Age-Related Changes in Response of Brain Stem Vessels to Opening of ATP-Sensitive Potassium Channels
Background and Purpose This study was designed to determine regional differences and age-related changes in the contribution of ATP-sensitive potassium (KATP) channels to vasodilator responses in the brain stem circulation in vivo.
Methods Changes in diameter of the basilar artery (baseline diameter, 270±5 μm [mean±SEM]), its large branch (112±5 μm), and its small branch (49±2 μm) in response to KATP channel openers levcromakalim and Y-26763 were measured through a cranial window in anesthetized adult (4 to 6 months) and aged (24 to 26 months) Sprague-Dawley rats.
Results Topical application of levcromakalim and Y-26763 produced concentration-dependent vasodilation that was similar among the three vessel groups in adult rats. In aged rats, dilator responses of the branches, but not of the basilar artery, to the KATP channel openers were smaller than those in adult rats (P<.05). Glibenclamide, a selective KATP channel blocker, almost abolished this vasodilation in both groups of rats. Vasodilator responses to sodium nitroprusside were preserved in aged rats.
Conclusions In adult rats, there is no regional heterogeneity in vasodilator response to KATP channel openers in the brain stem circulation in vivo. In aged rats, although KATP channels are also functional in the brain stem circulation, dilator response of the microvessels but not of the large arteries to direct activation of KATP channels is impaired.
ATP-sensitive potassium (KATP) channels are present in both large cerebral arteries and arterioles, and opening of the channels is a major mechanism for cerebral vasodilation,1 2 3 4 as well as for an increase in cerebral blood flow.5 KATP channels may be unevenly distributed in cerebral circulation because openers of these channels fail to dilate some cerebral vessels in vitro.6 7 We have recently reported that there is a marked heterogeneity in the sensitivity of levcromakalim, an opener of KATP channels, in the rabbit vertebrobasilar system in vitro.8 It is not known, however, whether such heterogeneity results from the difference in vessel size. The first goal of this study was to determine whether dilator responses of the basilar artery and its branches to levcromakalim and Y-26763,9 both KATP channel openers, vary with vessel size in vivo. For this purpose, we used an open cranial window that permitted observation of several vessels with different diameters of the brain stem simultaneously.10
Dilator responses of cerebral vessels to activation of KATP channels appear to be impaired during several pathological conditions,4 ie, hypertension,5 11 diabetes mellitus,12 13 and hypercholesterolemia.14 Aging is another condition that causes alterations of cerebral vasodilator responses to several vasoactive stimuli.15 16 17 Thus, we anticipated that aging might also alter dilator responses of the cerebral arteries to KATP channel openers in vivo. The second goal of this study was to test the hypothesis that dilatation of the basilar artery and its branches in response to levcromakalim and Y-26763 is also impaired in aged rats compared with younger ones.
Materials and Methods
Experiments were performed in male Sprague-Dawley rats aged 4 to 6 months (adult, n=14; 513±24 g [mean±SEM]) and 24 to 26 months (aged, n=14; 655±19 g). Rats had been maintained in the Animal Research Center at Kyushu Dental College. Rats were anesthetized with 50 mg/kg IP amobarbital sodium followed by additional doses of 15 to 20 mg·kg−1·h−1 IV. The depth of anesthesia was examined often by observing the effects of pressure to the paw on heart rate and blood pressure. The trachea was cannulated, and each rat was mechanically ventilated with room air and supplemental oxygen after immobilization with 5 to 10 mg/kg d-tubocurarine chloride. Catheters were placed in both femoral arteries for the measurement of systemic arterial pressure (124±3 mm Hg in adult rats and 116±3 mm Hg in aged rats, P>.05) and for sampling of arterial blood. Levels of arterial pH, blood gases, and hematocrit were all within normal limits and did not differ among the subgroups (data not shown). A femoral vein was cannulated for the infusion of drugs. Rectal temperature was maintained at 37°C with a heating pad.
A craniotomy was prepared over the ventral brain stem as previously described in detail.10 18 19 Portions of the dura and pia mater and the arachnoid membrane were resected. The open cranial window was suffused with artificial CSF (composition in mmol/L: 132 NaCl, 2.95 KCl, 1.71 CaCl2, 0.65 MgCl2, 24.6 NaHCO3, and 3.69 d-glucose), warmed to 37°C, and continuously bubbled with a gas mixture of 5% CO2-95% N2. Suffusion rate of CSF using inlet and outlet ports over the exposed brain stem was 3.0 mL/min. In CSF sampled from the craniotomies, Pco2 was 35.6±0.3 mm Hg, Po2 was 126±3 mm Hg, and pH was 7.43±0.01. The diameters of the basilar artery, a large branch directly originating from the basilar artery with a baseline diameter ≥70 μm, and a small branch with a baseline diameter <70 μm were measured in each rat using a microscope equipped with a television camera coupled to a video monitor and width analyzer (C3161, Hamamatsu Photonics). Baseline diameter of the basilar artery was 262±8 μm in adult rats and 278±5 μm in aged ones (P>.05); diameter in the large branch was 106±6 μm and 118±7 μm (P>.05) and in the small branch 49±3 μm and 49±3 μm (P>.05), respectively. The images were recorded on videotape for later analysis.
We examined responses of the vessels to topical application of levcromakalim (10−7 to 10−5.5 mol/L) and sodium nitroprusside (10−8 to 10−6 mol/L) in 7 adult and 7 aged rats. Both agonists were dissolved in dimethyl sulfoxide (<0.05%), further diluted by artificial CSF, and suffused over the craniotomy for 5 minutes. Vessel diameters were measured immediately before application of each agonist and during the last minute of application. After application of each agonist, 10 minutes of washout was allowed before vessel diameters returned to baseline values. The application sequence of agonists was randomized. Finally, the effect of levcromakalim (10−6 mol/L) was tested in the presence of glibenclamide (10−6 mol/L), a selective KATP channel blocker. Glibenclamide was dissolved in dimethyl sulfoxide (<0.05%) further diluted by artificial CSF, and suffusion was started 10 minutes before application of levcromakalim.
We also examined responses of the vessels to topical application of Y-26763 (10−7.5 to 10−6 mol/L) and sodium nitroprusside (10−8 to 10−6 mol/L) in another 7 adult and 7 aged rats. Y-26763 was dissolved in dimethyl sulfoxide (<0.05%), further diluted by artificial CSF, and suffused over the craniotomy for 5 minutes. The effect of Y-26763 (10−6 mol/L) was also tested in the presence of glibenclamide (10−6 mol/L).
Values are expressed as mean±SEM. Two-way repeated measures ANOVA was used to compare whole concentration-response curves among three groups with different vessel size and between adult and aged rat groups. When a significant F value was found, post hoc analysis among the groups was made with Fisher's protected least significant difference test. An unpaired t test was used to compare responses in each concentration step, as well as baseline values between adult and aged rats. A value of P<.05 was accepted as statistically significant.
Responses to Levcromakalim in Adult Rats
Levcromakalim (10−7 to 10−5.5 mol/L) produced concentration-dependent increases in diameter of the vessels in adult rats (Fig 1⇓). Changes in diameters were similar among the three groups with different vessel size, with a maximum response of 52±6% in the basilar artery, 45±8% in the large branch, and 48±7% in the small branch (at 10−5.5 mol/L). Application of glibenclamide (10−6 mol/L) alone did not alter diameters of vessels of any size but almost abolished levcromakalim (10−6 mol/L)–induced dilatation of the vessels (data not shown).
Responses to Levcromakalim in Aged Rats
Levcromakalim (10−7 to 10−5.5 mol/L) also produced concentration-dependent dilatation of the vessels in aged rats, with a maximum response of 53±7% in the basilar artery, 33±8% in the large branch, and 35±7% in the small branch (at 10−5.5 mol/L) (Fig 2⇓). However, the response of the small branch was significantly smaller in aged rats than in adult rats (P<.05 by two-way ANOVA). Responses of the basilar artery and large branch to levcromakalim were not significantly different between adult and aged rats by two-way ANOVA. Comparison of concentration-response curves among three groups with different vessel size in aged rats also showed the significant difference between the basilar-artery and small-branch groups (P<.05 by two-way ANOVA). Glibenclamide (10−6 mol/L) alone did not alter diameters of vessels of any size but almost abolished levcromakalim (10−6 mol/L)–induced dilatation in aged rats (data not shown).
Responses to Y-26763 in Adult and Aged Rats
Y-26763 (10−7.5 to 10−6 mol/L) produced concentration-dependent increases in diameter of the vessels in adult rats (Fig 3⇓). Changes in diameters were similar among the three groups with different vessel size, with a maximum response of 59±5% in the basilar artery, 54±11% in the large branch, and 56±6% in the small branch (at 10−6 mol/L).
Y-26763 (10−7.5 to 10−6 mol/L) also produced concentration-dependent dilatation of the vessels in aged rats, with a maximum response of 42±7% in the basilar artery, 22±5% in the large branch, and 23±3% in the small branch (at 10−6 mol/L). The response of the large and small branches was significantly smaller in aged rats than in adult rats (P<.01 for the large branch and P<.02 for the small branch by two-way ANOVA), but that of the basilar artery was not significantly different between adult and aged rats. Comparison of concentration-response curves among three groups with different vessel size in aged rats showed significant differences between the basilar artery and each branch group (P<.05 by two-way ANOVA). Glibenclamide (10−6 mol/L) almost abolished Y-26763 (10−6 mol/L)–induced dilatation of the vessels both in adult and aged rats (data not shown).
Responses to Sodium Nitroprusside
Sodium nitroprusside (10−8 to 10−6 mol/L) increased diameters of the vessels in a concentration-dependent manner, and the concentration-response curves were similar between adult and aged rats irrespective of the vessel size in both the former and latter experiments; the former findings are shown in Fig 4⇓.
There are two major new findings in the present study. First, both levcromakalim and Y-26763, KATP channel openers, dilated the basilar artery and its branches to the same extent in adult rats, and the responses were virtually abolished by glibenclamide, a selective KATP channel blocker. Thus, there is no regional heterogeneity in vasodilator response to KATP channel openers in the brain stem circulation in vivo, at least in adult rats. Second, although levcromakalim and Y-26763 also produced glibenclamide-sensitive dilatation of the basilar artery and its branches in aged rats, the dilator response of the small branch (diameter of <70 μm) to levcromakalim and those of the large and small branches to Y-26763 were markedly impaired. In contrast, nitroprusside-induced vasodilation did not alter with aging. Thus, aging, or strictly speaking, maturation or senility, appears to attenuate the response to opening of KATP channels selectively in small vessels in the brain stem circulation in vivo.
Heterogeneity of the Distribution in the Function of KATP Channels
The vasodilator effect of KATP channel openers appears to be heterogenous along cerebral arteries in vitro. Cromakalim, a KATP channel opener, relaxes rat basilar7 and superior cerebellar arteries20 but not posterior cerebral arteries.7 Rabbit vertebral arteries were much more sensitive to levcromakalim than were superior cerebellar arteries.8 These findings presumably result from the difference in the density of KATP channels in these arteries. The vessels examined in the present study had a wide distribution of diameter, with a more than fivefold difference between the basilar artery and small branch. However, their responses to KATP channel openers were similar in adult rats. Thus, regional differences demonstrated in previous studies in the function of KATP channels in cerebral vessels may not be due to the difference in vessel size.
Age-Related Changes in Responses to KATP Channel Openers
Aging does not alter cerebral vasodilator responses to nitroglycerin16 or sodium nitroprusside,21 whereas it attenuates some responses to endothelium-dependent agonists such as acetylcholine, ATP, and bradykinin.15 16 17 These changes in endothelium-dependent vasodilation presumably result from morphological and functional alterations of the endothelium by aging.22 The present study demonstrated that cerebral vasodilator responses to KATP channel openers are impaired in small vessels in aged rats. In contrast, Faraci and Heistad21 reported that topical application of aprikalim, a KATP channel opener, at 10−6 and 10−5 mol/L dilated parietal arterioles similarly in adult and aged Fischer 344 rats. Differences in the strains of rats, agonists, examined vessels, and number of examined steps of agonist concentration may have produced discrepancies between their study results and ours. The concentration-response curve to KATP channel openers appears to be very steep in a certain narrow range of the drug concentration, and accordingly, narrow concentration steps are desirable for the experiment.
The diminished vasodilator response to KATP channel openers in aged rats is not due to nonspecific impairment of vasodilation because their responses to nitroprusside were preserved. Our results suggest that number or affinity of KATP channels in small vessels is decreased by aging. Alternatively, the binding of KATP channel openers to the channel or subsequent channel opening mechanisms may be impaired with aging.
Dilator responses of cerebral vessels are impaired in several pathological conditions. Dilatation of the basilar artery in response to aprikalim is impaired in spontaneously hypertensive rats in vivo.11 Y-26763 increased parietal cerebral blood flow in normotensive rats, whereas this increase was markedly diminished in spontaneously hypertensive rats.5 Thus, chronic hypertension appears to attenuate the function of KATP channels in large cerebral arteries as well as in microvessels. In rats with streptozotocin-induced diabetes, aprikalim-induced dilatation was impaired in both the basilar artery and parietal arterioles, but the impairment seemed to be greater in small vessels.12 13 The present study is unique in that cerebral vasodilator responses to KATP channel openers were impaired selectively in small vessels in aged rats. Thus, maturation or senility also alters cerebral vasodilator responses to KATP channel openers in a different manner according to the vessel size.
Consideration of Methods
Male Sprague-Dawley rats have been reported to have a median life span between 19.5 and 30 months, most often between 23.5 and 25.5 months.23 Thus, rats aged 24 to 26 months in this study are in the senile stage. The different findings observed between rats aged 4 to 6 months and aged 24 to 26 months therefore reflect alteration of cerebrovascular responses during aging or, strictly speaking, during maturation or senility.24 However, we could not accurately determine from the present findings whether these changes occurred during maturation or senility.
Dilator responses of the large or small vessels to agonists may affect those of other small or large vessels, and these effects may limit the interpretation of the findings. We consider, however, these interactions to be inevitable and important characteristics of the study in vivo. In addition, in the present study, it is unlikely that changes in pressure gradient along the basilar artery during the applications of the drugs produced different responses in the distal vessels between adult and aged rats because dilatation of the basilar artery was similar between the two groups.
In summary, size-dependent impairment of dilator responses of the brain stem vessels to opening of KATP channels was demonstrated in aged rats. Recently, KATP channels have been reported to be important modulators of cerebral blood flow autoregulation; eg, topical application of glibenclamide disturbed the autoregulatory dilatation of parietal pial arterioles of rats during hypotension.25 26 Cerebral vasodilator responses to hypoxia also seem to be mediated by activation of KATP channels.3 27 Thus, we speculate that the impaired responses of the brain stem vessels to KATP channel openers during aging may predispose the aged to cerebral ischemia and perhaps stroke under several physiological stimuli such as hypotension and hypoxia.
This study was supported by grant-in-aid 08770463 for scientific research from the Japanese Ministry of Education, Science, and Culture. Levcromakalim and Y-26763 were kindly provided by Yoshitomi Pharmaceuticals. The authors are grateful to the staffs in the Animal Research Center and Department of Dental Anesthesiology at Kyushu Dental College for technical advice.
- Received June 24, 1996.
- Revision received September 16, 1996.
- Accepted October 9, 1996.
- Copyright © 1997 by American Heart Association
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Change in activity of potassium channels is a major mechanism that regulates vascular tone.1R 2R The list of vasoactive stimuli that have been reported to relax cerebral vessels by mechanisms that involve activation of potassium channels is long and growing; it includes hypoxia, hypercapnia, reductions in blood pressure, nitric oxide, receptor-mediated agonists (vasoactive intestinal peptide, calcitonin gene–related peptide, adrenomedullin, prostacyclin, opioids, adenosine, norepinephrine), reactive oxygen species, 11,12-epoxyeicosatrienoic acid, and second messengers (cAMP and cGMP).3R 4R 5R 6R 7R
Aging is associated with functional changes in cerebral blood vessels, including impairment of endothelium-dependent relaxation and vasodilatation during hypercapnia.8R The present study examined the hypothesis that aging is also associated with impairment of dilatation of brain stem vessels to two activators of potassium channels. Dilatation of the basilar artery and its branches in response to activators of potassium channels was almost completely inhibited by glibenclamide, which suggests that activation of KATP channels mediated the response. The major finding of the study was that dilatation of branches of the basilar artery in response to activators of KATP channels, but not to nitroprusside, was markedly impaired in aged rats. These findings suggest that aging is associated with selective impairment of dilatation in response to activation of KATP channels in small vessels that supply the brain stem.
What are the implications of these findings? Activation of potassium channels appears to contribute to a variety of important regulatory mechanisms in the cerebral circulation. These regulatory mechanisms include autoregulatory, neurohumoral, and endothelium-dependent responses of cerebral blood vessels. Because resistance of small vessels in brain stem is greater than resistance of small vessels in cerebrum under normal conditions,9R impairment of function of potassium channels in small vessels may have greater consequences in brain stem. Impairment of such a key mechanism of vasodilatation may predispose to ischemia during aging.
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Kitazono T, Faraci FM, Taguchi H, Heistad DD. Role of potassium channels in cerebral blood vessels. Stroke. 1995;26:1713-1723.
Faraci FM, Sobey CG. Potassium channels and the cerebral circulation. Clin Exp Pharmacol Physiol. In press.
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Paternò R, Faraci FM, Heistad DD. Role of Ca2+-dependent K+ channels in cerebral vasodilatation induced by increases in cyclic GMP and cyclic AMP in the rat. Stroke.. 1996;27:1603-1608.
Lang MG, Paternò R, Faraci FM, Heistad DD. Mechanisms of adrenomedullin-induced dilatation of cerebral arterioles. Stroke. 1997;28:181-185.
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Faraci FM, Mayhan WG, Heistad DD. Segmental vascular responses to acute hypertension in cerebrum and brain stem. Am J Physiol.. 1987;252:H738-H742.