Reduced functional expression of K + channels in vascular smooth muscle cells from rats made hypertensive with N ω -nitro-l-arginine

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1 Articles in PresS. Am J Physiol Heart Circ Physiol (May 6, 2005). doi: /ajpheart Reduced functional expression of K + channels in vascular smooth muscle cells from rats made hypertensive with N ω -nitro-l-arginine Final Accepted Version H R1 Ian N. Bratz, Albert N. Swafford, Jr., Nancy L. Kanagy 1, and Gregory M. Dick* Department of Physiology Louisiana State University Health Sciences Center 1 Department of Cell Biology and Physiology University of New Mexico School of Medicine Short title: Hypertension reduces functional expression of K + channels *Please address correspondence to Gregory M. Dick, Ph.D. Department of Physiology LSU Health Sciences Center 1901 Perdido Street New Orleans, LA Phone (504) Fax (504) gdick@lsuhsc.edu Copyright 2005 by the American Physiological Society.

2 Abstract The membrane potential of vascular smooth muscle cells is determined, in large part, by K + channels. In the companion paper, we demonstrate that superior mesenteric arteries from rats made hypertensive with L-NNA (N ω -nitro-l-arginine) are depolarized compared to normotensive rats. Further, L-NNA hypertension is associated with reduced molecular expression of two K + channel proteins: K V 1.5 (voltage-sensitive delayed rectifier) and BK Ca α subunit (Ca 2+ /voltagesensitive). 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 cells. In whole-cell experiments using a Ca 2+ -free pipette solution, current at 0 mv, largely due to delayed rectifier K + channels, was reduced ~60% in smooth muscle cells from hypertensive rats (2.7 ± 0.4 vs. 1.1 ± 0.2 pa/pf). Current at +100 mv with 300 nm free Ca 2+, largely due to BK Ca channels, was reduced ~40% in smooth muscle cells from hypertensive rats (181 ± 24 vs. 101 ± 28 pa/pf). Current blocked by 3 mm 4-aminopyridine, an inhibitor of many K V channel types (including K V 1.5), was reduced ~50% in smooth muscle cells from hypertensive rats (1.0 ± 0.4 vs. 0.5 ± 0.2 pa/pf). Current blocked by 1 mm tetraethylammonium, an inhibitor of BK Ca channels, was reduced ~40% in myocytes from hypertensive rats (86 ± 14 vs. 53 ± 19 pa/pf). Differences in BK Ca current magnitude are not attributable to changes in single channel conductance or Ca 2+ /voltage-sensitivity. The data support the hypothesis that L-NNA-induced hypertension reduces macroscopic 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. Key Words: nitric oxide, membrane potential, Ca 2+ -activated K + channel, delayed rectifier K + channel, hypertension 2

3 Introduction Elevated arterial tone increases peripheral vascular resistance and blood pressure. Vascular smooth muscle tone is controlled, in large part, by the intracellular free Ca 2+ concentration, which in turn is influenced by voltage. The open probability of L-type Ca 2+ channels, a major pathway of Ca 2+ entry in vascular smooth muscle, is determined by membrane potential. Thus, smooth muscle membrane potential, intracellular Ca 2+, and contraction and 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 Ca 2+ 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 upon 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 have demonstrated previously a variety of vascular changes in male rats made hypertensive with the nitric oxide synthase inhibitor N ϖ -nitro-l-arginine (L-NNA). Smooth muscle changes associated with L-NNA-induced hypertension include increased contractility (22), enhanced Ca 2+ -sensitivity (6), and augmented responses to dihydropyridines (29). In the companion paper, we demonstrate that L-NNA-induced hypertension depolarizes the smooth muscle membrane potential and diminishes expression of K + channel proteins (3). 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 using whole-cell and single channel patch clamp techniques. The data support the notion that L-NNA-induced hypertension reduces whole-cell K + current in 3

4 vascular smooth muscle. Reduced functional expression of K + current is secondary to diminished molecular expression and these changes may underlie depolarization and augmented contractility. 4

5 Methods Hypertension induction and the isolation of smooth muscle cells: Male Sprague-Dawley rats ( g; Charles River; Wilmington, MA) were randomly assigned to control and L-NNA treated groups. Rats in the L-NNA group were provided tap water containing 0.5 mg/ml L-NNA for 2 weeks. Rats were anesthetized with sodium pentobarbital (65 mg/kg i.p.), intubated, 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 3 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 minutes. Tissue were then incubated for 20 minutes 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 solution three for minutes 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 minutes and then passed repeatedly through the fire-polished tip of a Pasteur pipette to liberate single myocytes. Cell suspension was kept at room temperature and patch clamp recordings were performed within 8 hours. PSS contained (mm) 125 NaCl, 5 KCl, 2 CaCl 2, 1 MgCl 2, 10 glucose, 20 mannitol, 10 HEPES, 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 Ca 2+ -free PSS that contained 2 mm MnCl 2 in place of CaCl 2. Single myocytes were approached with heat-polished pipettes having tip resistances between 2-4 MΩ when filled with solution containing (mm) 135 KCl, 10 HEPES, 5 Tris, 3 Mg- 5

6 ATP, 1 Na-GTP, 1 EGTA; ph 7.1. To create a pipette solution with 300 nm free Ca 2+, CaCl 2 was added to this 1 mm EGTA pipette solution (calculations performed with MAXCHELATOR software; Whole-cell K + currents were measured at room temperature using 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 mm, thus no adjustment for junction potentials was necessary. The bath solution was hypertonic ( 315 mosm) relative to the pipette ( 290 mosm) to reduce volumesensitive 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 HEDTA, and 10 HEPES; 5 Tris; ph 7.1 Solutions that were considered Ca 2+ -free or contained 300 nm free Ca 2+ were made with EGTA, while HEDTA was used to make a solution with 10 µm free Ca 2+. Data were analyzed with WinASCD software ( and BK Ca channel conductance was determined from all-points amplitude histograms. Statistical analysis: Data are reported as mean ± standard error from n number of rats. Current-voltage relationships were determined by measuring steady-state current (i.e., at the end of the 400 msec test pulse) and compared between the two groups. Data were analyzed by one or two-way ANOVA or t-test as indicated in the text and figure legends. Post hoc analyses were performed using Student Newman-Keuls (two-way ANOVA) or Holm-Sidak (one-way ANOVA) tests. Differences were considered significant for p <

7 Results Male Sprague-Dawley rats were divided into 2 groups given either normal tap water or tap water containing 0.5 mg/ml L-NNA. Treatment was for two weeks and blood pressure was measured under pentobarbital anesthesia on the day of sacrifice. 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 using the conventional whole-cell patch clamp technique. The bath solution was nominally Ca 2+ -free and the cells were dialyzed with a Ca 2+ -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). Wholecell currents under these conditions were generally small (< mv) and composed of two apparent conductances, BK Ca and K V, 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) in order to negate any possible differences in cell size (although we did not detect any change in cell capacitance; 16.2 ± 1.3 vs ± 1.0 pf for myocytes from normotensive and hypertensive rats, respectively). A comparison of the current-voltage relationships demonstrated the curves had similar shapes; however, differences in magnitude were observed (Fig. 2B). Under these Ca 2+ -free conditions, whole-cell currents were reduced in smooth muscle cells from hypertensive rats, suggesting a reduction in K V current. Smooth muscle cells from normotensive and hypertensive rats were also studied after dialyzing them with a pipette solution buffered to 300 nm Ca 2+. Whole-cell currents under these 7

8 conditions were larger than those observed with the Ca 2+ -free pipette (> mv) and BK Ca channel current became more prominent (Fig. 2C). Similar to results with the Ca 2+ -free pipette solution, the current-voltage relationship was depressed in cells from hypertensive rats compared to normotensive rats (Fig. 2D). The most notable differences in current density were at positive membrane potentials, where BK Ca channels are active (> +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 to control, regardless of the Ca 2+ concentration (Fig. 3). These data suggest a decrease in K V or delayed rectifier current, as well as a reduction in BK Ca current. A reduction in K V 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 Ca 2+ -free pipette solution (Fig. 3A) or one containing 300 nm Ca 2+ (Fig. 3B). These data with different intracellular Ca 2+ concentrations suggest hypertension reduces both voltage- and Ca 2+ -dependent whole-cell K + current. In order to further assess whether reductions in whole-cell current were more specifically attributable to K V or BK Ca channels, responses to tetraethylammonium (TEA) and 4- aminopyridine (4-AP) were determined (Fig. 4). In the companion paper, we demonstrated reduced expression of two K + channel proteins in smooth muscle from hypertensive rats: Kv1.5 and the BK Ca α subunit (3). K V 1.5 is among the delayed recitifiers inhibited by 4-AP (38). In contrast, BK Ca 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 1 mm TEA primarily inhibits BK Ca current, and not K V 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 K V current (including K V 1.5), but 8

9 does not inhibit BK Ca 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. 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 K V 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 BK Ca 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 BK Ca, we determined whether experiments with a more selective BK Ca blocker, such as iberiotoxin, would be necessary. We studied cells dialyzed with a pipette solution containing 300 nm Ca 2+ 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 BK Ca channels. Further, the data with TEA suggest that the differences in TEA-sensitive current between normotensive and hypertensive rats is due to BK Ca channels. 9

10 We determined whether the reduction in whole-cell BK Ca current could be attributed to changes in single channel conductance or Ca 2+ /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). Since the activity of BK Ca channels is regulated by Ca 2+ concentration, we determined whether changes in whole-cell BK Ca current were due to changes in the sensitivity of the channels to Ca 2+. Using excised inside-out membrane patches, activation curves were constructed in solutions that were Ca 2+ -free or contained 10 µm free Ca 2+. The Ca 2+ /voltage-sensitivity of BK Ca channels was not different between normotensive and hypertensive rats (Fig. 6D). 10

11 Discussion This study addresses whether functional expression of whole-cell K + current parallels reduced molecular expression of BK Ca and K V 1.5 channel proteins in the superior mesenteric arteries of rats made hypertensive with L-NNA (3). Further, this study aims to determine 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 where we demonstrated smooth muscle depolarization, augmented vascular contractility, and reduced molecular expression of BK Ca and K V 1.5 channel proteins in arteries from L-NNA hypertensive rats. Importantly, however, the question whether functional expression of K + channels in smooth muscle was affected by L- NNA-induced hypertension remained unanswered. Using whole-cell patch clamp techniques, we tested the hypothesis directly and demonstrate reduced K + current in vascular myocytes from rats made hypertensive with L-NNA. These data are comparable to 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 which 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 K V 1.5 and BK Ca 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 Ca 2+ -free or buffered to 300 nm Ca 2+. 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 (K V ) current and current mediated by BK Ca channels. K V current is readily identified by using a Ca 2+ -free pipette, which limits the activation of BK Ca channels. Additionally, at least some K V channels (including K V 1.5) are 11

12 sensitive to 4-AP (38). Thus additional pharmacological evidence comes from our demonstration that 4-AP-sensitive K + current, measured with a Ca 2+ -free pipette solution, is reduced in smooth muscle cells from L-NNA-hypertensive rats. BK Ca channels are most easily identified by using a pipette with an elevated free Ca 2+ concentration. Further, BK Ca channels are sensitive to block by 1 mm TEA, a concentration which has little effect on most types of smooth muscle K V channels (24). We demonstrate, using a pipette solution containing 300 nm Ca 2+, that L-NNA-induced hypertension reduces the functional expression of Ca 2+ - and TEAsensitive K + current. These K V and BK Ca 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 K V and BK Ca channels would be expected to reproduce some aspects of the vascular phenotype observed in L-NNA-induced hypertensive rats (5; 33). Inhibition of K V (with 4-AP) or BK Ca (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 have 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 (SHR) rats. Particularly relevant to this discussion are documented changes in the expression and activity of L-type Ca 2+, K V, and BK Ca channels in the smooth muscle of SHR hypertensive rats compared to control Wistar-Kyoto (WKY) rats (e.g.,(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 Ca 2+ current (15; 32; 35), and increased intracellular free Ca 2+ (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 12

13 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 may serve to oppose depolarization and vasoconstriction. A gain-of-function mutation in BK Ca 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 BK Ca channels (1; 2; 5; 33). Although there exists some controversy, the literature generally reports increased BK Ca and decreased in K V channel expression in various models of hypertension (e.g., (28)). Wholecell BK Ca current is enhanced in smooth muscle cells from SHR and stroke-prone SHR compared to WKY rats (12; 34; 36). Particularly convincing evidence for increased BK Ca expression in hypertension comes from using both molecular and functional approaches (25; 26). In contrast, reduced BK Ca 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 BK Ca is reduced in smooth muscle from hypertensive rats. Unlike the studies of Amberg et al. (1; 2), demonstrating a reduction in BK Ca current secondary to diminished expression of the regulatory β1 subunit in genetic (SHR) and angiotensin II-induced hypertension, we found no difference in the Ca 2+ /voltage-sensitivity of BK Ca channels in smooth muscle cells from rats made hypertensive with L-NNA. Importantly, however, we show evidence of reduced BK Ca α subunit protein expression by Western blot, supporting the observed reduction in whole-cell current. 13

14 While fewer data are available regarding the functional and molecular expression levels of K V channels in hypertension (9), findings in the literature are more uniform. As a general rule, functional expression of K V channels in smooth muscle is reduced by hypertension (8; 10; 28). Cox and coworkers have reported decreased K V current in various smooth muscle cell types from SHR, including mesenteric artery (8; 10). Similarly, K V current density is reduced in smooth muscle cells from deoxycorticosterone acetate (DOCA) hypertensive rats (28). K V 1.2 and K V 1.5 contribute to the formation K V channels in smooth muscle cells from the rat mesenteric artery (27; 39). Sadanaga et al. demonstrated that endothelial cells from stroke-prone SHR rats are depolarized compared to those from WKY rats and associated with decreased expression of K V 1.5 (37). We therefore assessed the role of K V 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 K V channels play a role in regulating E m. In the companion study, we demonstrated that the molecular expression of K V 1.5 channel proteins was reduced in arterial smooth muscle from L-NNA hypertensive rats (3). In the present study, we demonstrate reduced functional expression of 4-AP-sensitive K V current. In conclusion, the data lead us to conclude that reduced molecular and functional expression of K + channels (both K V 1.5 and BK Ca ) contributes to the depolarization and augmented contraction of smooth muscle in hypertension. The present results suggest 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 Ca 2+ /voltage-sensitivity) of BK Ca channels. Rather, reduced whole-cell K V and BK Ca current is likely due to a decrease in BK Ca and K V 1.5 channel protein expression (3). Future studies are required to determined 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 14

15 reduced molecular and functional expression of BK Ca and K V 1.5 channel proteins in smooth muscle remain to be determined. 15

16 Acknowledgments Dr. Kanagy was supported by NIH HL03852, a Scientist Development Grant from the American Heart Association, and a Research Allocations Committee grant from the University of New Mexico. Dr. Dick was supported by a Beginning Grant-in-Aid from the American Heart Association and the LSU Center of Biomedical Research Excellence (COBRE; NIH P20 RR018766). 16

17 References 1. Amberg GC, Bonev AD, Rossow CF, Nelson MT and Santana LF. Modulation of the molecular composition of large conductance, Ca 2+ activated K + channels in vascular smooth muscle during hypertension. J Clin Invest 112: , Amberg GC and Santana LF. Downregulation of the BK channel β1 subunit in genetic hypertension. Circ Res 93: , Bratz IN, Dick GM, Partridge LD and Kanagy NL. Reduced molecular expression of K + channels in vascular smooth muscle cells from rats made hypertensive with N ω -nitro-larginine. (companion paper). 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, Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT and Aldrich RW. Vasoregulation by the β1 subunit of the calcium-activated potassium channel. Nature 407: , Carter RW and Kanagy NL. Mechanism of enhanced calcium sensitivity and α 2 -AR vasoreactivity in chronic NOS inhibition hypertension. Am J Physiol Heart Circ Physiol 284: H309-H316, Cole WC and Sanders KM. Characterization of macroscopic outward currents of canine colonic myocytes. Am J Physiol 257: C461-C469, Cox RH. Comparison of K + channel properties in freshly isolated myocytes from thoracic aorta of WKY and SHR. Am J Hypertens 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: ,

18 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: , Cox RH and Rusch NJ. New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure. Microcirculation 9: , England SK, Wooldridge TA, Stekiel WJ and Rusch NJ. Enhanced single-channel K + current in arterial membranes from genetically hypertensive rats. Am J Physiol 264: H1337-H1345, Erne P and Hermsmeyer K. Intracellular vascular muscle Ca 2+ modulation in genetic hypertension. Hypertension 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: , Gerzanich V, Ivanova S, Zhou H and Simard JM. Mislocalization of enos and upregulation of cerebral vascular Ca 2+ channel activity in angiotensin-hypertension. Hypertension 41: , Gollasch M, Tank J, Luft FC, Jordan J, Maass P, Krasko C, Sharma AM, Busjahn A and Bahring S. The BK channel β1 subunit gene is associated with human baroreflex and blood pressure regulation. J Hypertens 20: , 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: , Harder DR, Smeda J and Lombard J. Enhanced myogenic depolarization in hypertensive cerebral arterial muscle. Circ Res 57: , Hume JR and Leblanc N. Macroscopic K + currents in single smooth muscle cells of the rabbit portal vein. J Physiol 413: 49-73,

19 20. Jackson WF. Ion channels and vascular tone. Hypertension 35: , 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: , Kanagy NL. Increased vascular responsiveness to α 2 -adrenergic stimulation during NOS inhibition-induced hypertension. Am J Physiol 273: H2756-H2764, Kotlikoff MI. Potassium currents in canine airway smooth muscle cells. Am J Physiol 259: L384-L395, 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 260: H927-H934, Liu Y, Hudetz AG, Knaus HG and Rusch NJ. Increased expression of Ca 2+ -sensitive K + channels in the cerebral microcirculation of genetically hypertensive rats: evidence for their protection against cerebral vasospasm. Circ Res 82: , Liu Y, Pleyte K, Knaus HG and Rusch NJ. Increased expression of Ca 2+ -sensitive K + channels in aorta of hypertensive rats. Hypertension 30: , 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: , Martens JR and Gelband CH. Alterations in rat interlobar artery membrane potential and K + channels in genetic and nongenetic hypertension. Circ Res 79: , Mukundan H and Kanagy NL. Ca 2+ influx mediates enhanced α 2 -adrenergic contraction in aortas from rats treated with NOS inhibitor. Am J Physiol Heart Circ Physiol 281: H2233-H2240,

20 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 259: C3-18, Papageorgiou P and Morgan KG. Intracellular free Ca 2+ is elevated in hypertrophic aortic muscle from hypertensive rats. Am J Physiol 260: H507-H515, Pesic A, Madden JA, Pesic M and Rusch NJ. High blood pressure upregulates arterial L-type Ca 2+ channels: is membrane depolarization the signal? Circ Res 94: e97-104, 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 β1 subunit gene feature abnormal Ca 2+ spark/stoc coupling and elevated blood pressure. Circ Res 87: E53-E60, Rusch NJ, De Lucena RG, Wooldridge TA, England SK and Cowley AW, Jr. A Ca 2+ - dependent K + current is enhanced in arterial membranes of hypertensive rats. Hypertension 19: , Rusch NJ and Hermsmeyer K. Calcium currents are altered in the vascular muscle cell membrane of spontaneously hypertensive rats. Circ Res 63: , Rusch NJ and Runnells AM. Remission of high blood pressure reverses arterial potassium channel alterations. Hypertension 23: , 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: , 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: , 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 277: G1055-G1063,

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22 Fig. 1 Blood pressure of control and L-NNA-treated rats. Panel A shows a 10 second recording of blood pressure from a representative control rat. Panel B contains a representative blood pressure tracing from a rat treated 2 weeks with L-NNA. Panel C shows group data for mean arterial pressure in 7 control and 8 L-NNA-treated rats; asterisk indicates p < 0.05 by unpaired t-test. 22

23 Fig. 2 Whole-cell K + current is reduced in vascular smooth muscle cells from hypertensive rats regardless of the intracellular free Ca 2+ concentration. Myocytes were bathed in Ca 2+ -free PSS and dialyzed with a either a Ca 2+ -free pipette solution (1 mm EGTA) or one buffered to 300 nm Ca 2+. The Ca 2+ -free pipette solution was designed to maximize K V current, while the 300 nm Ca 2+ pipette solution was designed to enhance BK Ca. Cells were held at -80 mv and stepped from -100 mv to +100 mv in 20 mv increments. Panel A contains representative current traces with a Ca 2+ -free pipette. Panel B shows group data for the I-V relationship (inset is expanded to appreciate differences in magnitude). Outward current was larger in smooth muscle cells from normotensive (n = 7) compared to hypertensive rats (n = 6). Panel C contains representative current traces with a pipette solution containing 300 nm Ca 2+. Panel D contains the group I-V relationship. Outward current was larger in smooth muscle cells from normotensive (n = 6) compared to hypertensive rats (n = 6). Two-way ANOVA indicated that I-V curves are different; asterisks indicate a value of p < 0.05 at specific voltages (Student- Newman-Keuls post-hoc analysis). 23

24 Fig. 3 Difference currents: normotensive minus hypertensive. Panel A shows the difference current obtained by subtracting the hypertensive current from the mean normotensive current when cells were dialyzed with a Ca 2+ -free pipette solution (from Fig. 2B). Panel B contains the difference current from cells dialyzed with 300 nm free Ca 2+ (from Fig. 2D). The insets of panels 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. Asterisks indicate voltages where the two groups differed. 24

25 Fig. 4 Block of K + currents by TEA- and 4-AP in cells dialyzed with Ca 2+ -free pipette solution. Panel A Representative traces demonstrate the effect of 1 mm TEA, 3 mm 4-AP, and the combination of the two 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. Panel B, C, and D Whole-cell current which remains in the presence of each inhibitor (inset shows current that was blocked by each inhibitor). 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 (asterisks indicate p < 0.05 at specific voltages; Student-Newman-Keuls post-hoc analysis). 25

26 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 Ca 2+ ; 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 shows current inhibited by 1 mm TEA and the two concentrations of iberiotoxin. 26

27 Fig. 6 Reduced whole-cell BK Ca current is not due to a change in single channel or Ca 2+ /voltage-sensitivity. Panel A contains a representative current trace of BK Ca channel activity at +80 mv. Single channel currents were recorded in symmetrical 140 mm K +. Panel B shows group data for the single channel I-V relationship for BK Ca channels from 5 normotensive and 6 hypertensive rats. Panel 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 Ca 2+ (left) or 1 mm HEDTA buffered to 10 µm free Ca 2+ (right). Membrane potential was stepped from -200 to +200 mv in 20 mv increments. Currents were converted to conductance (G), normalized to the maximum, and plotted vs. voltage (Panel D). No differences in Ca 2+ /voltage-sensitivity were detected between normotensive (n = 5) and hypertensive rats (n = 6). 27