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Am J Physiol Heart Circ Physiol 289: H1277-H1283, 2005. First published March 25, 2005; doi:10.1152/ajpheart.01052.2004
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Reduced molecular expression of K+ channel proteins in vascular smooth muscle from rats made hypertensive with N{omega}-nitro-L-arginine

Ian N. Bratz,1 Gregory M. Dick,1 L. Donald Partridge,3 and Nancy L. Kanagy2

1Department of Physiology, Louisiana State University Health Science Center, New Orleans, Louisiana; and 2Cell Biology and Physiology Department and 3Neurosciences Department, University of New Mexico School of Medicine, Albuquerque, New Mexico

Submitted 13 October 2004 ; accepted in final form 14 March 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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Smooth muscle membrane potential (Em) depends on K+ channels, and arteries from rats made hypertensive with N{omega}-nitro-L-arginine (LHR) are depolarized compared with control. We hypothesized that decreased K+ channel function, due to decreased K+ channel protein expression, underlies Em depolarization. Furthermore, K+ channel blockers should move control Em (–46 ± 1 mV) toward that in LHR (–37 ± 2 mV) and normalize contraction. The Em vs. K+ relationship was less steep in LHR (23 ± 2 vs. 28 ± 1 mV/log K+ concentration), and contractile sensitivity to K+ was increased (EC50 = 37 ± 1 vs. 23 ± 1 mM). Iberiotoxin (10 nM), an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels, depolarized control and LHR Em to –35 ± 1 and –30 ± 2 mV, respectively; however, effects on K+ sensitivity were more profound in LHR (EC50 = 25 ± 2 vs. 15 ± 3 mM). The voltage-dependent K+ (KV) channel blocker 4-aminopyridine (3 mM) depolarized control Em to the level of LHR (–28 ± 1 vs. –28 ± 1 mV); however, effects on K+ sensitivity were greater in LHR (EC50 = 17 ± 4 vs. 4 ± 4 mM). Western blots revealed reduced BKCa and KV1.5 channel expression in LHR arteries. The findings suggest that diminished expression of K+ channels contributes to depolarization and enhanced contractile sensitivity. These conclusions are supported by direct electrophysiological assessment of BKCa and KV channel function in control and LHR smooth muscle cells [see companion paper (Bratz IN, Swafford AN Jr, Kanagy NL, and Dick GM. Am J Physiol Heart Circ Physiol 289: H1284–H1290, 2005)].

nitric oxide; membrane potential; iberiotoxin; 4-aminopyridine

IN HYPERTENSION, exaggerated smooth muscle tone increases peripheral vascular resistance and elevates blood pressure. Arterial tone is, in many ways, a reflection of the intracellular Ca2+ concentration in vascular smooth muscle cells. Sources of activator Ca2+ include release from intracellular stores or influx from the extracellular space, the latter mediated primarily by L-type Ca2+ channels. L-type Ca2+ channels can be activated, and thus smooth muscle contraction initiated, by depolarization of the membrane potential (Em) (e.g., increasing extracellular K+). K+-induced contractions are augmented in arteries from male Sprague-Dawley rats (LHR) made hypertensive for 2 wk with the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine (L-NNA; Ref. 25). Other smooth muscle changes associated with L-NNA-induced hypertension include depolarization (6) and enhanced responsiveness to dihydropyridines (BAY K 8644, an activator, and nifedipine, an inhibitor, of L-type Ca2+ channels; Ref. 31). The affinity of L-type Ca2+ channels for dihydropyridines is regulated by Em (4), which in turn is determined largely by K+ channels (14, 23, 32). These data suggest that enhanced vasoconstriction in L-NNA hypertension may be due to smooth muscle depolarization secondary to reduced K+ channel expression and/or function. In the present study, we tested the hypothesis that decreased K+ channel function, due to decreased K+ channel protein expression, underlies membrane depolarization in LHR vascular smooth muscle. We used isometric tension and intracellular recording techniques to determine whether K+ channel antagonists cause depolarization, increase contractile sensitivity to K+, and normalize differences between control and LHR smooth muscle. Western blot techniques were used to determine the molecular expression level of K+ channel proteins in smooth muscle from LHR and control rats. The results of this study are compared with those of the companion paper (7) to this article, in which we used patch-clamp electrophysiology techniques to determine directly the functional expression of K+ channels in smooth muscle cells from LHR and control rats.


   METHODS
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Animals. All procedures were approved by the Animal Use Committee of the University of New Mexico and conform to National Institutes of Health guidelines. Male Sprague-Dawley rats (250–300 g) were divided into two groups, L-NNA treated and control. The L-NNA-treated group drank water containing L-NNA (0.5 g/l), whereas control rats drank normal tap water. Daily water intake was not different between groups. Systolic blood pressure was measured by tail-cuff plethysmography (IITC, Woodland Hills, CA). After 2 wk of treatment, systolic blood pressure was elevated in the L-NNA-treated rats compared with control animals (195 ± 8 vs. 132 ± 8 mmHg; P < 0.05). On the day of the study, rats were anesthetized with pentobarbital sodium (150 mg/kg) and exsanguinated. The superior mesenteric artery, from the junction with the abdominal aorta to the second branch artery, was rapidly removed.

Em recordings. Arterial segments were cut open longitudinally and pinned to the bottom of a temperature-controlled chamber (37°C; Warner Instruments, Hamden, CT). Tissues were superfused with physiological saline solution (PSS) bubbled with 95% O2-5% CO2. PSS contained (mM) 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 14.9 NaHCO3, 5.5 dextrose, 0.026 CaNa2 EDTA, and 1.6 CaCl2 (pH 7.4). This normal PSS contained 5.9 mM K+. In PSS with elevated K+, NaCl was replaced with equimolar KCl. Arteries were impaled with sharp microelectrodes (tip resistances 60–100 M{Omega}) filled with 3 M KCl. Em was measured with a high-input impedance amplifier (Electro 705; World Precision Instruments, Sarasota, FL), filtered at 100 Hz, and sent to either a chart recorder (Gould Scientific, Cleveland, OH) or Axoscope software (Axon Instruments, Union City, CA). Intracellular recordings were accepted only if 1) Em measurements exhibited sharp negative deflections on cell penetration; 2) a stable recording was held for at least 1 min before experimental manipulations; and 3) an abrupt return to baseline was observed on withdrawal of the electrode. Em was measured continuously both before and during addition of vehicle or drug.

Contractile studies. Superior mesenteric arteries were cleaned of connective tissue and surrounding fat and cut into 3-mm segments. In some experiments, endothelial cells were removed by rubbing the lumen gently with the tips of fine forceps. Tissue rings were suspended in a bath containing PSS maintained at 37°C and bubbled with 95% O2-5% CO2. Measurements were made with FT03 force transducers (Grass-Telefactor, West Warwick, RI) connected to a Gould chart recorder. Rings were stretched to 800 mg of passive tension and equilibrated for 60 min. After equilibration, viability was confirmed by contraction to norepinephrine (10–7 M). The presence or absence of functional endothelium was assessed by relaxation to acetylcholine (10–6 M) in rings contracted with norepinephrine. Segments relaxing at least 80% were considered endothelium intact, whereas those relaxing <5% were considered endothelium denuded.

Western blot analysis. The expression level of large-conductance Ca2+-activated K+ (BKCa), voltage-dependent (KV)1.2, and KV1.5 channel proteins was evaluated with standard immunoblotting methods (5). Superior mesenteric arteries were collected, cleaned of fat and connective tissue, and frozen in liquid nitrogen. Tissue was homogenized by sonication in ice-cold buffer containing 255 mM sucrose, 50 mM Tris·HCl, 10 mM EDTA, 50 µg/ml phenylmethylsulfonyl fluoride, 1.6 mg/ml benzamidine, and 30 µl/ml Complete protease inhibitor (Roche Mannheim). The resulting homogenate was centrifuged at 13,000 g for 10 min at 4°C. Protein concentration of the samples was assayed by the Lowry method, and 15 µg of protein from each sample was separated on 4–20% gradient polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes and blocked 1 h with Tris-buffered saline containing 0.1% Tween 20, 5% milk, and 3% bovine serum albumin. Blots were incubated overnight at 4°C with 1:1,000 anti-BKCa ({alpha}-subunit), anti-KV1.2, and anti-KV1.5, followed by incubation with a horseradish peroxidase-labeled secondary antibody at room temperature for 2 h. Blots were stained with Coomassie blue to ensure equivalent protein loading. K+ channel protein expression levels were estimated by densitometric analysis with SigmaGel software (SPSS, Chicago, IL).

Chemicals. Reagents for Western blot analysis were purchased from Bio-Rad (Hercules, CA), and ECL reagent was obtained from Amersham (Little Chalfont, UK). The protein assay kit was purchased from Pierce (Rockford, IL), and K+ channel antibodies were obtained from Alomone Labs (Jerusalem, Israel). All other reagents, including K+ antagonists, were purchased from Sigma (St. Louis, MO).

Data analysis and statistics. Data are reported as means ± SE. Data were analyzed by two-way ANOVA or t-test as appropriate. Post hoc analysis was performed with Student-Newman-Keuls tests. The n value represents the number of animals used. The level for significance was chosen as P < 0.05.


   RESULTS
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Dependence of Em on K+ differs between control and LHR. Increasing the extracellular K+ concentration ([K+]) decreases the driving for K+ efflux, moving the Nernst equilibrium potential for K+ to more positive values. Intracellular recording techniques were used to determine whether smooth muscle from control and L-NNA-treated rats demonstrates the same dependence of Em on K+ (Fig. 1). Extracellular K+ was changed by equimolar replacement of NaCl with KCl while KH2PO4 concentration (1.2 mM) was fixed to keep pH constant. Em was recorded at [K+] = 1.2, 3.0, 5.9, 10, 30 and 45 mM. Increasing extracellular K+ depolarized smooth muscle linearly above 3.0 mM, and the relationship between Em and log[K+] was fit with least-squares linear regression. The relationship between Em and extracellular K+ was less steep in smooth muscle from LHR. Removing the endothelium changed the relationship between Em and K+ in control rats (i.e., made the slope less steep) but had no effect in LHR. The slope of the relationship for denuded arteries from control rats and LHR was 27.5 ± 1.8 vs. 22.7 ± 0.8 mV/log[K+], respectively (P < 0.05). These data suggest that the contribution of K+ channels to Em is reduced in LHR.



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  		Fig. 1. Relationship between K+ and membrane potential (Em): effects of hypertension and the endothelium. Increasing extracellular K+ depolarized Em as predicted by the Nernst equation. *Difference between control rats and rats made hypertensive with N{omega}-nitro-L-arginine (LHR); #significant effect of endothelium removal in control rats. Reduced slope in LHR suggests that K+ channels make less of a contribution to Em. Denuding the endothelium only affected Em in arteries from control rats. [KCl], KCl concentration.

 
Depolarization and enhanced sensitivity to K+ in LHR. Previous studies in our laboratory (25) demonstrated enhanced contraction to K+ in denuded aortas from LHR. In the present study, we determined K+-induced contractions and Em in endothelium-intact and endothelium-denuded superior mesenteric arteries. Isometric tension recordings were made with endothelium-intact artery segments from LHR and control rats (Fig. 2). Increasing extracellular K+ elicited contractions in arteries from both groups; however, contractile sensitivity to K+ was enhanced in LHR compared with control rats (EC50 = 23.3 ± 0.7 vs. 36.9 ± 0.3 mM; P < 0.05). Intracellular recordings were made in endothelium-intact artery segments from LHR and control rats (Fig. 2). Em was depolarized in endothelium-intact arteries from LHR compared with control rats (–46.2 ± 0.9 vs. –36.9 ± 0.8 mV; P < 0.05). Presence of the endothelium had different effects on contractility and Em of arteries from LHR and control rats (Fig. 3). Endothelium removal depolarized Em (–40.8 ± 2.0 mV; P < 0.05 vs. control intact) and caused a leftward shift in the contractile response to K+ in arteries from control rats (25.6 ± 0.4 mM; P < 0.05 vs. control intact). Removing the endothelium had no effect on Em (–36.0 ± 2.6 mV) or K+ sensitivity (21.4 ± 0.5 mM) in arteries from LHR. Differences in Em and K+ sensitivity persisted in endothelium-denuded artery segments from LHR and control rats (P < 0.05). These data indicate that the endothelium plays little or no role in regulating Em or contractile sensitivity to K+ in LHR, as arteries from LHR remain depolarized and demonstrate enhanced contractile sensitivity compared with those from control rats. Furthermore, the data suggest that changes in smooth muscle K+ channels may underlie differences in Em between LHR and control rats.



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  		Fig. 2. Membrane depolarization and augmented K+-induced contraction in endothelium-intact artery segments from LHR: cumulative concentration-response curves for K+-induced contraction in superior mesenteric arteries from LHR (n = 29) and control rats (n = 29). Artery rings from LHR exhibit enhanced contractile sensitivity. Inset, smooth muscle Em from endothelium- intact arteries (extracellular [K+] = 5.9 mM). Arterial segments from LHR (n = 14) were depolarized compared with control (n = 14). *Differences between groups.

 


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  		Fig. 3. Membrane depolarization and augmented K+-induced contraction in endothelium-denuded artery segments from LHR. Cumulative concentration-response curves for K+-induced contraction in endothelium-denuded artery segments from LHR (n = 31) and control rats (n = 41). Endothelium removal increased contractile sensitivity in artery segments from control rats but had no effect on LHR (compare to Fig. 2). Endothelium removal depolarized artery segments from control rats but had no effect on Em in LHR (compare to Fig. 2). Inset, Em in denuded arteries. *Significant differences from control.

 
Effects of K+ channel antagonists on Em and contraction. Because Em in LHR smooth muscle is depolarized relative to control, the functional and/or molecular expression of K+ channels may be reduced. Isometric tension and intracellular recordings were made to determine whether blocking specific K+ channels would normalize differences between endothelium-denuded arteries from LHR and control rats. Measurements in the presence of K+ channel antagonists were used to evaluate the role of specific types of K+ channels in establishing the resting Em and determining contractile sensitivity to K+. Inhibition of BKCa channels with iberiotoxin (10 nM) depolarized Em in both groups (Fig. 4). After addition of iberiotoxin, control Em (–34.9 ± 1.3 mV) did not differ from the baseline Em of LHR in the absence of iberiotoxin (–36.0 ± 2.6 mV); however, Em in the presence of iberiotoxin was still different between groups (–34.9 ± 1.3 vs. –30.3 ± 1.7 mV; P < 0.05). Iberiotoxin increased the sensitivity to K+-induced contraction in both groups; however, a difference between LHR and control rats persisted in the presence of iberiotoxin (24.7 ± 1.7 vs. 15.3 ± 2.9 mM). Contractile sensitivity to K+ of arteries from control rats in the presence of iberiotoxin was similar to that in the untreated arteries from LHR (21.4 ± 0.5 mM), suggesting that decreased BKCa activity may enhance sensitivity to K+-induced contraction in LHR.



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  		Fig. 4. Effects of iberiotoxin on Em and contraction in endothelium-denuded arteries. Contractile response to K+ in the presence of the large-conductance Ca2+-activated K+ (BKCa) channel antagonist iberiotoxin (10 nM). Iberiotoxin depolarized artery segments from control (n = 6) and LHR (n = 6), but a difference between groups persisted. Iberiotoxin enhanced the sensitivity to K+-induced contraction in LHR. Inset, Em in the presence of iberiotoxin. *Differences between groups.

 
An inhibitor of KV channels, 4-aminopyridine, depolarized Em in both groups (Fig. 5). In the presence of 3 mM 4-aminopyridine, control and LHR Em were not different at –28.3 ± 1.1 vs. –27.5 ± 0.6 mV. Inhibiting KV channels with 4-aminopyridine significantly increased contractile sensitivity to K+ in both groups, but a difference in sensitivity to K+ persisted (16.5 ± 3.6 vs. 3.9 ± 4.0 mM; P < 0.05). These data suggest that KV channels have a large effect on Em and on K+-induced contraction; therefore, reduced expression of KV channels may, in part, explain the differences in Em and contractile responses between LHR and control rats. Neither apamin (100 nM), an inhibitor of small-conductance Ca2+-activated K+ (SKCa) channels, nor glibenclamide (10 µM), an inhibitor of ATP-dependent K+ (KATP) channels, caused depolarization or enhanced the sensitivity of contraction to K+ in either group (data not shown). Thus BKCa and KV channels appear to be more important regulators of Em and contraction than SKCa or KATP channels.



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  		Fig. 5. Effects of 4-aminopyridine on Em and contraction in endothelium-denuded arteries. Contractile response to K+ in the presence of the voltage-dependent K+ (KV) channel antagonist 4-aminopyridine (3 mM). 4-Aminopyridine depolarized artery segments from control (n = 8) and LHR (n = 8) and abrogated the difference in Em between groups. 4-Aminopyridine enhanced the sensitivity to K+-induced contraction in both groups. Inset, Em in the presence of 4-aminopyridine. *Differences between groups.

 
Western blot analysis of K+ channel protein expression. Figure 6 is a summary of the effects of K+ channel antagonists on Em and contractile sensitivity to K+. Inhibition of BKCa channels with iberiotoxin renders smooth muscle contraction from control rats as sensitive to K+ as that in untreated LHR smooth muscle. Furthermore, iberiotoxin depolarizes smooth muscle from control rats to the level seen in untreated smooth muscle from LHR. Similarly, inhibition of KV channels with 4-aminopyridine depolarizes smooth muscle and renders arteries from control rats as sensitive to K+-induced contraction as untreated tissues from LHR. Thus reduced molecular expression of BKCa and/or KV channels may be responsible for depolarization and increased contractile sensitivity to K+ in LHR smooth muscle.



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  		Fig. 6. Summary of EC50 values of K+-induced contraction (left) and Em measurements (right). *Differences between control and LHR; #differences (from denuded) within the control group; {dagger}differences (from denuded) within the LHR group.

 
To determine the relative expression level of K+ channel proteins, Western blot analysis was performed. Smooth muscle from LHR expressed less BKCa {alpha}-subunit protein compared with control (Fig. 7). BKCa channels are sensitive to block by iberiotoxin (18), and inhibiting them in arteries from control rats reproduces the phenotype of arteries from LHR. KV1.2 and KV1.5 are the most likely contributors to the native delayed rectifier K+ current in rat mesenteric artery smooth muscle (12). LHR expressed less Kv1.5 protein in smooth muscle compared with control rats (Fig. 8). KV1.5 channels are sensitive to block by 4-aminopyridine (26, 38), and inhibiting them in arteries from control rats reproduces the phenotype of arteries from LHR. We found no difference in KV1.2 protein expression between groups (data not shown). The findings suggest that diminished expression of BKCa and KV1.5 channels in LHR smooth muscle contributes to depolarization and enhanced contractile sensitivity to K+. In the companion paper (7), we used patch-clamp electrophysiology techniques to determine directly whether functional expression of K+ channels is reduced in smooth muscle from LHR.



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  		Fig. 7. Reduced BKCa channel protein expression in smooth muscle from LHR. Top: representative Western blot for BKCa channel {alpha}-subunit protein from 4 control and 4 LHR arteries. Bottom: group data of the densitometry analysis. *Significant reduction in BKCa channel protein in LHR.

 


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  		Fig. 8. Reduced KV1.5 channel protein expression in smooth muscle from LHR. Top: representative Western blot for KV1.5 channel protein from 4 control and 4 LHR arteries. Bottom: group data of the densitometry analysis. *Significant reduction in KV1.5 channel protein in LHR.

 

   DISCUSSION
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 ABSTRACT
 METHODS
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GRANTS
 REFERENCES
 
This study addresses potential molecular mechanisms underlying depolarization of the resting Em and enhanced contractile sensitivity to K+ in superior mesenteric arteries from LHR. Using intracellular recording techniques, we demonstrated depolarization and reduced dependence of Em on K+ in smooth muscle from LHR, suggesting that K+ channels make less of a contribution to Em compared with control. The presence of functional endothelium determines, in part, the dependence of Em on K+ in control but not LHR arteries. By measuring Em and isometric tension in endothelium-denuded arteries, we showed that iberiotoxin-sensitive BKCa and 4-aminopyridine-sensitive KV channels contribute importantly to the resting Em and sensitivity of K+-induced contraction of smooth muscle from both LHR and control rats. Furthermore, inhibition of KV or BKCa channels causes smooth muscle from control rats to respond electrically and functionally more like that in LHR. Importantly, however, inhibition of BKCa and KV channels cannot completely normalize smooth muscle contraction between control and LHR, as arteries from LHR also demonstrate increased Ca2+ sensitivity (9). Finally, Western blot analysis indicated that diminished BKCa and KV channel protein expression is a likely contributor to the depolarized resting Em and enhanced K+-induced contraction of smooth muscle from LHR. These conclusions about molecular expression of K+ channel proteins are supported by the companion paper (7), in which we used patch-clamp electrophysiology techniques to demonstrate that functional expression of BKCa and KV channels is diminished in smooth muscle cells isolated from LHR.

Alterations in ion channel activity have previously been associated with increased vascular reactivity in hypertension (14, 23). Depolarization of the resting Em (21, 22), augmented Ca2+ current (19, 35, 37), and a resulting increase in intracellular free Ca2+ (16, 24, 34) are all generally agreed to play a role in the enhanced arterial contraction of hypertension. The effects of hypertension on smooth muscle K+ channels—and the role of smooth muscle K+ channels in opposing hypertension—are less clear. There are seemingly incongruous reports regarding the role of K+ channels in hypertension, but two general hypotheses have been advanced in explanation. First, if the expression and/or functional activity of K+ channels were upregulated, then it would be logical to propose that K+ channels are increased to oppose depolarization and vasoconstriction. Examples of K+ channels serving to oppose hypertension have been provided in polymorphisms and a gain-of-function mutation in BKCa channels (17, 20), as well as in the therapeutic value of K+ channel openers (40). Conversely, if the expression and/or functional activity of K+ channels were downregulated, then the loss of K+ channels could be considered a contributing factor in producing depolarization and augmenting vasoconstriction. Several examples of this second scenario have been provided, particularly with regard to BKCa channels (1, 3, 8, 36).

Our data indicate that expression of BKCa and KV1.5 channel proteins is reduced in smooth muscle from LHR rats. Furthermore, we demonstrate in the following study that the functional expression of K+ channels is reduced in smooth muscle cells from LHR. Together, these data lead us to conclude that this contributes to the depolarized Em and augmented contraction of smooth muscle in LHR. These data generally agree with the studies of Amberg and coworkers (1, 3), who demonstrated that genetic and angiotensin II-induced hypertension are associated with a reduction in BKCa current secondary to diminished expression of the regulatory {beta}1-subunit. These investigators observed no difference in the expression level of mRNA for the BKCa {alpha}-subunit but a trend for reduced BKCa {alpha}-subunit immunofluorescence in hypertension. We, using a different model of hypertension, demonstrated reduced BKCa {alpha}-subunit protein expression by Western blot. In contrast, using both molecular and functional approaches, Rusch and coworkers (28, 29) demonstrated that BKCa is upregulated in hypertension. Differences in tissues and models of hypertension may serve to explain contrasting findings regarding BKCa expression. Whether BKCa channels are upregulated or downregulated in response to hypertension may depend on the molecular nature (i.e., splice variant) of the channel in a particular tissue. It is particularly important whether the vessels in question are conduit or resistance arteries. For example, in the spontaneously hypertensive rat (SHR) cerebral microcirculation, hypertension increases expression of the pore-forming BKCa subunit fourfold, with no change in Ca2+/voltage sensitivity (28). In contrast, in the SHR aorta, there is increased Ca2+/voltage sensitivity (15). BKCa channels from the cerebral microcirculation may be a unique splice variant, as the voltage of half-activation with 1 µM Ca2+ averaged +56–58 mV—unusually depolarized. In contrast, in our studies of the rat mesenteric artery (companion paper; Ref. 7), expression of BKCa channel protein was increased but there was no change in Ca2+/voltage sensitivity. When functional measurements of BKCa channels are made, current magnitude will depend on whether dialyzed whole cell or perforated patch methods are used, as intracellular free Ca2+ is higher in smooth muscle from hypertensive animals (16, 24, 34). Thus demonstrations of increased BKCa current (13) may be a reflection of altered Ca2+ homeostasis. Fewer data are available regarding molecular expression levels of KV channels in hypertension (12); however, functional expression has been shown to be reduced (11, 13, 30). Our Western blot data indicate reduced expression of KV1.5 in smooth muscle from LHR.

How an increase in blood pressure translates to decreased K+ channel expression is not understood. Furthermore, it has been suggested that decreased K+ expression leads to hypertension (1, 3, 8, 36); therefore, cause-and-effect relationships among K+ expression, Em, smooth muscle contraction, and hypertension remain to be elucidated. Hemodynamic shear may be a mechanism altering K+ channel expression, as shear stress has been shown to activate a cascade of signaling events leading to an altered phenotype in vascular smooth muscle (39). Similarly, stretch of the smooth muscle membrane may be a mechanism for altering smooth muscle phenotype (33). The absence of NO, and thus altered cGMP signaling, is another possible mechanism for altering K+ channel expression (27). Elevated intracellular free Ca2+, as seen in hypertension, could also regulate transcription factors in smooth muscle including cAMP response element binding protein (10) and nuclear factor of activated T cells (2). Specific signaling mechanisms leading to reduced expression of BKCa and KV channel proteins in LHR smooth muscle remain to be determined.

In summary, we demonstrate that Em is depolarized and K+-induced contraction is augmented in mesenteric arteries from L-NNA-treated rats. Depolarization and enhanced contraction are associated with reduced expression of BKCa and KV channel proteins in smooth muscle from hypertensive rats. These molecular studies are supported by results of the companion paper (7), in which we assessed functional expression of smooth muscle K+ channels directly with patch-clamp techniques. Together, the results of the two studies suggest that endothelial dysfunction (in the form of reduced NO production) reduces the molecular and functional expression of K+ channels in smooth muscle, a factor likely to contribute to the depolarization and augmented vascular reactivity in hypertension.


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N. L. Kanagy was supported by National Institutes of Health (NIH) Grant HL-03852, a Scientist Development Award from the American Heart Association, and a Research Allocations Committee grant from the University of New Mexico. G. M. Dick was supported by a Beginning Grant-in-Aid from the American Heart Association and the Louisiana State University Center of Biomedical Research Excellence (COBRE; NIH P20-RR-018766-02).


   ACKNOWLEDGMENTS
 
The authors give special thanks to Pam Allgood for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. Dick, Dept. of Physiology, LSU Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112 (E-mail: gdick{at}lsuhsc.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.


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