(Hypertension. 1996;28:143-146.)
© 1996 American Heart Association, Inc.
Articles |
Altered Cerebrovascular Response to a Potassium Channel Opener in Hypertensive Rats
the Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Correspondence to Hitonori Takaba, MD, Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-82, Japan.
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Abstract |
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We examined whether the effect of Y-26763, an ATP-sensitive potassium channel opener, on cerebral blood flow is altered in stroke-prone spontaneously hypertensive rats (SHRSP) and, if altered, whether long-term antihypertensive treatment with cilazapril, an angiotensin-converting enzyme inhibitor, is capable of preventing the change. Cerebral blood flow during intracarotid infusion of Y-26763 was measured in anesthetized SHRSP and normotensive Wistar-Kyoto rats (WKY) as control. Y-26763 increased cerebral blood flow in a dose-dependent manner in WKY, and glibenclamide, a selective inhibitor of ATP-sensitive potassium channels, inhibited the Y-26763–induced increase in cerebral blood flow. In contrast, the response to Y-26763 in SHRSP was significantly impaired compared with that in WKY. Antihypertensive treatment with cilazapril lowered blood pressure toward normal and prevented the impaired response in cerebral blood flow to Y-26763 in SHRSP. These findings suggest that (1) ATP-sensitive potassium channels contribute to the regulation of cerebral blood flow in rats, (2) the response to an ATP-sensitive potassium channel opener is markedly diminished in hypertensive rats, and (3) the altered response to an ATP-sensitive potassium channel opener during chronic hypertension can be prevented by long-term antihypertensive treatment.
Key Words: potassium channels • hypertension, experimental • rats • cerebrovascular disorders • antihypertensive therapy
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Introduction |
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The activation of ATP-sensitive potassium channels hyperpolarizes and relaxes vascular smooth muscle.1 2 Synthetic activators of ATP-sensitive potassium channels relax cerebral arteries in vitro3 4 5 and dilate basilar arteries6 and cerebral arterioles7 8 9 in vivo. However, little is known about the effects of ATP-sensitive potassium channel openers on cerebral microcirculation and CBF. Our first goal in this study was to determine the fundamental effects of an ATP-sensitive potassium channel opener on CBF in rats. We examined whether intracarotid infusion of Y-26763, an ATP-sensitive potassium channel opener,10 modulates CBF.
Endothelium-dependent relaxation of vascular smooth muscle is impaired during chronic hypertension,11 12 13 14 but the treatment of hypertension restores endothelium-dependent relaxation toward normal in vitro11 15 16 and in vivo.17 In contrast, endothelium-independent vasodilator responses generally remain unaffected in hypertensive animals.12 15 18 19 In the case of ATP-sensitive potassium channels, the dilatation of the basilar artery in response to aprikalim, an opener of this class of potassium channel, is reported to be impaired in SHRSP in vivo.20 In clear contrast to this observation, the response to ATP-sensitive potassium channel openers is enhanced in carotid21 and tail22 arteries from SHRSP. Our second goal in this study was to examine whether the effect of Y-26763 on CBF is altered during chronic hypertension and if altered, whether long-term antihypertensive treatment with cilazapril, an ACE inhibitor, prevents the alternation in CBF in SHRSP.
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Methods |
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Animal Preparation
Experiments were conducted on female WKY (n=12) and SHRSP (n=14) that were bred in the Kyushu University Animal Center. At the age of 4 months, SHRSP were randomly divided into two groups: an untreated group that received tap water (n=7) and a treated group that received cilazapril (500 mg/kg) in the drinking water (n=7). Systolic pressure was measured with the tail-cuff method at 1-month intervals in SHRSP. WKY were allowed free access to tap water. Experiments were conducted when the rats were approximately 7 months of age.
Rats were anesthetized with amobarbital (100 mg/kg IP). After tracheotomy, rats were ventilated mechanically with room air. Skeletal muscle paralysis was produced with d-tubocurarine (4.5 mg/kg IV). Body temperature was maintained at 37°C with a heating pad. Catheters were placed in femoral arteries on both sides to measure systemic arterial pressure and obtain arterial blood samples. A femoral vein was cannulated for infusion of glibenclamide or vehicle. Arterial blood gases were monitored and maintained within normal limits throughout the experiments. Another catheter was inserted retrogradely through the right external carotid artery to the bifurcation of the right common carotid artery for infusion of Y-26763. The right external carotid artery was then ligated. Rats were mounted on a stereotaxic head-holder in a sphinx position. One burr hole (4 mm posterior and 2 mm lateral to the bregma, 5 mm in diameter) was made in the parietal bone, with the dura mater left intact, under an operating microscope for CBF measurement. CBF at the parietal cortex was continuously monitored with laser-Doppler flowmetry (Periflux PF3, Perimed) according to the method of Dirnagl et al.23
Experimental Protocol
We examined changes in CBF induced by intracarotid infusion of Y-26763. After more than 20 minutes of a stabilizing period, glibenclamide (20 mg/kg in 5 WKY) or vehicle (0.5 mL/kg dimethyl sulfoxide in the other rats) was injected intravenously. Thirty minutes after injection, Y-26763 was administered into the right internal carotid artery with a constant infusion pump (EP-60, Eicom Co) at rates of 0.1, 0.2, 0.4, 0.8, and 1.6 µg/kg per minute for 1 minute at 15-minute intervals. Because vehicle infusion at different rates did not change CBF, a fixed concentration of Y-26763 (25 mg/L) was infused at different rates to change the dose of Y-26763 administered. This was possible because the body weight of the rats varied little (230 to 270 g). In preliminary experiments, infusion of Y-26763 induced the following response in CBF (Fig 1
): CBF started to increase immediately after infusion, reaching the maximum within 1 minute. Thereafter, CBF fell gradually to baseline within a few minutes, exhibiting different decreasing patterns in each rat. In the following experiments, the initial peak (the maximal value) in CBF was determined as the change induced by Y-26763 because it should reflect the direct effect of Y-26763 on CBF. Y-26763 and glibenclamide were dissolved in dimethyl sulfoxide.
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Statistical Analysis
All values are expressed as mean±SE. One-way ANOVA followed by Scheffe's F test for significance was used for comparison of physiological variables. An unpaired t test was used for comparison of the change in systolic pressure between untreated and cilazapril-treated SHRSP. The Mann-Whitney U test and Kruskal-Wallis test were used for comparisons of the changes in CBF at a given dose of Y-26763. A value of P<.05 was considered statistically significant.
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Results |
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Long-term treatment with cilazapril decreased systolic pressure in SHRSP to a level similar to that in WKY throughout the experimental period (Fig 2
). Arterial blood gases and hematocrit were at constant levels, and no significant differences were observed among the groups throughout the experiment (Table).
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Intracarotid infusion of Y-26763 increased CBF in a dose-dependent manner in WKY (Fig 3). Systemic arterial blood pressure did not change significantly during infusion of Y-26763. In WKY, Y-26763 (1.6 µg/kg per minute) increased CBF by 77.3±9.9% of the baseline value (Fig 3). Glibenclamide had no effect on baseline CBF (data not shown) but inhibited the increase in CBF induced by Y-26763 (P<.01).
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In untreated SHRSP, the responses in CBF to Y-26763 were smaller than those in WKY (Fig 4). Y-26763 (0.4 and 0.8 µg/kg per minute) increased CBF by 20.4±1.7% and 58.2±13.9% of the baseline value in WKY but only by 8.3±1.5% and 8.3±4.6% in untreated SHRSP (P<.01 versus WKY). The response to the highest concentration of Y-26763 (1.6 µg/kg per minute), however, was similar in untreated SHRSP and WKY. In SHRSP treated with cilazapril, Y-26763 (0.4 and 0.8 µg/kg per minute) increased CBF by 18.5±2.0% and 42.3±9.2% of the baseline value (P<.01 and P<.05 versus untreated SHRSP) (Fig 4). Thus, long-term treatment with cilazapril prevented the impaired response to Y-26763.
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Discussion |
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There are three major new findings in the present study. First, intracarotid infusion of Y-26763 increased CBF in both WKY and SHRSP. Second, the Y-26763–induced increase in CBF was impaired in SHRSP. Thus, the activity of ATP-sensitive potassium channels may be altered during chronic hypertension. Third, long-term antihypertensive treatment with cilazapril prevented the impaired response to Y-26763 in SHRSP.
Role of ATP-Sensitive Potassium Channels in CBF Regulation
Y-26763 is a novel opener of ATP-sensitive potassium channels without any other known vasodilator mechanism.10 In the present study, intracarotid infusion of Y-26763 increased CBF without a significant change in systemic arterial pressure, and this response was inhibited by glibenclamide, a selective inhibitor of ATP-sensitive potassium channels.1 2 Thus, the increase in CBF produced by Y-26763 is likely to be mediated by the activation of ATP-sensitive potassium channels in cerebral arteries. Since Y-26763 is lipophilic and has a low molecular weight, it may cross the blood-brain barrier and reach the vascular muscle.
In most previous studies, the effect of ATP-sensitive potassium channel openers was examined with a cranial window technique (measuring arterial diameter)6 7 9 20 or an in vitro tension recording system.3 4 Although these methods provide valuable information, they have not clarified whether ATP-sensitive potassium channel openers increase CBF. In rats, isolated middle cerebral arteries are resistant to the vasorelaxant effects of ATP-sensitive potassium channel openers, including cromakalim, pinancidil, and nicorandil.4 Thus, Y-26763 probably increases CBF by activating ATP-sensitive potassium channels on small cerebral arteries or arterioles. Since glibenclamide did not affect baseline CBF, the channel may not participate in the determination of CBF at rest in rats.
It may be possible that Y-26763 acted on neurons and thereby increased CBF because ATP-sensitive potassium channels are reported to be present in neuronal cells.24 25 26 However, if Y-26763 activated ATP-sensitive potassium channels in neurons, the response would be inhibitory rather than excitatory because of hyperpolarization. Such an effect would result in a decrease rather than an increase in CBF. Hence, the Y-26763–induced increase in CBF is not likely to be a secondary effect on neurons.
Effects of Hypertension on ATP-Sensitive Potassium Channels
Dilatation of the basilar artery in response to aprikalim is impaired in SHRSP in vivo.20 The present study has confirmed and extended this previous finding. However, it can be argued that the impaired response in CBF to the ATP-sensitive potassium channel opener could be attributed to the inability of vascular smooth muscle to respond to any kind of vasodilator stimulus. We tested the effects of sodium nitroprusside and nilvadipine, a calcium antagonist, on CBF in our preparation. The agents showed little change in CBF, probably because of poor permeability to the blood-brain barrier. Accordingly, we have no direct evidence against general suppression of dilator capacity in cerebral arteries in SHRSP. However, previous studies12 20 27 confirmed that endothelium-independent vasodilator responses to nitrovasodilators and calcium antagonists are unaffected or even enhanced during hypertension both in vitro and in vivo. Furthermore, in patch-clamp experiments, currents activated by an ATP-sensitive potassium channel opener, levcromakalim, were significantly reduced in spontaneously hypertensive rats (SHR) compared with those in WKY, and this impairment in SHR could be prevented by long-term treatment with hydralazine.28 Therefore, impaired responses to Y-26763 are not likely to be due to nonspecific alternation of vascular smooth muscle during chronic hypertension. Another possible explanation for the altered cerebrovascular responses to Y-26763 is that the blood pressure levels just before application of Y-26763 could be a determining factor; the higher the blood pressure, the less vasodilation. However, the dilatation of isolated basilar arteries in response to levcromakalim is impaired in SHRSP (unpublished observation, 1996). Moreover, as mentioned above, in patch-clamp experiments, currents activated by levcromakalim were significantly reduced in SHR compared with those in WKY.28 Because the stretch imposed on the smooth muscle is similar in both rat strains in these in vitro experiments, impaired cerebral vasodilator responses to Y-26763 in the present study are not likely to be attributed to the level of arterial stretching (in other words, blood pressure) but rather to chronic hypertension. The precise mechanism that accounts for the impaired response to Y-26763 in SHRSP in the present study is not clear. It is possible that the number, function, or both of ATP-sensitive potassium channels are reduced in SHRSP. Alternatively, the binding of ATP-sensitive potassium channel openers to the channel and/or the subsequent channel opening mechanisms may be impaired in SHRSP.
In contrast to the present study, the response to ATP-sensitive potassium channel openers is reported to be enhanced in carotid21 and tail22 arteries from SHRSP. Although the discrepancy between these previous studies and the present study is not clear, methodological differences (in vitro versus in vivo), regional differences, and differences in the ages of examined rats could be contributing factors.
Effects of Antihypertensive Treatment on ATP-Sensitive Potassium Channels
Long-term treatment with cilazapril prevented the impaired response to Y-26763 in SHRSP. Some different mechanisms may account for this finding. First, it is possible that reduction in blood pressure per se contributes to the prevention. Second, some unique effects of ACE inhibitors unrelated to the reduction of blood pressure may have influenced the responsiveness of vascular smooth muscle to the activation of ATP-sensitive potassium channels. For example, treatment with cilazapril but not hydralazine prevents remodeling of pial arterioles in SHRSP.29 Furthermore, ACE inhibitors appear to affect the calcium influx through calcium channels in smooth muscle and thereby change vessel tone in hypertensive rats.30 These structural and functional changes by ACE inhibitors in the vessel wall may influence vascular responses. However, the latter explanation is less likely because the impaired response to levcromakalim in mesenteric arteries could be prevented not only by an ACE inhibitor but also by a calcium antagonist (unpublished observation, 1996) and hydralazine.28
Because impaired endothelium-dependent responses in SHRSP are restored to normal after short-term treatment with cilazapril,31 short-term treatment with cilazapril might also prevent impaired responses in CBF to an opener of ATP-sensitive potassium channels in SHRSP. This is of clinical interest and warrants further study.
In summary, ATP-sensitive potassium channels may contribute to the regulation of CBF in rats. In addition, the increase in CBF induced by Y-26763 is impaired in hypertensive rats. This altered activity of ATP-sensitive potassium channels during chronic hypertension can be prevented by long-term antihypertensive treatment with cilazapril.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Y-26763 was kindly provided by Yoshitomi Pharmaceutical Industries, Ltd, Fukuoka, Japan, and cilazapril by Nippon Roche KK, Tokyo, Japan.
Received September 20, 1995;
first decision October 19, 1995; first decision March 7, 1996;
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