AJP - Heart Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 289: H1284-H1290, 2005. First published May 6, 2005; doi:10.1152/ajpheart.01053.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/3/H1284    most recent
01053.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bratz, I. N.
Right arrow Articles by Dick, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bratz, I. N.
Right arrow Articles by Dick, G. M.

Reduced functional expression of K+ channels in vascular smooth muscle cells from rats made hypertensive with N{omega}-nitro-L-arginine

Ian N. Bratz,1 Albert N. Swafford, Jr.,1 Nancy L. Kanagy,2 and Gregory M. Dick1

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

Submitted 13 October 2004 ; accepted in final form 4 May 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Smooth muscle membrane potential is determined, in part, by K+ channels. In the companion paper to this article (Bratz IN, Dick GM, Partridge LD, and Kanagy NL. Am J Physiol Heart Circ Physiol 289: H1277–H1283, 2005), we demonstrated that superior mesenteric arteries from rats made hypertensive with N{omega}-nitro-L-arginine (L-NNA) are depolarized and express less K+ channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that L-NNA-induced hypertension reduces the functional expression of K+ channels in smooth muscle. In whole cell experiments using a Ca2+-free pipette solution, current at 0 mV, largely due to voltage-dependent K+ (KV) channels, was reduced ~60% by hypertension (2.7 ± 0.4 vs. 1.1 ± 0.2 pA/pF). Current at +100 mV with 300 nM free Ca2+, largely due to large-conductance Ca2+-activated K+ (BKCa) channels, was reduced ~40% by hypertension (181 ± 24 vs. 101 ± 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many KV channel types, was reduced ~50% by hypertension (1.0 ± 0.4 vs. 0.5 ± 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BKCa channels, was reduced ~40% by hypertension (86 ± 14 vs. 53 ± 19 pA/pF). Differences in BKCa current magnitude are not attributable to changes in single-channel conductance or Ca2+/voltage sensitivity. The data support the hypothesis that L-NNA-induced hypertension reduces K+ current in vascular smooth muscle. Reduced molecular and functional expression of K+ channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from L-NNA-treated rats.

nitric oxide; membrane potential; Ca2+-activated K+ channel; delayed rectifier K+ channel; hypertension

ELEVATED ARTERIAL TONE increases peripheral vascular resistance and blood pressure. Vascular smooth muscle tone is controlled, in large part, by the intracellular free Ca2+ concentration, which in turn is influenced by voltage. The open probability of L-type Ca2+ channels, a major pathway of Ca2+ entry in vascular smooth muscle, is determined by membrane potential. Thus smooth muscle membrane potential, intracellular Ca2+, and contraction are intimately intertwined in a phenomenon referred to as electromechanical coupling (20, 30). K+ channels play an important role in electromechanical coupling, functioning to set a negative membrane potential and limit the activation of L-type Ca2+ channels. The loss of proper K+ channel function results in altered vascular reactivity and hypertension (5, 33). Conversely, hypertension alters the expression of ion channels involved in the electromechanical coupling of smooth muscle (11, 20). Recently, interesting light has been shed on concepts of cause and effect between hypertension and alterations in smooth muscle K+ channels (1, 2), and it remains to be determined in what scenarios ion channel changes precede hypertension and vice versa.

We previously demonstrated a variety of vascular changes in male rats made hypertensive with the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine (L-NNA). Smooth muscle changes associated with L-NNA-induced hypertension include increased contractility (22), enhanced Ca2+ sensitivity (6), and augmented responses to dihydropyridines (29). In the companion paper to this article (3), we demonstrated that L-NNA-induced hypertension depolarizes the smooth muscle membrane potential and diminishes expression of K+ channel proteins. In the present study, we tested the hypothesis that L-NNA-induced hypertension reduces the functional expression of K+ channels in vascular myocytes from hypertensive rats. Smooth muscle cells were isolated from the superior mesenteric artery of normotensive and hypertensive rats and studied with whole cell and single-channel patch-clamp techniques. The data support the notion that L-NNA-induced hypertension reduces whole cell K+ current in vascular smooth muscle. Reduced functional expression of K+ current is secondary to diminished molecular expression, and these changes may underlie depolarization and augmented contractility.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypertension induction and isolation of smooth muscle cells. All procedures were approved by the Animal Use Committee of Louisiana State University Health Sciences Center and conform to National Institutes of Health guidelines. Male Sprague-Dawley rats (200–250 g; Charles River, Wilmington, MA) were randomly assigned to control and L-NNA-treated groups. Rats in the L-NNA-treated group were provided tap water containing 0.5 mg/ml L-NNA for 2 wk. Rats were anesthetized with pentobarbital sodium (65 mg/kg ip), intubated, and ventilated with room air, and blood pressure was measured through a carotid catheter. After exsanguination, the superior mesenteric artery, between the abdominal aorta and the second mesenteric branch, was removed. Arteries were cleaned of fat and connective tissue and denuded of endothelial cells by rotating on a pair of fine forceps. Arterial segments (~1 mm) were cut and treated in three solutions for tissue digestion. The first solution was physiological saline solution (PSS; see below) with 0.5 mg/ml fatty acid-free bovine serum albumin; treatment was at room temperature for 10 min. Tissues were then incubated for 20 min at 37°C in a second PSS solution containing dithiothreitol (0.5 mg/ml) and papain (1.0 mg/ml). Tissues were next incubated in the third solution for 10–15 min at 37°C; this solution contained collagenase XI (1.5 mg/ml), trypsin inhibitor (1.0 mg/ml), and elastase (1.0 mg/ml). Tissue was transferred to 4 ml of cold PSS on ice for 10 min and then passed repeatedly through the fire-polished tip of a Pasteur pipette to liberate single myocytes. The cell suspension was kept at room temperature, and patch-clamp recordings were performed within 8 h. PSS contained (mM) 125 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 20 mannitol, 10 HEPES, and 5 Tris (pH 7.4). All chemicals and enzymes were purchased from Sigma (St. Louis, MO).

Electrophysiology. Drops of cell suspension were added to a recording chamber mounted on an inverted microscope. After cells adhered to the glass bottom of the chamber, the chamber was perfused with nominally Ca2+-free PSS that contained 2 mM MnCl2 in place of CaCl2. Single myocytes were approached with heat-polished pipettes having tip resistances between 2 and 4 M{Omega} when filled with solution containing (mM) 135 KCl, 10 HEPES, 5 Tris, 3 Mg-ATP, 1 Na-GTP, and 1 EGTA (pH 7.1). To create a pipette solution with 300 nM free Ca2+, CaCl2 was added to this 1 mM EGTA pipette solution (calculations performed with MAXCHELATOR software; http://www.stanford.edu/~cpatton/maxc.html). Whole cell K+ currents were measured at room temperature with the conventional dialyzed configuration of the patch-clamp technique. Series resistance (~70%) and membrane capacitance were compensated (WPC-100 amplifier; E.S.F. Electronic; Goettingen, Germany). Intracellular and extracellular Cl– were equivalent at 135–136 mM; thus no adjustment for junction potentials was necessary. The bath solution was hypertonic (~315 mosM) relative to the pipette (~290 mosM) to reduce volume-sensitive Cl– current. Currents were digitized at 5 kHz (Digidata 1322A; Axon Instruments; Union City, CA) and low-pass filtered at 1 kHz. For single-channel experiments, the pipette and bath solutions contained (mM) 140 KCl, 1 EGTA or 1 N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), 10 HEPES, and 5 Tris (pH 7.1). Solutions that were considered Ca2+ free or contained 300 nM free Ca2+ were made with EGTA, whereas HEDTA was used to make a solution with 10 µM free Ca2+. Data were analyzed with WinASCD software (http://ftp.cc.kuleuven.ac.be/pub/droogmans/winascd.zip), and large-conductance Ca2+-activated K+ (BKCa) channel conductance was determined from all-points amplitude histograms.

Statistical analysis. Data are reported as means ± SE from n rats. Current-voltage relationships were determined by measuring steady-state current (i.e., at the end of the 400-ms test pulse) and compared between the two groups. Data were analyzed by one- or two-way ANOVA or t-test as indicated. Post hoc analyses were performed with Student-Newman-Keuls (2-way ANOVA) or Holm-Sidak (1-way ANOVA) tests. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Male Sprague-Dawley rats were divided into two groups given either normal tap water or tap water containing 0.5 mg/ml L-NNA. Treatment was for 2 wk, and blood pressure was measured under pentobarbital anesthesia on the day of death. Animals were intubated and ventilated with room air while blood pressure was measured through a catheter placed in the carotid artery. Systolic and diastolic blood pressures in control rats were 110 ± 8 and 84 ± 7 mmHg, respectively (n = 7). Systolic and diastolic pressures in rats drinking water with L-NNA were 176 ± 10 and 135 ± 6 mmHg, respectively (n = 8; P < 0.05 for systolic and diastolic pressure in L-NNA treated vs. control by unpaired t-test). Mean arterial blood pressure was significantly higher in rats treated with L-NNA (Fig. 1C). Smooth muscle cells were isolated from the superior mesenteric artery of these normotensive and hypertensive rats and studied with the conventional whole cell patch-clamp technique. The bath solution was nominally Ca2+ free, and the cells were dialyzed with a Ca2+-free pipette solution (1 mM EGTA). Cells were held at –80 mV and stepped from –100 mV to +100 mV in 20-mV increments (Fig. 2A). Whole cell currents under these conditions were generally small (<1 nA at +100 mV) and composed of two apparent conductances, BKCa and voltage-dependent K+ (KV), as reported previously in various smooth muscle cell types (7, 19, 23). Currents recorded from smooth muscle cells of normotensive and hypertensive rats were normalized to membrane capacitance (pA/pF) to negate any possible differences in cell size (although we did not detect any change in cell capacitance; 16.2 ± 1.3 vs. 16.4 ± 1.0 pF for myocytes from normotensive and hypertensive rats, respectively). A comparison of the current-voltage relationships demonstrated that the curves had similar shapes; however, differences in magnitude were observed (Fig. 2B). Under these Ca2+-free conditions, whole cell currents were reduced in smooth muscle cells from hypertensive rats, suggesting a reduction in KV current.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. Blood pressure of control and N{omega}-nitro-L-arginine (L-NNA)-treated rats. A: 10-s recording of blood pressure from a representative control rat. B: representative blood pressure tracing from a rat treated for 2 wk with L-NNA. C: group data for mean arterial pressure in 7 control and 8 L-NNA-treated rats; *P < 0.05 by unpaired t-test.

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 2. Whole cell K+ current is reduced in vascular smooth muscle cells from hypertensive rats regardless of the intracellular free Ca2+ concentration. Myocytes were bathed in Ca2+-free physiological saline solution and dialyzed with a either a Ca2+-free pipette solution (1 mM EGTA) or a solution buffered to 300 nM Ca2+. The Ca2+-free pipette solution was designed to maximize voltage-dependent K+ (KV) current, whereas the 300 nM Ca2+ pipette solution was designed to enhance large-conductance Ca2+-activated K+ (BKCa) current. Cells were held at –80 mV and stepped from –100 mV to +100 mV in 20-mV increments. A: representative current traces with a Ca2+-free pipette. B: group data for the current-voltage (I-V) relationship (inset is expanded to appreciate differences in magnitude). Outward current was larger in smooth muscle cells from normotensive (n = 7) compared with hypertensive (n = 6) rats. C: representative current traces with a pipette solution containing 300 nM Ca2+. D: group I-V relationship. Outward current was larger in smooth muscle cells from normotensive (n = 6) compared with hypertensive (n = 6) rats. Two-way ANOVA indicated that I-V curves are different; *P < 0.05 at specific voltages (Student-Newman-Keuls post hoc analysis).

 
Smooth muscle cells from normotensive and hypertensive rats were also studied after dialysis with a pipette solution buffered to 300 nM Ca2+. Whole cell currents under these conditions were larger than those observed with the Ca2+-free pipette (>1 nA at +100 mV), and BKCa channel current became more prominent (Fig. 2C). Similar to results with the Ca2+-free pipette solution, the current-voltage relationship was depressed in cells from hypertensive rats compared with normotensive rats (Fig. 2D). The most notable differences in current density were at positive membrane potentials, where BKCa channels are active (greater than +40 mV). Importantly, however, current density at more physiological membrane potentials (–40 and –20 mV) was reduced by as much as 79% in smooth muscle cells from hypertensive rats compared with control, regardless of the Ca2+ concentration (Fig. 3). These data suggest a decrease in KV or delayed rectifier current, as well as a reduction in BKCa current. A reduction in KV current was supported by comparing the difference between current densities in cells from normotensive and hypertensive rats. Subtraction revealed a significant reduction in whole cell current at negative voltages whether cells were dialyzed with a Ca2+-free pipette solution (Fig. 3A) or with a solution containing 300 nM Ca2+ (Fig. 3B). These data with different intracellular Ca2+ concentrations suggest that hypertension reduces both voltage- and Ca2+-dependent whole cell K+ current.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3. Difference currents: normotensive minus hypertensive. A: difference current obtained by subtracting the hypertensive current from the mean normotensive current when cells were dialyzed with a Ca2+-free pipette solution (from Fig. 2B). B: difference current from cells dialyzed with 300 nM free Ca2+ (from Fig. 2D). Insets in A and B show the current density from the normotensive and hypertensive groups on an expanded scale. Current near the physiological range of membrane potentials was reduced in smooth muscle cells from hypertensive rats. *Voltages at which the 2 groups differed.

 
To further assess whether reductions in whole cell current were more specifically attributable to KV or BKCa channels, responses to tetraethylammonium (TEA) and 4-aminopyridine (4-AP) were determined (Fig. 4). In the companion paper (3), we demonstrated reduced expression of two K+ channel proteins in smooth muscle from hypertensive rats: KV1.5 and the BKCa {alpha}-subunit. KV1.5 is among the delayed rectifiers inhibited by 4-AP (38). In contrast, BKCa channels are inhibited by TEA but not 4-AP. Thus our rationale was to determine whether differences in TEA- or 4-AP-sensitive current existed between smooth muscle cells from normotensive and hypertensive rats. TEA (1 mM) dramatically reduced whole cell K+ current, particularly "noisy" current at potentials positive to +40 mV (Fig. 4A). This suggests that 1 mM TEA primarily inhibits BKCa current and not KV current. 4-AP (3 mM) also substantially reduced whole cell current, leaving the noisy current at positive potentials (Fig. 4A). This suggests that 4-AP inhibits a large component of KV current (including KV1.5) but does not inhibit BKCa channels. The combination of TEA and 4-AP reduced, but did not eliminate, whole cell current. The ionic nature of this remaining current has not been determined, but preliminary pharmacology experiments indicate that it can be inhibited by increasing the TEA concentration one log order (10 mM; data not shown). Thus the residual current in the presence of 1 mM TEA and 3 mM 4-AP is likely mediated by K+ channels.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4. Block of K+ currents by tetraethylammonium (TEA) and 4-aminopyridine (4-AP) in cells dialyzed with Ca2+-free pipette solution. A: representative traces demonstrate the effect of 1 mM TEA, 3 mM 4-AP, and their combination to inhibit whole cell current. Cells were held at –80 mV and stepped from –100 to +100 mV in 20-mV increments before and after the addition of K+ channel antagonists. B–D: whole cell current that remains in the presence of each inhibitor and their combination (insets show current that was blocked by each inhibitor and the combination). Data are from 7 normotensive and 6 hypertensive rats, and the same cells were exposed sequentially to TEA, 4-AP, and TEA + 4-AP. Two-way ANOVA indicated significant differences (*P < 0.05 at specific voltages; Student-Newman-Keuls post hoc analysis).

 
The amount of current that persisted in the presence of 1 mM TEA was reduced in cells from hypertensive rats (Fig. 4B). Additionally, the TEA-sensitive current was smaller in myocytes from hypertensive rats (Fig. 4B, inset). Similarly, current persisting in the presence of 3 mM 4-AP was reduced in myocytes from hypertensive rats (Fig. 4C); the 4-AP-sensitive current was also less (Fig. 4C, inset). A reduction in KV current was supported by comparing the difference between current densities in cells from normotensive and hypertensive rats in the presence of 1 mM TEA. Subtraction revealed a significant reduction in whole cell current negative to +40 mV. A reduction in BKCa current was supported by comparing the difference in current densities in cells from normotensive and hypertensive rats in the presence of 3 mM 4-AP.

Because 1 mM TEA might inhibit K+ channels other than BKCa, we determined whether experiments with a more selective BKCa blocker, such as iberiotoxin, would be necessary. We studied cells dialyzed with a pipette solution containing 300 nM Ca2+ and measured current under control conditions and in the presence of 1 mM TEA, 10 nM iberiotoxin, and 100 nM iberiotoxin (Fig. 5). There were no statistical differences in currents persisting in the presence of TEA or the two concentrations of iberiotoxin. Additionally, there was no difference in the amount of current inhibited by TEA or the two concentrations of iberiotoxin (Fig. 5, inset). These data indicate that 1 mM TEA is a relatively selective inhibitor of BKCa channels. Furthermore, the data with TEA suggest that the differences in TEA-sensitive current between normotensive and hypertensive rats are due to BKCa channels.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Iberiotoxin and 1 mM TEA inhibit the same component of whole cell K+ current. Smooth muscle cells from normotensive rats were dialyzed with a pipette solution buffered to 300 nM free Ca2+; the holding potential was –80 mV, and cells were stepped from –100 to +100 mV in 20-mV increments. Current was measured before and after the addition of 1 mM TEA, 10 nM iberiotoxin, and 100 nM iberiotoxin (n = 6). Inset, current inhibited by 1 mM TEA and the 2 concentrations of iberiotoxin.

 
We determined whether the reduction in whole cell BKCa current could be attributed to changes in single-channel conductance or Ca2+/voltage sensitivity. We found that single-channel conductances were not different in patches from normotensive and hypertensive rats (215 ± 6 vs. 215 ± 2 pS; Fig. 6B). Because the activity of BKCa channels is regulated by Ca2+ concentration, we determined whether changes in whole cell BKCa current were due to changes in the sensitivity of the channels to Ca2+. With the use of excised inside-out membrane patches, activation curves were constructed in solutions that were Ca2+ free or contained 10 µM free Ca2+. The Ca2+/voltage sensitivity of BKCa channels was not different between normotensive and hypertensive rats (Fig. 6D).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 6. Reduced whole cell BKCa current is not due to a change in single-channel conductance or Ca2+/voltage sensitivity. A: representative current trace of BKCa channel activity at +80 mV. Single-channel currents were recorded in symmetrical 140 mM K+. B: group data for the single-channel I-V relationship for BKCa channels from 5 normotensive and 6 hypertensive rats. C: representative traces recorded from an inside-out patch of membrane from a normotensive rat. The patch was held at 0 mV in symmetrical 140 mM K+ solutions containing either 1 mM EGTA and no added Ca2+ (left) or 1 mM N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid buffered to 10 µM free Ca2+ (right). Membrane potential was stepped from –200 to +200 mV in 20-mV increments. D: currents were converted to conductance (G), normalized to the maximum (Gmax), and plotted vs. voltage. No differences in Ca2+/voltage sensitivity were detected between normotensive (n = 5) and hypertensive (n = 6) rats.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study addresses whether functional expression of whole cell K+ current parallels reduced molecular expression of BKCa and KV1.5 channel proteins in the superior mesenteric arteries of rats made hypertensive with L-NNA (3). Furthermore, this study was aimed at determining whether reduced functional expression of K+ channels is a mechanism for depolarization and enhanced contractility of smooth muscle from L-NNA hypertensive rats (3, 4, 22). Rationale for the study stems from the companion paper (3), in which we demonstrated smooth muscle depolarization, augmented vascular contractility, and reduced molecular expression of BKCa and KV1.5 channel proteins in arteries from L-NNA hypertensive rats. Importantly, however, the question of whether functional expression of K+ channels in smooth muscle is affected by L-NNA-induced hypertension remained unanswered. Using whole cell patch-clamp techniques, we tested the hypothesis directly and here demonstrate reduced K+ current in vascular myocytes from rats made hypertensive with L-NNA. These data are comparable to the findings of some studies of smooth muscle K+ current in hypertension, while they conflict with others. Differences and similarities between our studies and others may be due to factors such as model-specific diversity in the cause of hypertension; however, fundamental molecular mechanisms that may facilitate such comparisons remain to be identified. Regardless, at the present level of understanding, reduced whole cell K+ current in smooth muscle from L-NNA hypertensive rats is compatible with our studies demonstrating membrane depolarization, enhanced contractility, and diminished expression of KV1.5 and BKCa channel proteins (3).

A reduction in whole cell K+ current in L-NNA hypertension was evident whether myocytes were dialyzed with pipette solutions that were Ca2+ free or buffered to 300 nM Ca2+. These observations are in accordance with reduced expression of at least two components of whole cell K+ current (7, 19, 23), most likely, delayed rectifier (KV) current and current mediated by BKCa channels. KV current is readily identified by using a Ca2+-free pipette, which limits the activation of BKCa channels. Additionally, at least some KV channels (including KV1.5) are sensitive to 4-AP (38). Thus additional pharmacological evidence comes from our demonstration that 4-AP-sensitive K+ current, measured with a Ca2+-free pipette solution, is reduced in smooth muscle cells from L-NNA hypertensive rats. BKCa channels are most easily identified by using a pipette with an elevated free Ca2+ concentration. Furthermore, BKCa channels are sensitive to block by ≤1 mM TEA, a concentration that has little effect on most types of smooth muscle KV channels (24). We demonstrate, using a pipette solution containing 300 nM Ca2+, that L-NNA-induced hypertension reduces the functional expression of Ca2+- and TEA-sensitive K+ current. These KV and BKCa channels contribute importantly to the membrane potential and to the sensitivity of K+-induced contraction of arteries from both control and L-NNA-treated rats. Reduced expression of KV and BKCa channels would be expected to reproduce some aspects of the vascular phenotype observed in L-NNA-induced hypertensive rats (5, 33). Inhibition of KV (with 4-AP) or BKCa (with iberiotoxin) channels causes smooth muscle from control rats to respond electrically and functionally more like that in hypertensive rats; therefore, the results presented here fully support those of the companion paper (3). Together, the data suggest that reduced molecular and functional expression of K+ channels are mechanisms for depolarization and enhanced contraction of smooth muscle in hypertension.

Earlier studies demonstrated alterations in smooth muscle ion channels in hypertension. Perhaps the largest number of studies of this kind have been performed in the model of spontaneously hypertensive rats (SHR). Particularly relevant to this discussion are documented changes in the expression and activity of L-type Ca2+, KV, and BKCa channels in the smooth muscle of SHR compared with control Wistar-Kyoto (WKY) rats (see, e.g., Refs. 10, 32). Ion channel changes such as these are thought to underlie increased vascular reactivity in hypertension (11, 20). It is generally agreed that depolarization (17, 18), enhanced Ca2+ current (15, 32, 35), and increased intracellular free Ca2+ (13, 21, 31) contribute to augmented vasoconstriction in hypertension. Importantly, however, roles for smooth muscle K+ channels in producing or opposing hypertension are less clear. The activity/expression of smooth muscle K+ channels in hypertension may be increased or decreased depending on the type of K+ channel, artery, and model. Two general hypotheses can be used to support apparently disparate findings. First, if the expression and/or functional activity of K+ channels were increased by hypertension, then this might serve to oppose depolarization and vasoconstriction. A gain-of-function mutation in BKCa channels (14, 16) and the clinical utility of K+ channel openers (40) are examples of increased K+ channel activity as a mechanism opposing hypertension. Second, if K+ channel expression and/or functional activity were diminished by hypertension, then this could be considered a contributor to depolarization and vasoconstriction. Multiple examples of hypertension-induced K+ channel downregulation have been provided, especially for BKCa channels (1, 2, 5, 33).

Although some controversy exists, the literature generally reports increased BKCa and decreased KV channel expression in various models of hypertension (see, e.g., Ref. 28). Whole cell BKCa current is enhanced in smooth muscle cells from SHR and stroke-prone SHR compared with WKY rats (12, 34, 36). Particularly convincing evidence for increased BKCa expression in hypertension comes from the use of both molecular and functional approaches (25, 26). In contrast, reduced BKCa current has been reported by others in smooth muscle cells from SHR (2) and angiotensin II-induced hypertensive rats (1). Our data with L-NNA-induced hypertension are similar and indicate that functional expression of BKCa is reduced in smooth muscle from hypertensive rats. Unlike the studies of Amberg et al. (1, 2), demonstrating a reduction in BKCa current secondary to diminished expression of the regulatory {beta}1-subunit in genetic (SHR) and angiotensin II-induced hypertension, we found no difference in the Ca2+/voltage sensitivity of BKCa channels in smooth muscle cells from rats made hypertensive with L-NNA. Importantly, however, we show evidence of reduced BKCa {alpha}-subunit protein expression by Western blot, supporting the observed reduction in whole cell current.

Although fewer data are available regarding the functional and molecular expression levels of KV channels in hypertension (9), findings in the literature are more uniform. As a general rule, functional expression of KV channels in smooth muscle is reduced by hypertension (8, 10, 28). Cox and coworkers (8, 10) reported decreased KV current in various smooth muscle cell types from SHR, including mesenteric artery. Similarly, KV current density is reduced in smooth muscle cells from deoxycorticosterone acetate hypertensive rats (28). KV1.2 and KV1.5 contribute to the formation of KV channels in smooth muscle cells from the rat mesenteric artery (27, 39). Sadanaga et al. (37) demonstrated that endothelial cells from stroke-prone SHR are depolarized compared with those from WKY rats and associated with decreased expression of KV1.5. We therefore assessed the role of KV channels with 4-AP. The addition of 4-AP decreased outward current in both groups (Figs. 3 and 4). The decreased current in response to 4-AP in both groups suggests that KV channels play a role in regulating membrane potential. In the companion study (3), we demonstrated that the molecular expression of KV1.5 channel proteins was reduced in arterial smooth muscle from L-NNA hypertensive rats. In the present study, we demonstrate reduced functional expression of 4-AP-sensitive KV current.

In conclusion, the data lead us to conclude that reduced molecular and functional expression of K+ channels (both KV1.5 and BKCa) contributes to the depolarization and augmented contraction of smooth muscle in hypertension. The present results suggest that reduced macroscopic K+ current in smooth muscle cells from hypertensive rats is not related to changes in the biophysical properties (i.e., single-channel conductance or Ca2+/voltage sensitivity) of BKCa channels. Rather, reduced whole cell KV and BKCa current is likely due to a decrease in BKCa and KV1.5 channel protein expression (3). Future studies are required to determine how hypertension reduces the molecular and functional expression of K+ channels in vascular smooth muscle. Additionally, the suggestion that decreased K+ channel expression leads to hypertension needs to be considered (1, 2, 5, 33). Specific signaling mechanisms leading to reduced molecular and functional expression of BKCa and KV1.5 channel proteins in smooth muscle remain to be determined.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
N. L. Kanagy was supported by National Institutes of Health (NIH) Grant HL-03852, a Scientist Development Grant 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).


   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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amberg GC, Bonev AD, Rossow CF, Nelson MT, and Santana LF. Modulation of the molecular composition of large conductance, Ca2+ activated K+ channels in vascular smooth muscle during hypertension. J Clin Invest 112: 717–724, 2003.[CrossRef][Web of Science][Medline]
  2. Amberg GC and Santana LF. Downregulation of the BK channel {beta}1 subunit in genetic hypertension. Circ Res 93: 965–971, 2003.[Abstract/Free Full Text]
  3. Bratz IN, Dick GM, Partridge LD, and Kanagy NL. Reduced molecular expression of K+ channel proteins in vascular smooth muscle from rats made hypertensive with N{omega}-nitro-L-arginine. Am J Physiol Heart Circ Physiol 289: H1277–H1283, 2005.[Abstract/Free Full Text]
  4. Bratz IN, Falcon R, Partridge LD, and Kanagy NL. Vascular smooth muscle cell membrane depolarization after NOS inhibition hypertension. Am J Physiol Heart Circ Physiol 282: H1648–H1655, 2002.[Abstract/Free Full Text]
  5. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, and Aldrich RW. Vasoregulation by the {beta}1 subunit of the calcium-activated potassium channel. Nature 407: 870–876, 2000.[CrossRef][Medline]
  6. Carter RW and Kanagy NL. Mechanism of enhanced calcium sensitivity and {alpha}2-AR vasoreactivity in chronic NOS inhibition hypertension. Am J Physiol Heart Circ Physiol 284: H309–H316, 2003.[Abstract/Free Full Text]
  7. Cole WC and Sanders KM. Characterization of macroscopic outward currents of canine colonic myocytes. Am J Physiol Cell Physiol 257: C461–C469, 1989.[Abstract/Free Full Text]
  8. Cox RH. Comparison of K+ channel properties in freshly isolated myocytes from thoracic aorta of WKY and SHR. Am J Hypertens 9: 884–894, 1996.[CrossRef][Web of Science][Medline]
  9. Cox RH, Folander K, and Swanson R. Differential expression of voltage-gated K+ channel genes in arteries from spontaneously hypertensive and Wistar-Kyoto rats. Hypertension 37: 1315–1322, 2001.[Abstract/Free Full Text]
  10. Cox RH, Lozinskaya I, and Dietz NJ. Differences in K+ current components in mesenteric artery myocytes from WKY and SHR. Am J Hypertens 14: 897–907, 2001.[CrossRef][Web of Science][Medline]
  11. Cox RH and Rusch NJ. New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure. Microcirculation 9: 243–257, 2002.[CrossRef][Web of Science][Medline]
  12. England SK, Wooldridge TA, Stekiel WJ, and Rusch NJ. Enhanced single-channel K+ current in arterial membranes from genetically hypertensive rats. Am J Physiol Heart Circ Physiol 264: H1337–H1345, 1993.[Abstract/Free Full Text]
  13. Erne P and Hermsmeyer K. Intracellular vascular muscle Ca2+ modulation in genetic hypertension. Hypertension 14: 145–151, 1989.[Abstract/Free Full Text]
  14. Fernandez-Fernandez JM, Tomas M, Vazquez E, Orio P, Latorre R, Senti M, Marrugat J, and Valverde MA. Gain-of-function mutation in the KCNMB1 potassium channel subunit is associated with low prevalence of diastolic hypertension. J Clin Invest 113: 1032–1039, 2004.[CrossRef][Web of Science][Medline]
  15. Gerzanich V, Ivanova S, Zhou H, and Simard JM. Mislocalization of eNOS and upregulation of cerebral vascular Ca2+ channel activity in angiotensin-hypertension. Hypertension 41: 1124–1130, 2003.[Abstract/Free Full Text]
  16. Gollasch M, Tank J, Luft FC, Jordan J, Maass P, Krasko C, Sharma AM, Busjahn A, and Bahring S. The BK channel {beta}1 subunit gene is associated with human baroreflex and blood pressure regulation. J Hypertens 20: 927–933, 2002.[CrossRef][Web of Science][Medline]
  17. Harder DR, Brann L, and Halpern W. Altered membrane electrical properties of smooth muscle cells from small cerebral arteries of hypertensive rats. Blood Vessels 20: 154–160, 1983.[Web of Science][Medline]
  18. Harder DR, Smeda J, and Lombard J. Enhanced myogenic depolarization in hypertensive cerebral arterial muscle. Circ Res 57: 319–322, 1985.[Abstract/Free Full Text]
  19. Hume JR and Leblanc N. Macroscopic K+ currents in single smooth muscle cells of the rabbit portal vein. J Physiol 413: 49–73, 1989.[Abstract/Free Full Text]
  20. Jackson WF. Ion channels and vascular tone. Hypertension 35: 173–178, 2000.[Abstract/Free Full Text]
  21. Jelicks LA and Gupta RK. NMR measurement of cytosolic free calcium, free magnesium, and intracellular sodium in the aorta of the normal and spontaneously hypertensive rat. J Biol Chem 265: 1394–1400, 1990.[Abstract/Free Full Text]
  22. Kanagy NL. Increased vascular responsiveness to {alpha}2-adrenergic stimulation during NOS inhibition-induced hypertension. Am J Physiol Heart Circ Physiol 273: H2756–H2764, 1997.[Abstract/Free Full Text]
  23. Kotlikoff MI. Potassium currents in canine airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 259: L384–L395, 1990.[Abstract/Free Full Text]
  24. Langton PD, Nelson MT, Huang Y, and Standen NB. Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. Am J Physiol Heart Circ Physiol 260: H927–H934, 1991.[Abstract/Free Full Text]
  25. Liu Y, Hudetz AG, Knaus HG, and Rusch NJ. Increased expression of Ca2+-sensitive K+ channels in the cerebral microcirculation of genetically hypertensive rats: evidence for their protection against cerebral vasospasm. Circ Res 82: 729–737, 1998.[Abstract/Free Full Text]
  26. Liu Y, Pleyte K, Knaus HG, and Rusch NJ. Increased expression of Ca2+-sensitive K+ channels in aorta of hypertensive rats. Hypertension 30: 1403–1409, 1997.[Abstract/Free Full Text]
  27. Lu Y, Hanna ST, Tang G, and Wang R. Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K+ current in rat mesenteric artery smooth muscle cells. Life Sci 71: 1465–1473, 2002.[CrossRef][Web of Science][Medline]
  28. Martens JR and Gelband CH. Alterations in rat interlobar artery membrane potential and K+ channels in genetic and nongenetic hypertension. Circ Res 79: 295–301, 1996.[Abstract/Free Full Text]
  29. Mukundan H and Kanagy NL. Ca2+ influx mediates enhanced {alpha}2-adrenergic contraction in aortas from rats treated with NOS inhibitor. Am J Physiol Heart Circ Physiol 281: H2233–H2240, 2001.[Abstract/Free Full Text]
  30. Nelson MT, Patlak JB, Worley JF, and Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol Cell Physiol 259: C3–C18, 1990.[Abstract/Free Full Text]
  31. Papageorgiou P and Morgan KG. Intracellular free Ca2+ is elevated in hypertrophic aortic muscle from hypertensive rats. Am J Physiol Heart Circ Physiol 260: H507–H515, 1991.[Abstract/Free Full Text]
  32. Pesic A, Madden JA, Pesic M, and Rusch NJ. High blood pressure upregulates arterial L-type Ca2+ channels: is membrane depolarization the signal? Circ Res 94: e97-e104, 2004.[Abstract/Free Full Text]
  33. Pluger S, Faulhaber J, Furstenau M, Lohn M, Waldschutz R, Gollasch M, Haller H, Luft FC, Ehmke H, and Pongs O. Mice with disrupted BK channel {beta}1 subunit gene feature abnormal Ca2+ spark/STOC coupling and elevated blood pressure. Circ Res 87: E53–E60, 2000.[Abstract/Free Full Text]
  34. Rusch NJ, De Lucena RG, Wooldridge TA, England SK, and Cowley AW Jr. A Ca2+-dependent K+ current is enhanced in arterial membranes of hypertensive rats. Hypertension 19: 301–307, 1992.[Abstract/Free Full Text]
  35. Rusch NJ and Hermsmeyer K. Calcium currents are altered in the vascular muscle cell membrane of spontaneously hypertensive rats. Circ Res 63: 997–1002, 1988.[Abstract/Free Full Text]
  36. Rusch NJ and Runnells AM. Remission of high blood pressure reverses arterial potassium channel alterations. Hypertension 23: 941–945, 1994.[Abstract/Free Full Text]
  37. Sadanaga T, Ohya Y, Ohtsubo T, Goto K, Fujii K, and Abe I. Decreased 4-aminopyridine sensitive K+ currents in endothelial cells from hypertensive rats. Hypertens Res 25: 589–596, 2002.[CrossRef][Web of Science][Medline]
  38. Thorneloe KS, Chen TT, Kerr PM, Grier EF, Horowitz B, Cole WC, and Walsh MP. Molecular composition of 4-aminopyridine-sensitive voltage-gated K+ channels of vascular smooth muscle. Circ Res 89: 1030–1037, 2001.[Abstract/Free Full Text]
  39. Xu C, Lu Y, Tang G, and Wang R. Expression of voltage-dependent K+ channel genes in mesenteric artery smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 277: G1055–G1063, 1999.[Abstract/Free Full Text]
  40. Yokoshiki H, Sunagawa M, Seki T, and Sperelakis N. ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am J Physiol Cell Physiol 274: C25–C37, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 HypertensionHome page
H. Xu, W. F. Jackson, G. D. Fink, and J. J. Galligan
Activation of Potassium Channels by Tempol in Arterial Smooth Muscle Cells From Normotensive and Deoxycorticosterone Acetate-Salt Hypertensive Rats
Hypertension, December 1, 2006; 48(6): 1080 - 1087.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. N. Bratz, G. M. Dick, L. D. Partridge, and N. L. Kanagy
Reduced molecular expression of K+ channel proteins in vascular smooth muscle from rats made hypertensive with N{omega}-nitro-L-arginine
Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1277 - H1283.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/3/H1284    most recent
01053.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bratz, I. N.
Right arrow Articles by Dick, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bratz, I. N.
Right arrow Articles by Dick, G. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.