors were then reapplied to the tissue bath, and, after the contractile response reached a maximum level, varying concentrations of HYZ (10-8 to 10-3 M) or SNP (10 µM) were added to the bathing medium. The relaxation effect was allowed to reach a maximum level, and a second washout procedure was repeated. DTT was used to restore glutathione before the addition of all inhibitors. After the preparations reached baseline tension levels, 10-4 M ouabain, 10-5 M bumetanide, and 10-5 M GdCl3 were added as inhibitors of the Na-K pump and Na-K-2Cl cotransport and channel fluxes, respectively, from stock solutions to give the final concentrations indicated. The preparations were allowed to equilibrate again. To inhibit the BK and other K channels, 2 mM Ba, a known K channel inhibitor, and 1 mM TEA were added (16). These channel blockers were added later because they induce contraction (see Figs.7, 8, and 10). The bivalent cation ionophore A-23187 (10 µM) was added to increase intracellular Ca.

Statistical analysis. Data were expressed as means ± SE or SD or as a range. Data from tension studies were expressed as the percentage of maximum relaxation. Statistical significance between two groups was evaluated by Student's t-test for paired or unpaired data. A probability of <0.05 was considered as a statistically significant difference.


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MATERIALS AND METHODS
RESULTS
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VSMCs possess a functional K-Cl cotransport. Among the thiol reagents, NEM has been extensively used as an activator of K-Cl cotransport (20, 22). Furthermore, based on inhibition by okadaic acid and calyculin, a serine-threonine protein phosphatase 1 (ST-PP1) dephosphorylates and thus activates K-Cl cotransport, whereas an unidentified serine-threonine protein kinase phosphorylates and inactivates the carrier (7, 17). Genistein, a tyrosine kinase inhibitor, has been proposed to act on K-Cl cotransport either at the membrane level (12) or through the regulatory cascade (12). NEM activation is blocked by both calyculin and genistein, independently of the order of addition (12), indicating that NEM acts through the metabolic cascade. Thus we tested these inhibitors (calyculin and genistein) on NEM-, ISSB-, HYZ-, and swelling-activated K-Cl cotransport in LK SRBCs and cultured VSMCs.

Figure 1 shows the K efflux rate constant for control and calyculin- and genistein-treated SRBCs after ISSB, HYZ, and swelling activation. The experimental conditions are such that all other pathways for K transport except the K-Cl cotransport have been blocked with known inhibitors (16, 36) (see MATERIALS AND METHODS and Fig. 1 legend). Figure 1, A-C, contains three sets of data with four columns in each set representing, from left to right, the K efflux rate constant in Cl and NO3 for controls and for each activator (ISSB, HYZ, and swelling), respectively. The data show that calyculin abolished and genistein sharply reduced the activation induced by the three effectors.


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Fig. 1.   Effect of calyculin and genistein on K efflux activation by isosorbide mononitrate (ISSB) and hydralazine (HYZ) and by swelling in low-K sheep red blood cells (LK SRBCs). K efflux rate constant was calculated as described in MATERIALS AND METHODS. Calyculin and genistein were used at a final concentration of 20 nM and 100 µM, respectively. Treatment with these drugs was in Cl-free buffer (277 mosM) for 30 min at 37°C. Hatched bar, Cl control; crosshatched bar, NO3 control; ISSB (50 mM; A), HYZ (2.5 mM; B), and swelling (240 mosM; C) in Cl (solid bar) and in NO3 (open bar). Data are expressed as means ± SD for 5 time points and correspond to 1 of 2 experiments.

Figure 2A shows the Rb influx into cultured VSMCs obtained from rat aorta for isotonic control and NEM- and HYZ-treated cells. The data reveal that VSMCs possess K-Cl cotransport and that it is activated by both NEM and HYZ. NEM activated K-Cl cotransport by 13.2-fold and HYZ by 10.4-fold with respect to the control. Figure 2B shows a similar experiment as in Fig. 1 but for HYZ alone, where the characteristic inhibitors calyculin and genistein were tested to further assess the behavior of K-Cl cotransport in cultured VSMCs. In SRBCs, K-Cl cotransport can be measured in the absence of inhibitors for K/Rb transport. However, in most of the cultured cells we studied so far, due to the lack of a specific inhibitor for K-Cl cotransport, all the pathways for Rb(K) except K-Cl cotransport were blocked with known inhibitors (16, 36) (see MATERIALS AND METHODS). HYZ activated K-Cl cotransport 3.7-fold, whereas calyculin and genistein inhibited by 70% and 100%, respectively. The conclusion from Fig. 2 is that VSMCs possess a K-Cl cotransport system that behaves like that of LK SRBCs in terms of its response to ST-PP1 and tyrosine kinase inhibitors, suggesting that similar regulatory pathways are present in these model cells. Furthermore, as a control experiment and to insure that sulfamate was a good replacement for Cl in this tissue, isolated porcine coronary rings were washed and incubated in the presence of Cl or sulfamate (see MATERIALS AND METHODS for further details), and the Cl concentration was measured in all the samples. This procedure was done in three denuded and two intact rings for each anion. No Cl was detectable in the samples incubated in sulfamate (results not shown), indicating that, even in the intact tissue, Cl was fully exchangeable with sulfamate.


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Fig. 2.   Effect of N-ethylmaleimide (NEM) and HYZ, and of calyculin and genistein on Rb influx in cultured rat aortic smooth muscle cells (RASMCs). Cell culturing and Rb influx was as described in MATERIALS AND METHODS. A: after the initial wash, cells were preincubated in isotonic medium for 15 min at 37°C with NEM (0.5 mM) and HYZ (1.75 mM). Rb uptake was measured for 10 min at 37°C in isotonic medium (ISO) and in the absence of NEM and HYZ. Data represent means ± SD, n = 3. One of two experiments is shown. Solid bar, Cl; open bar, sulfamate. B: after the initial wash, cells were preincubated for 30 min in isotonic buffer saline salt (BSS; 300 mosM; see MATERIALS AND METHODS), Cl, or sulfamate containing 0.1% BSA ±20 nM calyculin (HYCAL) or 200 µM genistein (HYGEN), pH 7.4 at 37°C. Afterward, cells were fluxed in BSS-BSA (see MATERIALS AND METHODS) for 10 min. Genistein (200 µM) was present throughout the flux. Data represent means ± SD, n = 3. One of two experiments is shown. Solid bar, Cl; open bar, sulfamate. Cells were at passages 9 (A) and 13 (B); prot, protein.

Vasodilators activate K-Cl cotransport in LK SRBCs and VSMCs. The studies shown above were designed to determine whether VSMCs possess a functional K-Cl cotransport and whether its properties resemble those described in LK SRBCs. Furthermore, nitrovasodilators induce relaxation of VSM through activation of the cGMP pathway (6, 9, 19, 29). Thus the following studies were designed to test whether the effect of vasodilators on K-Cl cotransport may involve the cGMP in LK SRBCs as it occurs in VSMCs and whether activation of K-Cl cotransport by vasodilators may induce VSM relaxation.

Figure 3A shows the rate constants of K efflux from LK SRBCs in Cl and NO3 media and in the presence and absence of the indicated vasodilators. Cells were incubated in either isotonic or hypotonic buffer or in hypotonic medium containing HYZ, NO-2, SNP, ISSB, and PE. The activation of K-Cl cotransport by the vasodilators shown in Fig. 3B was highly significant (3.5- to 12-fold). The sequence of K-Cl cotransport activation was isotonic (1.0-fold) < PE (3.5-fold) < hypotonic (3.8-fold) < HYZ (5.1-fold) < ISSB (8.2-fold) < SNP (10.5-fold) < NO-2 (12.0-fold). The apparently lower PE activation than hypotonicity may be explained by the difference in the number of determinations (hypotonic, n = 16; PE, n = 4). However, comparing the effects of hypotonic and PE in the same experiments (n = 4), PE increased K-Cl cotransport by 50% above hypotonic. Thus all vasodilators tested activated erythrocyte K-Cl cotransport.


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Fig. 3.   Effect of hypotonicity and vasodilators on K efflux in LK SRBCs. A: K efflux rate constant calculated as described in MATERIALS AND METHODS. Cl medium composition was (in mM) 94 NaCl, 5 NaPO4, and sucrose to attain 295 mosM (isotonic; ISO) or 240 mosM (hypotonic; HY). Osmolalities of Cl-free medium (where NO3 replaced Cl) were: 277 mosM (isotonic) and 225 mosM (hypotonic). Vasodilator [HYZ, sodium nitrite (NO2), sodium nitroprusside (NP), ISSB, and pentaerythritol (PE)] concentrations were 5 mM for HYZ and 25 mM for the rest. All vasodilators were tested in hypotonic medium. Solid bar, Cl; open bar, NO3. B: KCl efflux rate constant calculated as difference between K efflux in Cl and NO3, as described in MATERIALS AND METHODS. Data are represented as means ± SE or range (for n = 2); number of independent experiments: 2 (NO-2 and ISSB) < n < 17.

Based on previous studies with NO-2, the drugs were tested at a concentration of 25 mM with the exception of HYZ (5 mM). Thus, to determine whether activation would occur at the concentrations previously reported by others for tension studies (in the micromolar range) (33), dose-response curves were determined for HYZ and SNP in LK SRBCs. Figure 4 shows K-Cl efflux rate constants as a function of HYZ concentration varied between 0 and 2.5 mM. The activation by HYZ was bimodal, with a maximum at 1.75 mM and declining at higher concentrations, indicating a complex relationship with more than one site of action available for HYZ. A closer look at the lower concentrations (Fig. 4, inset) showed a hyperbolic activation of K-Cl cotransport up to a concentration of 200 µM, and the estimated EC50 was ~30 µM (~20 µM in an independent experiment). Furthermore, at 20 µM, HYZ induced a 2.6-fold (or 162%) increase in K-Cl cotransport. These results indicate that HYZ activates LK SRBC K-Cl cotransport at the concentrations previously reported by others for tension studies (33).


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Fig. 4.   K-Cl efflux rate constants as a function of HYZ concentration in LK SRBCs. K efflux was determined as described in MATERIALS AND METHODS. HYZ was added only during the equilibration period at the concentrations indicated (0-2.5 mM). Points were fitted by a 4th order polynomial function. Inset: expanded scale for the lower HYZ concentrations. Fitting by a 2nd order polynomial function. Estimated EC50 for insert: ~30 µM. Data represent 1 of 2 independent experiments.

Figure 5 shows that the K-Cl efflux rate constants in LK SRBCs as a function of SNP concentration varied between 0 and 50 mM. The activation by SNP increased with the concentration, reaching a maximum at 25 mM but tended to decrease first and then remain constant between 40 and 50 mM, indicating a complex relationship with more than one site of action available for SNP. As for HYZ, a closer look at the lower concentrations (Fig. 5, inset) showed a tendency for K-Cl cotransport to first decrease in the low micromolar range and then increase up to 1 mM to further saturate between 1 to 4 mM. At 800 µM, SNP activated K-Cl cotransport by 1.5-fold (90%) and at 1 mM by 2.6-fold (160%). Thus, although higher concentrations for activation of the cotransporter are required for SNP than for HYZ, a significant activation by the two drugs was observed in the micromolar range.


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Fig. 5.   K-Cl efflux rate constants as a function of sodium nitroprusside (SNP) concentration in LK SRBCs. K efflux was determined as described in MATERIALS AND METHODS. SNP was added only during the flux period at the concentrations indicated (0-50 mM). Points were fitted by a 4th order polynomial function. Inset: expanded scale for the lower SNP concentrations. Fitting by a spline function. Data are average of 2 experiments where each rate constant is determined with 5 time points. For inset, only 1 experiment is shown where each rate constant is determined with 5 time points.

It is interesting to note that one of the enzymes involved in the NO pathway, guanylyl cyclase, like Hb, has a porphyrin group containing Fe (24). In addition, smooth muscle contains myoglobin that can be oxidized to metmyoglobin like Hb to met-Hb, and the different cytochromes also participate in oxidative stress by serving as electron carriers (23). Thus it is possible that heme containing macro molecules such as Hb or myoglobin and cytochromes (8, 11, 14, 18) may act as sinks or buffers for NO and modulate its effects. We searched for the possibility of NO buffering in LK SRBCs by varying the hematocrit (Hct) of the flux medium and determining the effect of well-established NO donors (ISSB and SNP) and of HYZ on K-Cl cotransport activation. For HYZ, we did not observe much of an effect. Also, note that the concentrations of HYZ were one order of magnitude lower than those of ISSB (Figs. 1-4). In contrast, for ISSB and SNP, changes in Hct had complex effects on K-Cl cotransport activation. Thus, when ISSB was tested at 25 mM in two independent experiments, the ISSB-stimulated KCl efflux rate constant decreased by 45% and 16% as the Hct increased from 1 to 3%. However, this relationship was complex at lower ISSB concentrations (results not shown). In experiments with 0.5 mM SNP, the mean activation (n = 2) was 17% (13% and 28%) and 42% (41% and 42%) at 5% and 0.5% Hct, respectively. Thus it seems that the relationship between drug effect and Hct depends on the type and concentration of the drug used and is more complex than expected (see DISCUSSION). However, at 25 mM ISSB and 0.5 mM SNP, it is clear that decreasing the Hct (and the available Hb) increased the activation by ISSB and SNP, whereas no effect was observed for HYZ.

To assess whether the activation of K-Cl cotransport by vasodilators involves the cGMP pathway, we tested the effect of KT-5823, a specific inhibitor of protein kinase G (the last step of the cGMP pathway) on SNP-activated K efflux (the initial step of the pathway) in swollen LK SRBCs. In addition, this experiment would suggest whether a complete cGMP pathway was present in LK SRBCs, as in human RBCs (31). Figure 6 shows the SNP-stimulated K efflux rate constant in hypotonic Cl and NO3 media and the calculated K-Cl efflux in the absence and presence of KT-5823. The inhibitor was tested at 0.3, 0.6, and 1 µM. At all the concentrations tested, KT-5823 abolished the SNP-dependent K-Cl cotransport, and thus the values at these three concentrations were pooled as shown in the Fig. 6. In another experiment, KT-5823 inhibited K-Cl cotransport by 80% at 0.3 µM. The results suggest the presence of a complete cGMP pathway in LK SRBCs and possible SNP activation of K-Cl cotransport through this pathway. Furthermore, these results exclude the possibility of a direct inhibition of KT-5823 on K-Cl cotransport, since only the SNP-stimulated but not the swelling-stimulated flux was inhibited by the drug (results not shown).


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Fig. 6.   Effect of KT-5823 on SNP-dependent K efflux rate constants in swollen LK SRBCs. K efflux was determined as described in MATERIALS AND METHODS, in hypotonic Cl and NO3 (composition as described in legend to Fig. 3). Pretreatment with 0, 0.3, 0.6, and 1 µM KT-5823 in hypotonic NO3 for 15 min at 37°C. SNP concentration, 10 mM. K-Cl efflux was calculated as the difference between the K efflux rate constant in Cl minus that in NO3. Data (means ± SD) represent the SNP-dependent flux in the absence (solid bars) and in the presence of (0.3-1.0 µM) KT-5823 (open bars). K effluxes in the presence of KT-5823 (0.3-1 µM) were pooled together (n = 3), and each individual concentration was assayed in duplicate.

K-Cl cotransport and vasodilation. Figure 7 shows the isometric contractile force recordings from one endothelium-denuded (Fig. 7A) and one intact (Fig. 7B) porcine coronary artery precontracted with 10 µM histamine and relaxed with increasing concentrations of HYZ (100 and 600 µM). The responses to HYZ were concentration dependent, albeit only two concentrations were applied in these experiments. On average, arteries were relaxed 50 ± 5% by 100 µM HYZ (n = 8), whereas increasing the HYZ concentration to 600 µM induced complete (100%) relaxation (n = 4). When all transport pathways for K/Rb transport except K-Cl cotransport were inhibited (10-4 M ouabain, 10-5 M bumetanide, 10-5 M gadolinium, 2 mM BaCl2 , and 1 mM TEA), HYZ (600 µM) was still able to induce ~50% relaxation of the arteries (n = 4). Identical results were obtained for all denuded and intact arteries studied. The ionophore A-23187 is a receptor-independent agonist that increases intracellular Ca and activates the constitutive NO synthase (cNOS) in porcine cultured endothelial cells, thereby increasing NO production (35). Thus addition of A-23187 was expected to induce further relaxation in intact arteries (as shown in Fig. 7B) and either to have no effect or to elicit contraction in the denuded ones (in contrast to results shown in Fig. 7A). See DISCUSSION for interpretation of these results.


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Fig. 7.   Isometric contractile force recordings from porcine coronary arteries in the absence (A) or presence (B) of an intact endothelium. In both preparations, initial precontraction was achieved with 10 µM histamine (Hist), and HYZ was added as indicated by arrows. After washout of all compounds, preparations were allowed to reequilibrate for 30 min in the presence of 10-4 M ouabain, 10 -5 M bumetanide (Bumet), 10 -5 M gadolinium (Gad), and 10-3 M dithiotreitol (DTT). After 30 min, arteries were then contracted by adding 2 mM BaCl2 and 1 mM tetraethylammonium (TEA), and HYZ (600 µM) was then added as indicated by arrow. The Ca ionophore A-23187 was added as indicated.

Wei et al. (33) reported an endothelium-dependent relaxation of porcine coronary arteries by HYZ with an EC50 of 1.6 × 10-6 M in intact and 3.8 × 10-5 M in denuded arteries. Based on these studies, we tested maximal concentrations of HYZ (Fig. 7). However, it was necessary to show that the relaxation induced by K-Cl cotransport activation was also dependent on the concentration of HYZ. Figure 8 shows the isometric contractile force recordings from an endothelium-intact coronary artery precontracted with 10 µM PGF2alpha and relaxed with increasing concentrations of HYZ (10-8 to 10-4 M). As in Fig. 7, the responses to HYZ were concentration dependent. When all transport pathways for K/Rb transport except K-Cl cotransport were inhibited (10-4 M ouabain, 10-5 M bumetanide, 10-5 M gadolinium, 2 mM BaCl2, and 1 mM TEA), HYZ (10-7 to 10-3 M) was still able to induce a dose-dependent relaxation of the arteries (n = 4). Identical results were obtained for all denuded and intact arteries studied. Note that the contraction elicited by PGF2alpha was initially unstable and stabilized afterward, albeit at a lower level compared with the second contraction. The difference in contractile response before and after addition of inhibitors may be due to the different vasoconstrictors used (PGF2alpha vs. Ba and TEA) and to the presence of DTT added to recover the metabolic state of the arteries.


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Fig. 8.   Typical isometric contractile force recording of HYZ-induced relaxation of an endothelium-intact coronary artery. The artery was contracted with 10 µM PGF2alpha , and HYZ was added cumulatively at the concentrations indicated by arrows. After the control response was obtained, preparations were incubated for 30 min with the blocking agents at the concentrations indicated. Arteries were contracted with Ba and TEA, and the response to HYZ was recorded again. BUM, bumetanide.

Figure 9 summarizes the calculated percentage of relaxation as a function of the logarithm of HYZ concentrations from 10-8 to 10-3 M. As expected, the maximum relaxation response to HYZ was greater in endothelium-intact vessels (~50%; Fig. 9, A and B), compared with endothelium-denuded vessels (~30%; Fig. 9, C and D), with an apparent increase in the EC50 value of one log unit occurring on removal of the endothelium (1 µM, intact; 10 µM, denuded; compare Fig. 9 panels A and B with C and D). In intact arteries in the presence of inhibitors, the relaxation response to HYZ required higher concentrations, as indicated by the rightward shift in the concentration-response relationship (Fig. 9, A and B). However, the maximum relaxation response to HYZ was not depressed. In conclusion, we confirmed the findings of Wei et al. of an endothelium-dependent relaxation of porcine coronary arteries and obtained similar EC50 values for HYZ in intact and denuded arteries. In addition, our study adds the new finding of a putative HYZ-induced activation of K-Cl cotransport through an endothelium-independent mechanism.


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Fig. 9.   Complete concentration-response relationships for HYZ-induced relaxation of coronary arteries from endothelium-intact (A, B) or endothelium-denuded (C, D) preparations before (solid lines) and 30 min after addition of 10-4 M ouabain, 10-5 M bumetanide, 10-5 M gadolinium, 10-3 M DTT, 2 mM BaCl2, and 1 mM TEA (dashed lines). Eeffect of HYZ was measured by a digital acquisition system that directly feeds the tension measurements to a computer, and a computer program calculates directly the values as percent relaxation. This system provides more accurate measurements than those obtained solely by visual interpretation.

To see whether activation of K-Cl cotransport in VSM involved the cGMP pathway, we tested the effects of SNP, known to act through a cGMP-dependent mechanism in this tissue, and DIOA, a more specific inhibitor of K-Cl cotransport in RBCs but not in human embryonic kidney (HEK-293) cells (14), after addition of the inhibitors of all non-K-Cl cotransport pathways. Figure 10 shows the isometric contractile force recordings from an endothelium-denuded porcine coronary artery precontracted with 10 µM PGF2alpha and relaxed with 10 µM SNP. Because SNP is an endothelium-independent vasodilator, all the arteries studied had the endothelium removed. DIOA (50 µM) was added with the rest of the inhibitors of K/Rb transport pathways (10-4 M ouabain, 10-5 M bumetanide, 10-5 M gadolinium, 2 mM BaCl2 , and 1 mM TEA). SNP induced a nearly complete relaxation of arteries in the presence (50-600 µM) or absence of DIOA (n = 4). Identical results were obtained for all the denuded arteries studied.


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Fig. 10.   Typical relaxation response of coronary arteries to 10 µM SNP (arrow) before and 30 min after addition of inhibitors. Effect of cumulative addition of [(dihydroindenyl)oxy]alkanoic acid (DIOA) on SNP-induced relaxation is also given (arrows). However, initially, DIOA was added 20 min before TEA and Ba.

To further test the hypothesis of a different isoform of K-Cl cotransport in VSMCs, we measured K-Cl cotransport in cultured RASMCs in the presence and absence of 0.5 mM NEM, furosemide, and DIOA. NEM activated K-Cl cotransport by 3- to 10-fold (results not shown). In addition, as previously published (28), little or no inhibition was observed with 1 mM furosemide. In contrast, DIOA at 300 µM inhibited between 50 and 100%.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Functional K-Cl cotransport in VSMCs. Previously, we reported that hypotonic swelling of VSMCs sharply increases the rate of bumetanide (20 µM)- and furosemide (1 mM)-resistant 86Rb efflux and influx in these cells, suggesting the presence of either swelling-activated K channels or of a new furosemide-resistant isoform of K-Cl cotransport (28). In the present study, the latter alternative was supported by demonstration of a Cl-dependent Rb influx in VSMCs in the presence of available inhibitors of other Rb/K transport pathways (Fig. 2). Gillen et al. (13) reported on K-Cl cotransport mRNA expression from a multiple tissue source where the highest expression appears in human muscle. Although the authors did not specify in which type of muscle, the source appears to be skeletal muscle. Here, we show that VSMCs possess a functional K-Cl cotransport. Based on the activation elicited by NEM and HYZ (Fig. 2A) and inhibition by calyculin and genistein (Fig. 2B), the regulation of the system in VSM appears to be similar to that of SRBCs. Thus the presence of K-Cl cotransport in VSM is supported by 1) the presence of Cl-dependent K(Rb) influx, 2) its activation by NEM, 3) its inhibition by calyculin and genistein, and 4) our previous data suggesting the presence of a bumetanide- and furosemide-resistant K-Cl cotransport influx and efflux in VSMCs (28). This study also reports, for the first time, an activation of K-Cl cotransport by vasodilators in SRBCs (Figs. 1 and 3-6) as well as in cultured VSMCs (Fig. 2) and, based on the novel observation with KT-5823, suggests a cGMP-mediated mechanism of activation (Fig. 6). Lack of inhibition by furosemide in cultured RASMCs (28) and by DIOA in porcine coronary arteries (Fig. 10) suggested the presence of a new isoform of K-Cl cotransport in VSM. However, when DIOA was tested in RASMCs in the present study, the drug did inhibit the NEM-activated K-Cl cotransport. Although these latest results are not consistent with the hypothesis of a different isoform of K-Cl cotransport in VSMCs, the lack of inhibition of VSM relaxation by DIOA during the tension studies may be explained by species' differences (murine vs. porcine), by preparation differences (cultured rat aortic cells vs. porcine coronary arteries), or by DIOA effects on the contractile machinery that cancel out those on K-Cl cotransport. Furthermore, inhibition by DIOA does not completely rule out the possibility of a different isoform of K-Cl cotransport with similar pharmacological properties to KCC1 and KCC2. This hypothesis needs further investigation.

Activation of K-Cl cotransport by vasodilators in LK SRBCs and VSMCs and its implication in vasodilation. As in VSM, the effect of nitrovasodilators in SRBCs may involve the release of NO, since RBCs appear to possess the enzymes required for NO synthesis as well as soluble and particulate guanylyl cyclase and cGMP (see Ref. 30 and Fig. 6). In addition, the relaxation observed in isolated porcine coronary rings on addition of HYZ (Figs. 7-9) and SNP (Fig. 10) and presumable stimulation of K-Cl cotransport suggests that this transporter plays a role in vasodilation. However, the metabolic pathways for Ca may differ between RBCs and VSMCs. In the latter, it is possible that vasodilators increase cGMP and, through a series of steps involving the inositol phosphate cycle, inhibit the entry of Ca as well as the mechanism of internal Ca release (6, 9). Thus these drugs could activate K-Cl cotransport by relieving the intracellular Ca inhibition through the cGMP pathway. However, a direct interaction between the vasodilators and the transporter cannot be excluded at this point. Furthermore, it is possible that the drugs tested in this study have more than one mechanism of action perhaps involving cGMP-dependent and -independent pathways. In conclusion, our data (Figs. 7-10) suggest that activation of K-Cl cotransport may be linked to vasodilation through either direct or indirect mechanisms (signal transduction). However, because DIOA inhibited K-Cl cotransport in cultured rat aortic smooth muscle cells but slightly or no inhibition was observed in the tension studies with porcine coronary arteries, the precise role of K-Cl cotransport in vasodilation remains to be further investigated.

The concentrations of vasodilators to produce an effect in SRBCs were initially tested in the high millimolar range (Figs. 1 and 3). We rationalized that these high drug concentrations were required because of the presence of ~5-7 mM hemoglobin (Hb) in SRBCs. Normally, concentrations as low as 3 µM of oxy-Hb or 10 µM of methylene blue are required to significantly inhibit NO action (11), and, under certain conditions, NO rapidly binds to Hb in a 4:1 ratio (14). In experiments designed to study the relationship between RBC Hb content and the vasodilator effect on K-Cl cotransport, it was found that activation by ISSB and SNP was dependent on the Hct of the flux suspension: the higher the Hct the lower the activation. However, this effect was complex and dependent on the type of drug and its concentration.

HYZ and ISSB induced a five- and eightfold activation of K-Cl cotransport, respectively (Fig. 3B). However, the concentration of HYZ was fivefold lower than that of ISSB. This finding may be explained by a different mechanism of action for HYZ. Interestingly, in the micromolar concentration range (0-200 µM), HYZ did not change the color of the RBCs, whereas above 200 µM the cells became brown (signaling met-Hb formation). This is in agreement with preliminary experiments in progress on the effect of HYZ on RBC metabolism.

In VSMCs, HYZ was tested at the concentration giving maximal activation in SRBCs (Fig. 2). In the tension studies (Figs. 7-9), the HYZ concentrations required to relax the arteries were in agreement with those reported by Wei et al. (33). The experiments reported in Figs. 4 and 5 indicate that HYZ and SNP are able to activate LK SRBC K-Cl cotransport in (HYZ) or near (SNP) the range of concentrations used in the tension studies.

The BK channel is the major K channel involved in vasodilation of porcine coronary arteries (10, 34). This conclusion is supported by two approaches. First, the lack of relaxation of precontracted porcine coronary arteries when depolarized by incubation in high-K medium, and second, in single-channel studies, the BK channel dominates membrane electrical activity (10, 34). The present study shows that, under the conditions where only the K-Cl cotransporter is operating and all other known transport pathways for K/Rb, including the BK channel are excluded (16, 36), stimulation of precontracted isolated porcine coronary rings by HYZ reduces VSM tension (Figs. 7-9). In contrast to the BK channel, the K-Cl cotransport system is electroneutral (21). This property makes the system undetectable by the patch-clamp technique, whereas the tension studies, described above (Fig. 7-9), lend themselves to the investigation of the role K-Cl cotransport might play in vasodilation.

A surprising result was the response to A-23187 in denuded arteries (Fig. 7A). This ionophore, a receptor-independent agonist that increases intracellular Ca, was added to increase NO production in endothelial cells by activation of cNOS (35). Our results of a decrease in contraction in intact arteries confirm the study by Xu et al. (35). However, the results in denuded arteries are unexpected, because SMCs are not known to possess cNOS. One possibility is that some endothelial function remained in the denuded arteries. Alternatively, the presence of inhibitors of monovalent cation pathways or A-23187 itself may have altered the ionic gradients and secondarily the function of Ca transport pathways.

In conclusion, our studies in SRBCs and in VSMCs indicate that vasodilators do activate K-Cl cotransport and that K-Cl cotransport appears to be involved in vasodilation. Although the detailed mechanism of K-Cl cotransport activation by vasodilators awaits further elucidation, our findings prompt two major questions: 1) Is K-Cl cotransport regulation by vasodilators a universal mechanism operating in all cells or is it tissue specific? 2) Does K-Cl cotransport affect VSM contractility without modifying the membrane potential, perhaps through changes in cell volume or intracellular Cl? There is a need for future efforts to be directed to answer these questions.


    ACKNOWLEDGEMENTS

The excellent technical assistance of Kathleen Rainey is highly appreciated. We also thank Dr. Abdala El-Mowafy, from the Department of Physiology and Biophysics, Wright State University, for kind assistance with the coronary artery preparations before the experiments of Cl and sulfamate equilibration and Jing Zhang, PhD student from the BioMedical Sciences program at Wright State University, for assisting with the hematocrit experiments.


    FOOTNOTES

This work was supported by American Heart Association Ohio-West Virginia Affiliate Grant MV-95-01-S and by National Heart, Lung, and Blood Institute Grant HL-54844.

Part of this work was published in abstract form (3, 4).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. C. Adragna, Dept. of Pharmacology and Toxicology, Wright State Univ., School of Medicine, Dayton, OH 45435 (E-mail: norma.adragna{at}wright.edu).

Received 14 May 1999; accepted in final form 17 September 1999.


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DISCUSSION
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