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(Stroke. 1999;30:2713.)
© 1999 American Heart Association, Inc.

Original Contributions

Mitochondrial Potassium Channel Opener Diazoxide Preserves Neuronal-Vascular Function After Cerebral Ischemia in Newborn Pigs

Ferenc Domoki, MD; James V. Perciaccante, MD; Roland Veltkamp, MD; Ferenc Bari, PhD David W. Busija, PhD

From the Department of Physiology and Pharmacology (F.D., R.V., F.B., D.W.B.), Department of Pediatrics (J.V.P.), and Stroke Research Center (R.V.), Wake Forest University School of Medicine, Winston-Salem, NC; and Department of Physiology, Albert Szent-Györgyi Medical University, Szeged, Hungary (F.D., F.B.).

Correspondence to David W. Busija, PhD, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1010. E-mail dbusija{at}wfubmc.edu

	Abstract

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Background and Purpose—N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K_ATP) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K_ATP channels (mitoK_ATP) are involved in the ischemic preconditioning-like effect of K⁺ channel openers. We determined whether the selective mitoK_ATP channel opener diazoxide preserves the vascular dilation to NMDA after I/R.

Methods—Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 µmol/L diazoxide; and coapplication of 100 µmol/L 5-hydroxydecanoic acid (5-HD), a K_ATP antagonist with diazoxide.

Results—NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 µmol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 µmol/L NMDA–induced vasodilation after I/R was 53±19% (mean±SEM, n=8) in vehicle-treated controls versus 55±10%, 85±5%, and 99±15% in animals pretreated with 1, 5, and 10 µmol/L diazoxide (n=8, n=8, and n=12, respectively) and 60±15% in the group treated with 5-HD+diazoxide (n=5).

Conclusions—The mitoK_ATP channel opener diazoxide in vivo preserves neuronal function after I/R, shown by pial arteriolar responses to NMDA, in a dose-dependent manner. Thus, activation of mitoK_ATP channels may play a role in mediating the protective effect of other K⁺ channel openers.

Key Words: cerebral ischemia, global • N-methyl-D-aspartate • potassium channels • reperfusion injury • pigs

	Introduction

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Glutamate elicits cerebral arteriolar vasodilation in piglets via a multistep process, involving activation of neuronal N-methyl-D-aspartate (NMDA) receptors, stimulation of nitric oxide (NO) production by neuronal NO synthase, and actions of NO on vascular smooth muscle cells.^{1 2 3} This sequence of events may represent an important mechanism coupling local blood flow to metabolism and neuronal activity.

NMDA-induced vasodilation is attenuated by hypoxia and ischemia/reperfusion (I/R) in a dose- and time-dependent manner.^{4 5 6} For example, 10 minutes of global ischemia followed by reperfusion reduces NMDA-induced vasodilation by 50%. However, arteriolar dilator responses to exogenously applied NO are intact,^{5 6} thereby implying that the attenuation of the vascular response to NMDA is due to effects of ischemia at the level of the neurons. Furthermore, results from other laboratories as well as our own indicate that dysfunction of the NMDA receptor rather than of general neuronal injury is the primary reason for attenuated arteriolar responsiveness to NMDA.^{5 7} The mechanisms involved in attenuated arteriolar dilation to NMDA are not known with certainty but appear to involve actions of reactive oxygen species (ROS), such as superoxide anion. Thus, pharmacological agents that prevent production of superoxide anion or that scavenge this radical prevent attenuation of NMDA-induced dilator responses.^{4 5 8}

In our laboratory, NMDA-induced vasodilation has been used as a sensitive bioassay to assess the functional integrity of the neuronal-vascular axis. For instance, we have shown that activation of ATP-sensitive potassium (K_ATP) channels with aprikalim for a short period immediately before combined hypoxic/ischemic stress preserves pial arteriolar dilation to NMDA.⁶ Possible mechanisms of action of K_ATP activation may be via hyperpolarization of neurons through plasmolemmal K_ATP channels, which may result in (1) reduced glutamate release, (2) smaller increases in intracellular Ca²⁺ levels during ischemia, or possibly (3) less ROS production during reperfusion. However, intracellular sites of action of K⁺ channel activators have not been considered previously.

Mitochondrial K_ATP (mitoK_ATP) channels have been found in the inner membrane of mitochondria⁹ and represent a pharmacologically distinct population of K_ATP channels.¹⁰ There is increasing evidence about the diverse functions of mitoK_ATP channels in the regulation of mitochondrial matrix volume, ATP production, and Ca²⁺ homeostasis in mitochondria, essential factors determining the outcome of ischemic stress on cellular function and survival.^{11 12 13 14} In fact, several K⁺ channel openers can mimic ischemic preconditioning (IPC) in the heart,¹⁵ and mitoK_ATP channels are certainly involved in mediating these effects.^{16 17 18} However, no study has investigated the possible beneficial role of mitoK_ATP channel activation in vivo in the brain and the cerebral circulation.

In this study our purpose was to determine whether diazoxide, a selective mitoK_ATP channel opener, would preserve the NMDA-induced arteriolar dilation 1 hour after 10 minutes of global cerebral ischemia. Additionally, we investigated whether 5-hydroxydecanoic acid (5-HD), a relatively selective inhibitor of mitoK_ATP channels, would reduce the effect of diazoxide.

	Materials and Methods

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Animals
Newborn piglets of either sex (age, 1 to 7 days; body weight, 1 to 2 kg) were used. All procedures were approved by the Institutional Animal Care and Use Committee. The animals were anesthetized with sodium thiopental (30 to 40 mg/kg IP) followed by injection of -chloralose (75 mg/kg IV). Supplemental doses of -chloralose were given to maintain a stable level of anesthesia. The right femoral artery and vein were catheterized to record blood pressure and to administer drugs and fluids, respectively. The piglets were intubated via tracheotomy and artificially ventilated with room air. The ventilation rate (20/min) and tidal volume (20 mL) were adjusted to maintain arterial blood gas values and pH in the physiological range. Body temperature was maintained at 37°C to 38°C by a water-circulating heating pad. Body temperature, arterial pH, and blood gases were also in the normal ranges and did not vary significantly among different groups. For instance, in group 5, the values were as follows: body temperature, 37.9±0.2°C; pH, 7.51±0.03; PCO₂, 33.3±1.9 mm Hg; and PO₂, 97±4 mm Hg.

The head of the piglet was fixed in a stereotaxic frame. The scalp was incised and removed along with the connective tissue over the calvaria. A circular (19 mm in diameter) craniotomy was made in the left parietal bone. The dura was cut and reflected over the skull. A stainless steel cranial window with 3 needle ports was placed into the craniotomy, sealed with bone wax, and cemented with cyanoacrylate ester (Super Glue) and dental acrylic.

The closed window was filled with artificial cerebrospinal fluid (aCSF) warmed to 37°C and equilibrated with 6% O₂ and 6.5% CO₂ in balance N₂ to give pH=7.33, PCO₂=46 mm Hg, and PO₂=43 mm Hg. The aCSF consisted of the following (mmol/L): NaCl 132, KCl 2.9, CaCl₂ 1.2, MgCl₂ 1.4, NaHCO₃ 24.6, urea 6.7, and glucose 3.7. Diameters of pial arterioles were measured with a microscope (Wild M36) equipped with a video camera (Panasonic) and a video micro scaler (IV-550, For-A-Co). After surgery, the cranial window was gently perfused with aCSF until a stable baseline was obtained. At the end of the experiments, the animals were killed while anesthetized with an intravenous bolus of KCl.

Cerebral Ischemia
To induce global cerebral ischemia, a 3-mm hole was made by an electric drill with a toothless bit, and the dura was exposed. A hollow brass bolt was inserted in the left frontal cranium rostral to the cranial window and secured in place with cyanoacrylate ester and dental acrylic. Cerebral ischemia was produced by infusion of aCSF to raise intracranial pressure above arterial pressure. Ischemia was verified by the cessation of blood flow in the observed vessels. Previously, we have shown using microspheres that cerebral blood flow is virtually zero in all brain areas examined during the ischemic period.¹⁹ Venous blood was withdrawn as necessary to maintain mean arterial blood pressure near normal values. At the end of the ischemic period, the infusion tube was clamped, and the intracranial pressure returned to preischemic values. The heparinized blood was reinfused intravenously.

Experimental Design
After obtaining stable baseline arteriolar diameters, we examined the responses of cerebral arterioles to NMDA (10, 50, 100 µmol/L, except in group 7). NMDA and all other drugs were dissolved in aCSF and administered topically through the injectable ports of the cranial window onto the brain surface with single application. Arteriolar diameters were measured continuously for 5 to 7 minutes for each dose of NMDA. Then the window was flushed with aCSF, and the arteriolar diameters returned to baseline values. Instrumented piglets (n=49) were then divided into 7 groups, as follows.

Group 1 (n=4)
To assess whether diazoxide may have direct effect on NMDA-induced vasodilation, in the first group the animals were treated with 10 µmol/L diazoxide for 10 minutes but did not undergo ischemia. NMDA challenge was repeated 1 hour after treatment with diazoxide.

Group 2 (n=8)
To repeat our previous findings on attenuation of NMDA-induced vasodilation by I/R, in this group the piglets received vehicle (aCSF) and were exposed to 10 minutes of global cerebral ischemia followed by reperfusion. In all ischemia groups, NMDA-induced changes in pial arteriolar diameters were reexamined after the first hour of reperfusion. We have shown that attenuation of cerebral vasodilation to NMDA is greatest 1 hour after I/R (1 hour is also the shortest time after I/R at which the measurements are technically feasible).

Groups 3 to 5 (n=8, n=8, and n=12, Respectively)
To investigate the effect of diazoxide on preservation of NMDA-induced vasodilation, in these groups the piglets were pretreated with 1, 5, and 10 µmol/L diazoxide, respectively, for 10 minutes before the initiation of 10 minutes of global cerebral ischemia. The diazoxide was removed by flushing the window with aCSF just before the initiation of ischemia.

Group 6 (n=5)
To investigate the inhibitory effect of 5-HD on K_ATP channels activated by diazoxide, the piglets were pretreated with 100 µmol/L 5-HD for 5 minutes, followed by coapplication of 100 µmol/L 5-HD and 10 µmol/L diazoxide for 10 minutes before 10 minutes of ischemia. The diazoxide and 5-HD were removed by flushing the window with aCSF just before the initiation of ischemia.

Group 7 (n=4)
To study the effect of 5-HD on the sarcolemmal K_ATP channels and the vascular response to NMDA, we examined the cerebral arteriolar responses to the nonselective K_ATP channel opener aprikalim (10 µmol/L) followed by 100 µmol/L NMDA. Then we coapplied 10 µmol/L aprikalim and 100 µmol/L 5-HD for 10 minutes. We repeated the NMDA challenge 1 hour after pretreatment with 5-HD+aprikalim. Previously we have shown that aprikalim treatment does not affect the vascular response to NMDA.⁶ Between each drug application we flushed the window several times with aCSF, until arteriolar diameters returned to baseline values.

	Drugs

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The drugs used in this study were NMDA (Sigma), diazoxide (Sigma), 5-HD (H135, Research Biochemicals International), and aprikalim (Rhone-Roulenc-Rohrer).

	Statistical Analysis

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Data are expressed as mean±SEM. Pial arteriolar diameter data were analyzed with repeated-measures ANOVA, followed by pairwise comparisons using the Student-Newman-Keuls test when appropriate. Percent preservations of preischemic vasodilation data were analyzed with 1-tailed t test. P values of <0.05 were considered statistically significant.

	Results

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Arterial blood pressure was in the normal range and was not significantly different before and 1 hour after ischemia; for instance, in group 5 arterial pressure was 70±4 mm Hg before and 68±4 mm Hg after I/R (n=12).

Topical application of diazoxide did not affect pial vascular diameters significantly. Typically, there was only a transient dilation immediately on application of diazoxide. Percent changes from baseline diameters were as follows: group 3, no vasoactivity was observed; group 4, 2±1%; and group 5, 9±3%. Vascular diameters quickly returned to baseline values in 2 to 3 minutes, and none of these changes were significantly different from baseline values.

NMDA elicited dose-dependent pial arteriolar vasodilation (Figures 1 and 2). In group 1, 10 µmol/L diazoxide did not potentiate or attenuate vascular dilations to NMDA 1 hour after diazoxide treatment (Figure 1). Baseline arteriolar diameters were 100±2 µm before and 100±6 µm 1 hour after diazoxide treatment. Percent changes in pial arteriolar diameter from baseline to 10, 50, and 100 µmol/L NMDA (before versus 1 hour after diazoxide treatment) were 3±1% versus 4±1%, 28±7% versus 26±9%, and 50±8% versus 47±8%, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of 10 µmol/L diazoxide on pial arteriolar responses to NMDA. NMDA induced dose-dependent vasodilation that was unaffected 1 hour after topical application of diazoxide for 10 minutes (n=4). *P<0.05, significantly different from corresponding baseline diameter (base).

View larger version (22K): [in this window] [in a new window]   Figure 2. Changes in pial arteriolar diameters in response to NMDA 1 hour after 10 minutes of cerebral ischemia. Baseline diameters (base) did not change significantly in any groups after I/R. However, in the nontreated animals arteriolar responses to 50 and 100 µmol/L NMDA were severely reduced by 50%. Pretreatment with 1 µmol/L diazoxide (diaz) did not affect the reduction in NMDA-induced vascular dilation by I/R. In contrast, pretreatment with 5 or 10 µmol/L diazoxide resulted in preserved vascular responses; the changes in pial arteriolar diameters were not significantly different compared with preischemic values. Coapplication of 100 µmol/L 5-HD, a relatively specific inhibitor of mitoKATP channels with 10 µmol/L diazoxide attenuated the protective effect of diazoxide. *P<0.05, significantly different from corresponding preischemic value.

Global cerebral ischemia (10 minutes) followed by reperfusion significantly reduced pial arteriolar responses to NMDA (Figure 2). In group 2, baseline arteriolar diameters were 100±3 µm before and 103±4 µm 1 hour after ischemia. Percent changes in pial arteriolar diameter from baseline to 10, 50, and 100 µmol/L NMDA (before versus 1 hour after ischemia) were 6±2% versus 2±1%, 28±5% versus 9±3%, and 38±5% versus 16±4%, respectively. Thus, vascular dilations to 100 µmol/L NMDA were diminished by 50% (Figure 3).

View larger version (33K): [in this window] [in a new window]   Figure 3. Protective effect of diazoxide (diaz) on 100 µmol/L NMDA–induced pial arteriolar dilation. Data are expressed as percent preservation of dilation compared with preischemic values. Note the dose-dependent preservation of vascular responses in the diazoxide-treated groups; at 10 µmol/L diazoxide the vascular response was virtually identical to preischemic value. 5-HD antagonized the effect of diazoxide. *Significantly different from preischemic value.

Diazoxide exhibited a dose-dependent effect on preservation of NMDA-induced vasodilation after I/R. In group 3, decreases in pial arterial responsiveness to NMDA were similar to those observed in group 2 (Figures 2 and 3). In group 3, baseline arteriolar diameters were 102±3 µm before and 104±3 µm 1 hour after ischemia. Percent changes in pial arteriolar diameter from baseline to 10, 50, and 100 µmol/L NMDA (before versus 1 hour after ischemia) were 5±2% versus 3±1%, 20±7% versus 8±2%, and 38±5% versus 19±3%, respectively. In contrast, in groups 4 and 5 we found a dose-dependent preservation of pial vascular responses to NMDA (Figures 2 and 3). More specifically, in group 4, baseline arteriolar diameters were 95±3 µm before and 95±4 µm 1 hour after ischemia. Percent changes in pial arteriolar diameter from baseline to 10, 50, and 100 µmol/L NMDA (before versus 1 hour after ischemia) were 4±0% versus 7±2%, 30±10% versus 23±6%, and 45±6% versus 37±3%, respectively. In group 5, baseline arteriolar diameters were 102±6 µm before and 106±5 µm 1 hour after ischemia. Percent changes in pial arteriolar diameter from baseline to 10, 50, and 100 µmol/L NMDA (before versus 1 hour after ischemia) were 7±1% versus 6±2%, 28±5% versus 24±4%, and 36±5% versus 32±4%, respectively. Therefore, pretreatment with 10 µmol/L diazoxide resulted in virtually full preservation of pial arteriolar responses to NMDA 1 hour after I/R compared with preischemic values.

Topical application of the K_ATP channel antagonist 5-HD and coapplication of 5-HD with diazoxide did not alter pial arteriolar diameters. In addition, 5-HD treatment did not affect pial arteriolar responses to NMDA. In group 7, baseline arteriolar diameters were 105±9 µm before and 103±7 µm 1 hour after pretreatment with 5-HD. Percent changes in pial arteriolar diameter from baseline to 100 µmol/L NMDA (before versus 1 hour after 5-HD treatment) were 52±3% versus 56±7%. However, pretreatment with 5-HD and diazoxide abolished the protection on NMDA-induced vasodilation achieved by diazoxide alone (Figures 2 and 3). In group 6, baseline arteriolar diameters were 90±6 µm before and 92±6 µm 1 hour after ischemia. Percent changes in pial arteriolar diameter from baseline to 10, 50, and 100 µmol/L NMDA (before versus 1 hour after ischemia) were 3±1% versus 0±0%, 40±12% versus 19±6%, and 61±7% versus 33±5%, respectively. Interestingly, coapplication of 5-HD with aprikalim did not block the vasodilation elicited by aprikalim. In group 7, pial baseline arteriolar diameters were 102±8 µm before application of aprikalim alone and 101±7 µm before coapplication of aprikalim and 5-HD. Percent changes in pial arteriolar diameter from baseline to 10 µmol/L aprikalim were 65±6% versus 65±6% (aprikalim alone versus aprikalim+5-HD, respectively).

	Discussion

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The major finding of the present study is that the selective mitoK_ATP channel opener diazoxide dose-dependently preserves NMDA-induced cerebral arteriolar vasodilation after I/R in piglets. Since NMDA-induced vasodilation is dependent on intact neuronal function, we present evidence for the first time showing an in vivo protective effect of diazoxide after I/R in the central nervous system.

Previously, we found that the nonselective K_ATP channel opener aprikalim protected NMDA-induced vasodilation after combined hypoxia-ischemia.⁶ The protective effect of aprikalim was shown to be mediated by neuronal rather than vascular K_ATP channels and was independent of the vasodilation elicited by aprikalim. Our present data confirm that the protective effect of pretreatment with K⁺ channel openers is independent of vasodilation accompanied by the administration of such drugs: diazoxide showed no significant vasoactivity but preserved NMDA-induced dilation. The beneficial effects of K_ATP channel openers reducing injury by I/R have been most extensively studied in the heart. K_ATP channels serve as the final common pathway in the event of IPC, a phenomenon in which short periods of ischemia protect the heart from subsequent exposure of a more prolonged period of ischemia. K⁺ channel openers mimic IPC,^{15 16 17 18} and the protection by IPC is blocked by K_ATP channel inhibitors.^{20 21 22} The exact mechanism of this remarkable effect has not been elucidated.

The discovery of mitoK_ATP channels added further complexity to the interpretation of experimental data from pharmacological interventions on these channels. Unfortunately, there are no absolutely selective pharmacological tools to assess the mitoK_ATP channels in vivo. However, a consistent and unique feature of these channels is their remarkably selective sensitivity to opening by diazoxide. The mitoK_ATP channel was found to be >2000-fold more sensitive to diazoxide than the sarcolemmal K_ATP channel in bovine cardiac myocytes (K_1/2 was 0.4 µmol/L for mitoK_ATP channel versus 855 µmol/L for sarcolemmal K_ATP channel). In contrast, cromakalim was an equally potent opener of both mitochondrial and plasma membranes.¹⁰ Subsequently, mitoK_ATP channel selective concentrations (5 to 20 µmol/L) of diazoxide have been demonstrated to improve functional recovery in isolated rat hearts after I/R in a manner similar to that of a nonselective K_ATP channel opener, cromakalim. The cardioprotection by diazoxide was inhibited by K_ATP channel inhibitors glibenclamide and 5-HD, confirming the effect of diazoxide via K_ATP channels.¹⁷ In a different study, in intact rabbit ventricular myocytes, diazoxide induced mitochondrial depolarization, demonstrated by flavoprotein fluorescence with a K_1/2 of 27 µmol/L, but did not affect the simultaneously measured sarcolemmal K_ATP channel current.¹⁶ These findings and others in the literature (for recent review, see Reference 15¹⁵ ) strongly indicate the involvement of mitoK_ATP channels in the development of acute and perhaps delayed IPC in the heart.

In our present experiments we used topical diazoxide (1 to 10 µmol/L) in the mitoK_ATP channel–selective dose range. We did not test directly whether only mitoK_ATP channels were activated by diazoxide, but fortunately a good indication of selective activation was the absence of significant vasodilation accompanied by application of diazoxide. The vasodilatory effect of K⁺ channel openers on cerebral arterioles was directly mediated by the sarcolemmal K⁺ channels. Administration of 5 to 10 µmol/L diazoxide elicited only 2% to 9% arteriolar dilation, and the response was transient, ie, it did not last for >1 to 2 minutes. In contrast, we found that the nonselective K_ATP channel opener aprikalim (10 µmol/L) elicits 60% to 70% increases in vascular diameters, and the vasodilation does not wane. Moreover, the dose-dependent effect of diazoxide on preservation of the NMDA-induced vasodilation after I/R was inhibited by the selective K_ATP channel antagonist 5-HD, and 5-HD was found to be selective for mitoK_ATP channels, at least in some experimental designs.^{16 17 23} Additionally, in our experimental model 5-HD did not inhibit the vasodilation induced by aprikalim, suggesting minor effects on plasmolemmal K_ATP channel channels. These observations, together with those of the literature, lead us to conclude that the protective effect of diazoxide on neuronal-vascular function after I/R is probably mediated by activation of mitoK_ATP channels.

The mechanism by which activation of mitoK_ATP channels may lead to increased resistance to I/R remains to be clarified. In our experimental model, NMDA-induced vascular response is severely attenuated at 1 hour after I/R, and responsiveness gradually returns over the time course of 2 to 4 hours.^{5 24} The duration of global cerebral ischemia (10 minutes) used in the present study has been thought to cause only reversible mitochondrial alterations, ie, mitochondria have been shown to recover full function 1 to 2 hours after reperfusion.^{25 26} Thus, the attenuation of the NMDA-mediated cerebral arteriolar response is not likely due to energy failure by inhibited mitochondrial function. This statement is further supported by our previous findings that kainate-induced vasodilation is resistant to ischemia in the same experimental model.²⁷ Additionally, neuronal NO synthase levels and activity are unchanged by I/R, and cerebral arterioles show normal responses to exogenous NO donors such as sodium nitroprusside after ischemia.^{5 6} Therefore, the primary target of I/R may be the NMDA receptor itself. The acute effect of ischemia on NMDA-induced pial arteriolar vasodilation has been amply demonstrated to be mediated by ROS (Figure 4). Thus, NMDA-induced vascular response has been found to be preserved by ROS scavengers and inhibitors of cyclooxygenase (COX) activity,^{4 5 8 24} a major source of ROS after I/R.²⁸ Our recent observations on the preservation of neural function with K⁺ channel openers after hypoxia-ischemia were somewhat at odds with the general scheme of the pathological mechanism of the effect of I/R on NMDA-induced neuronal-vascular sequence. However, our present results may link the beneficial effect of K⁺ channel openers on preservation of NMDA-induced vasodilation to reducing oxidative stress on the neurons involved in this response. We speculate that activation of mitoK_ATP channels by K⁺ channel openers may reduce mitochondrial ROS production.

View larger version (26K): [in this window] [in a new window]   Figure 4. Preservation of NMDA-induced cerebral vasodilation after I/R. The most sensitive component of this neuronal-vascular sequence to I/R may be the NMDA receptor itself. ROS produced by mitochondria and COX seem to play a pivotal role in attenuating the NMDA-induced vasodilation. Thus, pretreatment with ROS scavengers, the COX inhibitor indomethacin, or the protein (including COX) synthesis inhibitor cycloheximide shortly before I/R results in preserved vascular responsiveness to NMDA. NMDA-induced dilation is also preserved by pretreatment with diazoxide, a selective mitoKATP channel opener, and other K+ channel openers (KCO-s). Activation of mitoKATP channels may reduce generation of ROS in reperfusion. Reduction in either COX-derived or mitochondrial ROS production may be sufficient to preserve the vascular response to NMDA after I/R. However, a potentiating interaction between these sources of ROS is conceivable but unknown.

Currently, the physiological role of mitoK_ATP channels is still debated and mostly speculative. Briefly, mitoK_ATP channels seem to control the activity of the electron transport chain via regulating mitochondrial matrix volume by regulated K⁺ uptake. The physiological patterns of activation and inhibition of these channels are largely unknown, but ironically the physiological role of ATP as a regulator is unlikely.²⁹ In isolated mitochondria, K⁺ channel openers induce slight swelling, partially dissipate the transmembrane potential (, negative inside), but increase the activity of electron transport chain and hence the chemical proton gradient (pH, alkaline inside); thus, the total protonmotive hardly changes.^{10 11 12 13 14} However, the activity of numerous important transport mechanisms depends on either or pH. One such possibly crucial "metabolite" may be Ca²⁺. Mitochondria readily uptake Ca²⁺ when intracellular levels increase above a so-called mitochondrial buffer concentration. Ca²⁺ is transported through the mitochondrial inner membrane via the electrogenic Ca²⁺ uniporter down its electrochemical gradient, and thus the rate of this transport is dependent on .³⁰ Mitochondrial Ca²⁺ overload substantially influences the recovery of mitochondrial function after ischemic stress: for example, increased mitochondrial Ca²⁺ sequestration has been demonstrated to increase production of ROS.^{31 32} Opening of mitoK_ATP channels should decrease mitochondrial Ca²⁺ uptake by decreasing , and in fact K⁺ channel openers induce release of Ca²⁺ from Ca²⁺-preloaded mitochondria in vitro.¹¹

In summary, we conclude that diazoxide in a mitoK_ATP channel–selective range dose-dependently preserves neuronal function demonstrated by NMDA-induced arteriolar dilation after I/R. This acute effect of mitoK_ATP channel openers may be mediated by decreasing mitochondrial ROS production in the immediate reperfusion. This effect may be important in the protective effect of other nonspecific K⁺ channel openers as well. Our findings may offer the development of new therapies to reduce neuronal injury after global hypoxic-ischemic stress in the newborn.

	Acknowledgments

 
This study was supported by grants HL-30260, HL-46558, and HL-50587 from the National Institutes of Health and by grants from the T-026295 OTKA and ETT-T-07614/97 (Hungary). We gratefully acknowledge that aprikalim was a gift from Rhone-Roulenc-Rohrer.

Received June 16, 1999; revision received August 2, 1999; accepted August 31, 1999.

	References

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1. Busija DW, Leffler CW. Dilator effects of amino acid neurotransmitters on piglet pial arterioles. Am J Physiol. 1989;257:H1200–H1203.[Abstract/Free Full Text]
2. Meng W, Tobin JR, Busija DW. Glutamate-induced cerebral vasodilation is mediated by nitric oxide through N-methyl-D-aspartate receptors. Stroke. 1995;26:857–862.[Abstract/Free Full Text]
3. Faraci FM, Breese KR. Nitric oxide mediates vasodilatation in response to activation of N-methyl-D-aspartate receptors in brain. Circ Res. 1993;72:476–480.[Abstract/Free Full Text]
4. Bari F, Errico RA, Louis TM, Busija DW. Differential effects of short-term hypoxia and hypercapnia on N-methyl-D-aspartate–induced cerebral vasodilatation in piglets. Stroke. 1996;27:1634–1639.[Abstract/Free Full Text]
5. Busija DW, Meng W, Bari F, McGough PS, Errico RA, Tobin JR, Louis TM. Effects of ischemia on cerebrovascular responses to N-methyl-D-aspartate in piglets. Am J Physiol. 1996;270:H1225–H1230.[Abstract/Free Full Text]
6. Veltkamp R, Domoki F, Bari F, Busija DW. Potassium channel activators protect the N-methyl-D-aspartate–induced cerebral vascular dilation after combined hypoxia and ischemia in piglets. Stroke. 1998;29:837–843.[Abstract/Free Full Text]
7. Hoffman DJ, McGowan JE, Marro PJ, Mishra OP, Delivoria-Papadopoulos M. Hypoxia-induced modification of the N-methyl-D-aspartate receptor in the brain of the newborn piglet. Neurosci Lett. 1994;167:156–160.[Medline] [Order article via Infotrieve]
8. Pourcyrous M, Leffler CW, Bada HS, Korones SB, Busija DW. Brain superoxide anion generation in asphyxiated piglets and the effect of indomethacin at therapeutic dose. Pediatr Res. 1993;34:366–369.[Medline] [Order article via Infotrieve]
9. Inoue I, Nagase H, Kishi K, Higuti T. ATP-sensitive K⁺ channel in the mitochondrial inner membrane. Nature. 1991;352:244–247.[Medline] [Order article via Infotrieve]
10. Garlid KD, Paucek P, Yarov-Yarovoy V, Xiaocheng Sun, Schindler PA. The mitochondrial K_ATP channel as a receptor for potassium channel openers. J Biol Chem. 1996;271:8796–8799.[Abstract/Free Full Text]
11. Holmuhamedov EL, Jovanovic S, Dzeja PP, Jovanovic A, Terzic A. Mitochondrial ATP-sensitive K⁺ channels modulate cardiac mitochondrial function. Am J Physiol. 1998;275:H1567–H1576.[Abstract/Free Full Text]
12. Garlid KD. Cation transport in mitochondria. Biochim Biophys Acta. 1996;1275:123–126.[Medline] [Order article via Infotrieve]
13. Halestrap AP. Regulation of mitochondrial metabolism through changes in matrix volume. Biochem Soc Trans. 1994;22:522–529.[Medline] [Order article via Infotrieve]
14. Szewczyk A, Czyz A, Wojcik G, Wojczak L, Nalecz M. ATP-regulated K⁺ channel in mitochondria: pharmacology and function. J Bioenerg Biomembr. 1996;28:147–152.[Medline] [Order article via Infotrieve]
15. Gross GJ, Fryer R. Sarcolemmal versus mitochondrial ATP-sensitive K⁺ channels and myocardial preconditioning. Circ Res. 1999;84:973–979.[Abstract/Free Full Text]
16. Liu Y, Sato T, O’Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation. 1998;97:2463–2469.[Abstract/Free Full Text]
17. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K⁺ channels: possible mechanism of cardioprotection. Circ Res. 1997;81:1072–1082.[Abstract/Free Full Text]
18. Baines CP, Liu GS, Birincioglu M, Critz SD, Cohen MV, Downey JM. Ischemic preconditioning depends on interaction between mitochondrial K_ATP channels and actin cytoskeleton. Am J Physiol. 1999;276:H1361–H1368.[Abstract/Free Full Text]
19. Beasley TC, Bari F, Thore C, Thrikawala N, Louis TM, Busija DW. Cerebral ischemia/reperfusion increases endothelial nitric oxide synthase levels by an indomethacin-sensitive mechanism. J Cereb Blood Flow Metab. 1998;18:88–96.[Medline] [Order article via Infotrieve]
20. Auchampach J, Grover G, Gross G. Blockade of ischemic preconditioning in dogs by the novel ATP-dependent potassium channel antagonist sodium 5-hydroxydecanoate. Cardiovasc Res. 1992;26:1054–1062.[Abstract/Free Full Text]
21. Gross G, Auchampach J. Blockade of the ATP-sensitive potassium channels prevents myocardial preconditioning. Circ Res. 1992;70:223–233.[Abstract/Free Full Text]
22. Schulz R, Rose J, Heusch G. Involvement of activation of ATP-dependent potassium channels in ischemic preconditioning in swine. Am J Physiol. 1994;267:H1341–H1352.[Abstract/Free Full Text]
23. Sato T, O’Rourke B, Marban E. Modulation of mitochondrial ATP-dependent K⁺ channels by protein kinase C. Circ Res. 1998;83:110–114.[Abstract/Free Full Text]
24. Veltkamp R, Domoki F, Bari F, Louis TM, Busija DW. Inhibitors of protein synthesis preserve the N-methyl-D-aspartate–induced cerebral arteriolar dilation after ischemia in piglets. Stroke. 1999;30:148–152.[Abstract/Free Full Text]
25. Rechncrona S, Mela L, Siesjo BK. Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia. Stroke. 1979;10:437–446.[Free Full Text]
26. Sims NR. Selective impairment of respiration in mitochondria isolated from brain subregions following transient forebrain ischemia in the rat. J Neurochem. 1991;56:1836–1844.[Medline] [Order article via Infotrieve]
27. Bari F, Louis TM, Busija DW. Kainate-induced cerebrovascular dilation is resistant to ischemia in piglets. Stroke. 1997;28:1272–1276.[Abstract/Free Full Text]
28. Armstead WM, Mirro R, Busija DW, Leffler CW. Postischemic generation of superoxide anion by newborn pig brain. Am J Physiol. 1988;255:H401–H403.[Abstract/Free Full Text]
29. Paucek P, Yarov-Yarovoy V, Xiaocheng S, Garlid KD. Inhibition of the mitochondrial K_ATP channel by long chain acyl-CoA esters and activation by guanine nucleotides. J Biol Chem. 1996;271:32084–32088.[Abstract/Free Full Text]
30. Gunter TE, Gunter KK, Sheu S-S, Gavin CE. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol. 1994;267:C313–C339.[Abstract/Free Full Text]
31. Dugan LL, Sensi SL, Canzoniero LMT, Handran SD, Rothman SM, Lin T-S, Goldberg MP, Choi DW. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J Neurosci. 1995;15:6377–6388.[Abstract/Free Full Text]
32. Dykens JA. Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca²⁺ and Na⁺: implications for neurodegeneration. J Neurochem. 1994;63:584–591.[Medline] [Order article via Infotrieve]

Editorial Comment

Robert M. Bryan, Jr, PhD, Guest Editor

Department of Anesthesiology, Baylor College of Medicine, Houston, Texas

	Introduction

Top Abstract Introduction Materials and Methods Drugs Statistical Analysis Results Discussion References Introduction  References

 
The protection of tissues and organs, after I/R and other pathological conditions, is an extremely important but often disappointing area of investigation. Such protection is of particular importance in heart and brain, vital organs that are most vulnerable to I/R. Interestingly, a brief activation of K_ATP before an ischemic event has been reported to provide a reasonable degree of tissue protection.¹ Although the mechanism behind this protective effect of K_ATP stimulation was not known, a reasonable hypothesis stated that protection occurred through hyperpolarizing the plasma membrane. Hyperpolarization would reduce the probability and/or the duration of an action potential. Protection would be afforded in 2 ways. First, the energy requirement would be reduced at a time when energy supply in the form of ATP is likely to be compromised. Second, a decrease in the number and/or duration of action potentials would reduce influx of Ca⁺² into the cell and help to prevent intracellular Ca⁺² concentrations from reaching toxic levels.

However, several studies in heart demonstrated that the abbreviation of action potentials could be dissociated from protection conferred by K_ATP channel openers.^{2 3 4 5 6} With this knowledge in mind and the discovery of mitochondrial K_ATP channels, attention was turned away from the sarcolemma to K_ATP channels in the mitochondria. Indeed, a number of studies in heart strongly support the hypothesis that K_ATP openers confer protection through K_ATP channels located not on the sarcolemma membrane but on the inner mitochondrial membrane.^{2 4}

Would the same hold true for cerebral protection after I/R? Could this be demonstrated in vivo? Domoki and colleagues asked these questions in the study in the accompanying article. Convincingly, these authors showed that prior stimulation of mitochondrial K_ATP channels, not plasma membrane K_ATP channels, are involved with cerebral protection (as measured by preservation of dilator responses to NMDA) after I/R in the newborn pig. In their studies the authors reported that the selective mitochondrial K_ATP opener diazoxide restored dilator responses to NMDA that had been diminished by I/R. Furthermore, the selective mitochondrial K_ATP blocker 5-HD antagonized the protective effects of diazoxide. Although selectivity of these 2 agents had been demonstrated in heart,¹ there was no guarantee that this selectivity automatically transferred to brain. In a series of cleverly designed studies, Domoki et al demonstrated that diazoxide produced only minor and transient dilations (5% dilation that lasted 2 to 3 minutes) of the pial arteries, whereas stimulation of K_ATP channels in the plasma membrane (by aprikalim) produced large (65% dilation) and sustained dilations. Hence, diazoxide had practically no effect on dilation and thus plasma membrane K_ATP channels. Second, 5-HD had no effect on dilations elicited by opening K_ATP channels in the plasma membrane with aprikalim. If 5-HD was not blocking K_ATP channels in the plasma membrane, then it was reasonable to conclude that it was selectively blocking K_ATP channels in the mitochondria.

This study by Domoki and colleagues has 2 important implications. First, it demonstrates the importance of mitochondria in the developing pathophysiology after I/R. Second, this study provides a potentially important therapy for treatment of stroke in humans. Cerebral protection by openers of mitochondrial K_ATP channels and the study of mitochondria in the pathophysiology of cerebral insults promise to be important frontiers for future research.

Received June 16, 1999; revision received August 2, 1999; accepted August 31, 1999.

	References

Top Abstract Introduction Materials and Methods Drugs Statistical Analysis Results Discussion References Introduction  References

 

1. Szewczyk A, Marban E. Mitochondria: a new target for K channel openers? Trends Pharmacol Sci.. 1999;20:157–161.[Medline] [Order article via Infotrieve]
2. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K⁺ channels: possible mechanism of cardioprotection. Circ Res.. 1997;81:1072–1082.
3. Grover GJ, D’Alonzo AJ, Dzwonczyk S, Parham CS, Darbenzio RB. Preconditioning is not abolished by the delayed rectifier K⁺ blocker dofetilide. Am J Physiol.. 1996;271:H1207–H1214.[Abstract/Free Full Text]
4. Grover GJ, D’Alonzo AJ, Hess T, Sleph PG, Darbenzio RB. Glyburide-reversible cardioprotective effect of BMS-180448 is independent of action potential shortening. Cardiovasc Res.. 1995;30:731–738.[Medline] [Order article via Infotrieve]
5. Grover GJ, D’Alonzo AJ, Parham CS, Darbenzio RB. Cardioprotection with the KATP opener cromakalim is not correlated with ischemic myocardial action potential duration. J Cardiovasc Pharmacol.. 1995;26:145–152.[Medline] [Order article via Infotrieve]
6. Yao Z, Gross GJ. Effects of the KATP channel opener bimakalim on coronary blood flow, monophasic action potential duration, and infarct size in dogs. Circulation.. 1994;89:1769–1775.[Abstract/Free Full Text]

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