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(Hypertension. 1996;27:1234-1239.)
© 1996 American Heart Association, Inc.

Articles

Impaired Action of Levcromakalim on ATP-Sensitive K⁺ Channels in Mesenteric Artery Cells From Spontaneously Hypertensive Rats

Yusuke Ohya; Motoko Setoguchi; Koji Fujii; Tetsuhiko Nagao; Isao Abe; Masatoshi Fujishima

From the Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.

Correspondence to Yusuke Ohya, MD, PhD, Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan. E-mail ohya@intmed2.med.kyushu-u.ac.jp.

	Abstract

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Abstract The purpose of the present study was to test the hypothesis that properties of ATP-sensitive K⁺ channels are altered in arterial smooth muscle cells of hypertensive rats. Using a patch-clamp technique, we compared effects of a K⁺ channel opener, levcromakalim, on membrane currents in mesenteric artery cells from adult Wistar Kyoto rats (WKY) and age-matched spontaneously hypertensive rats (SHR) treated or not treated with hydralazine. Blood pressure was significantly higher in SHR than in WKY or hydralazine-treated SHR. Levcromakalim evoked a time-independent and voltage-insensitive current in a dose-dependent manner in the whole-cell clamp configuration. The reversal potential of the evoked current depended on extracellular K⁺ concentration. Application of 3 µmol/L glibenclamide, a specific blocker of ATP-sensitive K⁺ channels, abolished the levcromakalim-evoked current; however, the current was unaffected by either 1 mmol/L tetraethylammonium or 0.3 µmol/L charybdotoxin. These results suggest that the levcromakalim-evoked current was carried through ATP-sensitive K⁺ channels. In SHR cells, the maximal slope conductance of the levcromakalim-evoked current, normalized by cell capacitance, was decreased, and the dose-response curve was shifted to the right compared with WKY cells. The levcromakalim action was not impaired in cells from hydralazine-treated SHR. In conclusion, the action of levcromakalim on ATP-sensitive K⁺ channels in SHR mesenteric artery muscle cells was impaired compared with WKY cells. This impairment was corrected by long-term antihypertensive treatment.

Key Words: rats, inbred, SHR • patch clamp techniques • potassium channels • hydralazine • adenosine triphosphate

	Introduction

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Changes in the properties of ion channels in vascular smooth muscle cells from hypertensive animals have been revealed by the patch-clamp technique. The activity of L-type Ca²⁺ channels is increased in resistance mesenteric arteries of young spontaneously hypertensive rats (SHR), in cerebral arteries of adult stroke-prone SHR (SHRSP), and in the azygous vein of neonatal SHR compared with that in age-matched Wistar Kyoto rats (WKY).^{1 2 3} The activity of Ca²⁺-dependent K⁺ channels in aortic cells of hypertensive rats (SHR; two-kidney, one clip hypertensive rats; and rats with coarctation of the aorta) is also increased compared with that in normotensive controls.^{4 5} These alterations are thought to play an important role in the regulation of vascular tone in hypertension.

K⁺ channel openers activate K⁺ channels, hyperpolarize the cell membrane, and relax vascular smooth muscle.^{6 7} K⁺ channels activated by these compounds in vascular smooth muscle cells are thought to be ATP-sensitive K⁺ channels.^{8 9 10 11} Other types of K⁺ channels, such as Ca²⁺-dependent K⁺ channels, have also been shown to be possible targets of these agents.^{12 13} Because possible clinical uses of K⁺ channel openers include the treatment of hypertension, the effects of these drugs in hypertensive animals are of great interest. The in vivo administration of K⁺ channel openers such as cromakalim and levcromakalim effectively reduces the blood pressure of hypertensive patients and SHR.^{14 15 16} In contrast, with an open cranial window method, Kitazono et al¹⁷ showed that topical application of aprikalim induced a dose-dependent vasodilation in the basilar artery that was more marked in WKY than in SHRSP; the authors therefore suggested that the sensitivity of ATP-sensitive K⁺ channels to K⁺ channel openers is decreased in SHRSP. However, the electrophysiological properties of ATP-sensitive K⁺ channels in arterial cells have not been characterized in hypertensive animals. Our hypothesis in the present study was that properties of ATP-sensitive K⁺ channels in arterial smooth muscle cells are altered in hypertensive rats. With the use of the patch-clamp technique, we compared the characteristics of K⁺ channels evoked by levcromakalim in single smooth muscle cells from the mesenteric artery of WKY and SHR.

	Methods

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Animals
SHR and WKY (18 to 24 weeks old) that had been maintained in the Institute of Experimental Animals at Kyushu University were studied. The study protocol was approved by the Committee on Ethics of Animal Experimentation in the Faculty of Medicine, Kyushu University. SHR were divided into untreated and hydralazine-treated (hydralazine [Sigma Chemical Co] in drinking water at a concentration of 0.1 mg/mL) groups from 6 weeks of age. The rats had free access to standard rat chow and tap water. Systolic pressure and pulse rate were measured by the tail-cuff method.

Cell Dispersion
Single smooth muscle cells were obtained from the main trunk of the mesenteric artery by collagenase treatment. Procedures were basically the same as those described previously.^{2 18} In brief, rats were stunned by a blow to the head and decapitated. The main trunk of the mesenteric artery was dissected and connective tissue carefully removed. The artery was rinsed and incubated for about 15 minutes at 36°C in a Ca²⁺-free solution (145 mmol/L NaCl, 6 mmol/L KCl, 10 mmol/L glucose, 10 mmol/L HEPES-NaOH [pH 7.3]). The tissue was then incubated for 45 to 50 minutes at 36°C in the Ca²⁺-free solution containing 0.3% collagenase (Wako). The digested tissue was resuspended in Ca²⁺-free solution without collagenase and gently agitated with a glass pipette to disperse single cells. Cells were stored at 6° to 8°C in Ca²⁺-free solution containing 1 mmol/L MgCl₂ and 0.2% bovine serum albumin (essentially fatty acid free; Sigma) until use. Patch-clamp experiments were performed within 4 hours after cell dispersion.

Electrical Recording
Whole-cell voltage clamp was performed with a patch pipette through a voltage-clamp amplifier (Axopatch 1-D, Axon Instruments) by the method of Hamill et al.¹⁹ The conditions and procedures were basically the same as those we have described previously.^{2 18} Electrical recording was performed at room temperature (22° to 24°C). Membrane currents were low-pass–filtered at 2 kHz, digitized with a sampling frequency of 5 to 10 kHz, and stored in a personal computer for subsequent analysis. Cell capacitance was estimated when compensating the series resistance by the dial setting of the capacitive cancellation network in the voltage-clamp amplifier.¹⁹ The liquid junction potential was not corrected.

It has been reported that glibenclamide-sensitive background currents were detected in the absence of K⁺ channel openers in smooth muscle cells from rabbit portal vein and pulmonary artery, which were enhanced by the presence of GDP inside the cell.^{11 20 21 22} In our experiments, the background current stabilized within 5 minutes after the whole-cell configuration was achieved and was negligible in 92 of the 96 cells examined (2 of the 4 exceptions were from WKY and 2 from SHR). We did not use results from these 4 cells that showed large background currents.

Solutions and Chemicals
The bath solution for electrical recording contained (mmol/L) NaCl 150, KCl 6, CaCl₂ 2, MgCl₂ 2, glucose 10, and HEPES-NaOH 10 (pH 7.3). The pipette solution contained (mmol/L) KCl 150, MgCl₂ 1, ATP (disodium salt) 0.3, GDP (disodium salt) 1, EGTA 10, and HEPES-TrisOH 10 (pH 7.25). In some experiments, the NaCl in the bath solution was replaced with an equimolar concentration of KCl.

Levcromakalim (BRL 38227; a gift from Smith-Kline Beecham) was dissolved in 100% ethanol as a 30 mmol/L stock solution, and glibenclamide (Sigma) was dissolved in 100% dimethyl sulfoxide as a 100 mmol/L stock solution. The stock solutions were diluted at least 1000 times when used; ethanol or dimethyl sulfoxide at 0.1% had no effect on membrane currents.

Data Analysis
The slope conductance of the levcromakalim-evoked current was obtained at potentials from -100 to -40 mV in a ramp pulse. When the currents in WKY and SHR were compared, the slope conductance was normalized by the cell capacitance because we have previously shown that the ratio of cell capacitance to cell surface area (specific membrane capacitance) does not differ between SHR and WKY.² The levcromakalim dose-response curve was obtained by assuming 1:1 binding of the drug to the receptor according to the Michaelis-Menten equation. Data were fitted to the equation by the nonlinear least-squares method. Data are expressed as mean±SE. Statistical significance was determined by ANOVA followed by a multiple comparison test (Scheffé's test). A value of P<.05 was considered statistically significant.

	Results

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Blood pressure, pulse rate, and body weight of WKY, SHR, and hydralazine-treated SHR are shown in the Table. Systolic pressure and pulse rate at 18 to 24 weeks of age were significantly higher in SHR than in WKY. Hydralazine treatment reduced blood pressure in SHR. Body weight of treated SHR did not differ from that of untreated SHR but was smaller than that of WKY.

View this table: [in this window] [in a new window]   Table 1. Blood Pressure, Pulse Rate, and Body Weight of WKY, SHR, and Hydralazine-Treated SHR at 18 to 24 Weeks of Age

Membrane currents were recorded from single smooth muscle cells by the whole-cell voltage-clamp method. Depolarizing and hyperpolarizing command potentials were applied from a holding potential of -60 mV. Command potentials to less than -40 mV evoked only small background currents (Fig 1A). Depolarization to -30 mV or more positive potentials induced a delayed outward current that was sensitive to 4-aminopyridine (data not shown), as has been observed in other arterial smooth muscle cells.^{23 24} A Ca²⁺-dependent K⁺ current was not apparent because of the presence of 10 mmol/L EGTA in the pipette solution. A net inward current was not usually observed because of a large K⁺ outward current. Addition of 3 µmol/L levcromakalim to the bath solution induced an upward shift in the holding current and increased the current amplitudes induced by the command potentials (Fig 1B). The levcromakalim-evoked current isolated by subtraction apparently did not decay within 200 milliseconds and was therefore time independent during this period (Fig 1C).

View larger version (43K): [in this window] [in a new window]   Figure 1. Typical recordings show the effect of levcromakalim on whole-cell currents evoked by command potentials. A and B, Currents before (A) and currents after (B) application of 3 µmol/L levcromakalim. C, Levcromakalim-evoked current components obtained by subtracting traces in A from those in B. D, Command potentials. The cell was obtained from WKY mesenteric artery. Cell capacitance was 22 pF. Arrow indicates zero current level.

Current was recorded during application of a 200-millisecond voltage ramp from -100 to -10 mV (Fig 2). Before application of levcromakalim, only a small linear background current was recorded at potentials less than -30 mV. Levcromakalim induced a large increase in the current slope; the evoked current was nearly linear at less than -40 mV. Subsequent application of glibenclamide (3 µmol/L) abolished the levcromakalim-evoked current. The current traces obtained before and after application of levcromakalim intersect at about -80 mV, suggesting that the reversal potential of the levcromakalim-evoked current was approximately -80 mV.

View larger version (16K): [in this window] [in a new window]   Figure 2. Current traces evoked by a 200-millisecond voltage ramp from -100 to -10 mV before (Control) and after (Levcromakalim) application of 3 µmol/L levcromakalim as well as after a subsequent application of 3 µmol/L glibenclamide (+ Glibenclamide). The cell was obtained from WKY and had a capacitance of 20 pF. Bottom trace indicates voltage ramp.

To evaluate the ionic nature of the levcromakalim-evoked current, we examined the effect of changing the K⁺ concentration in the bath solution ([K⁺]_o) on the reversal potential. The levcromakalim-evoked currents at various [K⁺]_o values are shown in Fig 3A. The K⁺ equilibrium potential was calculated from the Nernst equation for various [K⁺]_o values and is shown as a solid line in Fig 3B. The measured reversal potentials did not deviate from the calculated K⁺ equilibrium potentials for both WKY and SHR. This [K⁺]_o dependence of the reversal potential suggests that the levcromakalim-evoked currents were carried through K⁺-selective channels. The equilibrium potentials of Na⁺ and Cl^- under the standard condition were 110 and 2 mV, respectively, markedly different from the observed reversal potentials.

View larger version (17K): [in this window] [in a new window]   Figure 3. A, Effect of extracellular K+ concentration (6 K, 6 mmol/L; 50 K, 50 mmol/L; 100 K, 100 mmol/L) on levcromakalim-evoked current. Currents were recorded with a 200-millisecond voltage ramp from -100 to -10 mV (or -5 mV). Traces were obtained by subtracting currents before from those after application of 3 µmol/L levcromakalim. The cell was obtained from WKY and had a capacitance of 22 pF. B, Reversal potentials of levcromakalim-evoked current in WKY () and SHR (). Data points represent mean±SE (n=4-14). Solid line represents theoretical equilibrium potential for K+ (Erev) obtained from the Nernst equation: Erev=-58.6xlog([K+]i/[K+]o), where [K+]i and [K+]o represent intracellular and extracellular K+ concentrations, respectively.

We investigated the effects of various K⁺ channel blockers on the levcromakalim-evoked currents. Blockers were applied after the current was stabilized with 3 µmol/L levcromakalim. As indicated above, glibenclamide (3 µmol/L), a specific blocker of ATP-sensitive K⁺ channels, abolished the levcromakalim-evoked current (Fig 2); the relative slope conductance after application of 3 µmol/L glibenclamide was 0.07±0.03 (n=12) for WKY and 0.05±0.05 (n=10) for SHR. Charybdotoxin, a specific blocker of large-conductance Ca²⁺-dependent K⁺ channels (BK channels), did not inhibit the levcromakalim-evoked current at concentrations up to 0.3 µmol/L; subsequent application of glibenclamide abolished the levcromakalim-evoked current (Fig 4A). The relative slope conductance in the presence of 0.3 µmol/L charybdotoxin was 0.96±0.03 (n=7) in WKY and 0.97±0.04 (n=5) in SHR. Low concentrations (1 mmol/L) of tetraethylammonium, which also blocks BK channels, apparently did not inhibit the levcromakalim-evoked current; higher tetraethylammonium concentrations (3 and 10 mmol/L) partially inhibited the levcromakalim-evoked current (Fig 4B). The relative slope conductance with 1 mmol/L tetraethylammonium was 0.93±0.02 (n=5) for WKY and 0.95±0.03 (n=5) for SHR. These results suggest that the levcromakalim-evoked current is carried through ATP-sensitive K⁺ channels and not BK channels in this preparation.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effects of K+ channel blockers on levcromakalim-evoked current. A, Effect of 0.3 µmol/L charybdotoxin (CTX); B, effects of 1, 3, and 10 mmol/L tetraethylammonium (TEA). The control current (Control) is the difference between currents induced before and after application of 3 µmol/L levcromakalim. Charybdotoxin and tetraethylammonium were applied after the levcromakalim-evoked current was stabilized. Glibenclamide (3 µmol/L) was applied after charybdotoxin or tetraethylammonium. Cells were from WKY and had capacitances of 24 pF (A) and 20 pF (B).

We compared the potencies of levcromakalim for evoking currents in cells from SHR and WKY (Fig 5A and 5B). Levcromakalim evoked the ATP-sensitive K⁺ current in a dose-dependent manner in both SHR and WKY cells; however, 0.1 µmol/L levcromakalim induced an apparent current only in WKY. The slope conductance of levcromakalim-induced currents was obtained and normalized relative to the cell capacitances (SHR, n=32 cells; WKY, n=26 cells) (Fig 6). The maximal slope conductance and dissociation constant (K_d) were estimated from the dose-response relation by fitting the data to the Michaelis-Menten equation. The maximal slope conductance was smaller in SHR (34.0 pS/pF) than in WKY (51.4 pS/pF). The dose-response relation for SHR was shifted to the right (K_d, 0.50 µmol/L for WKY and 1.70 µmol/L for SHR). These results suggest that both responsiveness and sensitivity of ATP-sensitive K⁺ channels to levcromakalim were decreased in SHR cells.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effect of various levcromakalim concentrations on K+ current in cells from WKY (A), SHR (B), and hydralazine-treated SHR (C). Currents evoked by levcromakalim at 0.1, 1, and 10 µmol/L are shown. Capacitances of cells from WKY, SHR, and treated SHR were 21, 22, and 21 pF, respectively.

View larger version (14K): [in this window] [in a new window]   Figure 6. Dose-response curves for levcromakalim action in WKY (), SHR (), and hydralazine-treated SHR (). Data are mean±SE from 21 to 32 cells. Slope conductance was obtained at potentials from -100 to -40 mV and was normalized by cell capacitance. Curves were drawn by fitting the data to the Michaelis-Menten equation: Slope Conductance=Bmax[drug]/([drug]+Kd), where Bmax is maximal slope conductance, [drug] is levcromakalim concentration, and Kd is the dissociation constant. From the curve fitting, Bmax values are 51.4, 34.0, and 45.8 pS/pF and Kd values are 0.50, 1.70, and 0.54 µmol/L for WKY, SHR, and hydralazine-treated SHR, respectively. *P<.05, **P<.01.

Long-term treatment of SHR with hydralazine shifted the dose-response relation for the effect of levcromakalim toward that of WKY; the estimated maximal slope conductance was 45.8 pS/pF and the K_d was 0.54 µmol/L (n=21) (Figs 5C and 6). Hydralazine applied acutely to single smooth muscle cells did not evoke significant currents (change in current conductance with 10 µmol/L hydralazine: SHR, 0.21±0.05 pS/pF, n=4; WKY, 0.27±0.06 pS/pF, n=4). Thus, antihypertensive treatment with hydralazine reduced blood pressure and attenuated the alteration in levcromakalim action on ATP-sensitive K⁺ channels in SHR.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
With the whole-cell voltage-clamp technique, we have shown that levcromakalim evoked the ATP-sensitive K⁺ channel current in smooth muscle cells from rat mesenteric arteries and that the sensitivity and responsiveness of ATP-sensitive K⁺ channels to levcromakalim were decreased in SHR compared with WKY. Antihypertensive treatment with hydralazine decreased blood pressure and attenuated the impairment of levcromakalim action in SHR.

Physiological and pharmacological characteristics of ATP-sensitive K⁺ channels have been examined in various vascular smooth muscle cells (rabbit portal vein, mesenteric artery, and pulmonary artery; rat portal vein; canine coronary artery)^{9 10 11 22 23 24} ; however, the properties of channels in rat arteries are less well characterized. These previous studies have shown that ATP-sensitive K⁺ channel currents are (1) inhibited by intracellular ATP, (2) time independent and voltage insensitive, and (3) sensitive to glibenclamide and 4-aminopyridine but insensitive to charybdotoxin. Furthermore, K⁺ channel openers have been shown to activate these same ATP-sensitive K⁺ channels.^{9 10 22 23 24} In the present study, levcromakalim evoked K⁺-selective currents that were time independent. The current-voltage relation was linear at less than -40 mV, suggesting that channel activation was voltage insensitive. The levcromakalim-evoked current was sensitive to glibenclamide but insensitive to charybdotoxin and low concentrations of tetraethylammonium. These characteristics indicate that the levcromakalim-evoked current in rat mesenteric artery cells is carried by ATP-sensitive K⁺ channels.

The dose-response curve for current activation by levcromakalim was shifted to the right in SHR relative to that in WKY (K_d was larger in SHR). This observation suggests that the sensitivity of ATP-sensitive K⁺ channels to K⁺ channel openers is decreased in hypertensive rats. A similar shift in the dose-response curve was observed for aprikalim-induced vasodilation in the basilar artery of SHRSP compared with WKY.¹⁷ Furthermore, in preliminary experiments with adult SHR and age-matched WKY, we have observed that levcromakalim in a dose-dependent manner relaxed mesenteric arteries that had been precontracted with 1 µmol/L phenylephrine and that the dose-response curve was shifted to the right in SHR compared with WKY (unpublished data, 1995). We consider that the impaired relaxation of the artery to K⁺ channel openers is due to the impaired membrane hyperpolarization. These alterations could be explained by the impaired activation of ATP-sensitive K⁺ channels by this drug. However, it is also possible that the membrane hyperpolarization by any hyperpolarizing stimulus might be impaired in arteries from hypertensive rats.

We have shown that the activation of ATP-sensitive K⁺ channels by levcromakalim is impaired in mesenteric arteries of SHR. Furspan and Webb²⁵ showed that the potencies of diazoxide in activating ATP-sensitive K⁺ channels in tail arteries were similar in SHRSP and WKY. This discrepancy with our data may be attributable to the difference in the arteries (tail artery of SHRSP versus mesenteric artery of SHR) or the drugs (diazoxide versus levcromakalim) studied.

In the present study, the maximal slope conductance normalized by the cell capacitance was smaller in SHR than in WKY, suggesting that the maximal response to levcromakalim was decreased in SHR. In contrast, the maximal dilation of the basilar artery by aprikalim was similar in SHRSP and WKY despite the decreased sensitivity to aprikalim action in SHRSP.¹⁷ In addition, the maximal hyperpolarization (to near the K⁺ equilibrium potential) of mesenteric arteries in response to 10 µmol/L cromakalim did not differ between SHR and WKY.²⁶ However, the similar maximal relaxation and hyperpolarization responses between WKY and SHR (or SHRSP) in these studies are not inconsistent with the present results, because the maximal relaxation or hyperpolarization does not necessarily require maximal channel opening. Indeed, complete relaxation of mesenteric arteries from WKY and SHR was observed at lower concentrations of levcromakalim (0.3 to 1 µmol/L) than those required for maximal channel opening (10 to 30 µmol/L).

Hydralazine relaxes the vascular bed and reduces blood pressure. In hydralazine-treated SHR, blood pressure did not differ significantly from that in WKY, and the action of levcromakalim on channel activation was not impaired. Prevention of the impairment in levcromakalim action was not attributable to a direct effect of hydralazine on K⁺ channels because hydralazine did not activate K⁺ channels when applied acutely to single cells and the drug was washed out during the isolation of single cells. In addition, we have recently observed that long-term administration of angiotensin-converting enzyme inhibitor and calcium antagonist also restored the vasodilating effect of K⁺ channel openers in SHR arteries (T. Nagao et al, unpublished data, 1995). It is thus likely that the impairment of levcromakalim action in SHR may be attributable to the sustained hypertension. However, we have not examined the effects of long-term treatment with hydralazine on the activation of ATP-sensitive K⁺ channels by levcromakalim in WKY. Thus, the possibility still exists that long-term hydralazine administration might directly restore the action of levcromakalim on ATP-sensitive K⁺ channels.

Levcromakalim effectively reduces the systemic blood pressure of hypertensive patients and animals.^{5 6} Thus, the impaired action of levcromakalim in the mesenteric artery of SHR in vitro does not appear to reflect the efficacy of this drug as an antihypertensive agent. One possible explanation for this discrepancy is that the vascular tone of hypertensive patients and animals may be more sensitive to changes in the membrane potential than that of normotensive controls. A second possible explanation is that levcromakalim action may not be impaired in resistance vessels; we examined only the main trunk of the mesenteric artery.

The physiological and pathophysiological relevance of the impaired action of levcromakalim on ATP-sensitive K⁺ channels in hypertension remains to be determined. However, if the dilatation of a certain artery to K⁺ channel openers was impaired in hypertensive animals, a concomitant decrease in systemic blood pressure by this drug might result in hypoperfusion of the tissue. In addition, if the vasodilating actions of endogenous K⁺ channel openers such as adenosine,²⁷ calcitonin gene–related peptide,²⁸ vasoactive intestinal peptide,⁸ and norepinephrine (ß-adrenergic stimulation)²⁸ are impaired as well as those of synthetic K⁺ channel openers, such a defect might contribute to the altered control of vascular tone in hypertensive patients and animals. Moreover, the present observation that antihypertensive treatment restored the effect of a K⁺ channel opener on ATP-sensitive K⁺ channels reinforces the importance of antihypertensive treatment in correcting the alteration in arterial smooth muscle cells.

In conclusion, the action of levcromakalim on ATP-sensitive K⁺ channels in mesenteric arteries was impaired in adult SHR compared with age-matched WKY. This impairment during chronic hypertension could be corrected by long-term antihypertensive treatment. It remains to be determined whether the receptor for K⁺ channel openers, ATP-sensitive K⁺ channels themselves, or the interaction between the two is altered in SHR.

	Acknowledgments

 
This work was supported by grants from the Ministry of Education, Science, and Culture, Japan (Nos. 06770497 and 07670788).

Received November 14, 1995; first decision December 18, 1995; accepted February 27, 1996.

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