From the Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000
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ABSTRACT |
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The mitochondrial KATP channel (mitoKATP) is hypothesized to be the receptor for the cardioprotective effects of K+ channel openers (KCO) and for the blocking of cardioprotection by glyburide and 5-hydroxydecanoate (5-HD). Studies on glyburide have indicated that this drug is inactive in isolated mitochondria. No studies of the effects of 5-HD on isolated mitochondria have been reported. This paper examines the effects of glyburide and 5-HD on K+ flux in isolated, respiring mitochondria. We show that mitoKATP is completely insensitive to glyburide and 5-HD under the experimental conditions in which the open state of the channel is induced by the absence of ATP and Mg2+. On the other hand, mitoKATP became highly sensitive to glyburide and 5-HD when the open state was induced by Mg2+, ATP, and a physiological opener, such as GTP, or a pharmacological opener, such as diazoxide. In these open states, glyburide (K1/2 values 1-6 µM) and 5-HD (K1/2 values 45-75 µM) inhibited specific, mitoKATP-mediated K+ flux in both heart and liver mitochondria from rat. These results are consistent with a role for mitoKATP in cardioprotection and show that different open states of mitoKATP, although catalyzing identical K+ fluxes, exhibit very different susceptibilities to channel inhibitors.
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INTRODUCTION |
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During steady-state respiration, K+ influx into mitochondria is balanced by K+ efflux on the K+/H+ antiporter, and steady-state volume is maintained. Opening the mitochondrial KATP channel (mitoKATP)1 will increase K+ influx into mitochondria and shift matrix volume to a higher steady state. The energetic costs of this futile cycle are small, between 100-150 nmol of H+/mg of protein·min at 25 °C, and we have concluded that the sole function of mitoKATP is to regulate matrix volume (1). It has been suggested that matrix expansion secondary to mitoKATP opening plays an important role in cell-signaling pathways calling for activation of electron transport and stimulation of fatty acid oxidation (2).
A pharmacological role for mitoKATP in cardiac ischemia seems clearer. During prolonged cardiac ischemia, myocyte ATP levels fall, and the heart does not survive reperfusion. Either pretreatment with K+ channel openers (KCO) or preconditioning with a brief period of ischemia protects the heart; during subsequent ischemia, ATP loss is reduced, and the heart recovers to nearly normal function upon reperfusion (3). Importantly, cardioprotection by either KCO or preconditioning is blocked by glyburide and 5-HD. This set of agents, KCO, glyburide, and 5-HD, identifies the receptor as a KATP channel, and pharmacological studies indicate that mitoKATP is the receptor for these effects (4).
A major problem with this hypothesis has been that glyburide appears to
be ineffective as a specific inhibitor of K+ flux in
intact, respiring mitochondria (5). Nonspecific inhibition of
K+ flux does occur (5); however, we have attributed this
effect, which occurs at high doses of glyburide, to low affinity
reactions with key energy-transducing enzymes (6-8). The effect of
these nonspecific actions is to reduce , the driving force for
K+ uptake, and has nothing to do with mitoKATP.
We have now verified this conclusion in experiments that examine the
effects of glyburide on both respiration and respiration-driven cation
uptake into mitochondria.
Failure to inhibit mitoKATP seemed to be a property of both glyburide and 5-HD. We recognized, however, that these drugs had only been studied under conditions when no other ligands of mitoKATP were present, a condition that never obtains in vivo. We now report that glyburide and 5-HD are potent blockers of K+ flux through mitoKATP in open states in which Mg2+, ATP, and physiological (GTP) or pharmacological (KCO) openers are present. In intact rat heart mitochondria, K1/2 values for glyburide and 5-HD are about 1 and 50 µM, respectively. We infer that susceptibility to glyburide and 5-HD requires a ligand-induced conformational change in the mitochondrial sulfonylurea receptor (mitoSUR). These results are consistent with a role for mitoKATP in cardioprotection (4, 9).2
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EXPERIMENTAL PROCEDURES |
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Preparations-- Rat liver mitochondria were prepared according to Pedersen et al. (10), and rat heart mitochondria were prepared by the Glass-TeflonTM homogenization procedure according to Matlib et al. (11). The final mitochondrial pellet was washed and resuspended at 50 mg/ml (liver) or 20 mg/ml (heart) in 0.22 M mannitol, 0.07 M sucrose, and potassium salts of 5 mM TES and 0.5 mM EGTA. Mitochondria were kept on ice at pH 7.2 during the experiments. MitoKATP was purified and reconstituted as described previously (12).
Assay of Ion Transport in Intact
Mitochondria--
K+ or TEA+ uptake was
assayed by following swelling, which accompanies net salt transport,
using previously established light-scattering techniques (13, 14).
Reciprocal absorbance (A-1) at 520 nm varies
linearly with matrix volume within three well defined regions,
separated by transitions at 115 and 68 milliosmolal (13). is a
dimensionless parameter that normalizes A-1 for
mitochondrial protein concentration, P (mg/ml),
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(Eq. 1) |
Measurement of Respiration-- Respiration was measured with a Yellow Springs oxygen electrode assembly in a hypotonic assay medium identical to the one used for measurement of K+ uptake, or in an isotonic assay medium consisting of K+ salts of chloride (120 mM), succinate (10 mM), phosphate (5 mM), TES (5 mM), and MgCl2 (0.1 mM), pH 7.4, supplemented with rotenone (2 µg/mg of protein). Mitochondria were assayed at 1 mg of protein/ml at 25 °C.
Assay of K+ Flux in Liposomes-- MitoKATP was partially purified from rat liver mitochondria and reconstituted into proteoliposomes loaded with PBFI according to previously published protocols (15). Internal medium contained TEA+ salts of sulfate (100 mM), EGTA (1 mM), and HEPES (25 mM) at pH 6.8. 15 µl of stock proteoliposomes (50 mg of phospholipids/ml) were added to 2 ml of external medium containing 150 mM KCl and TEA+ salts of EGTA (1 mM) and HEPES (25 mM), at pH 7.4. Temperature was maintained at 25 °C during assays. Electrophoretic K+ flux was initiated by the addition of 1 µM FCCP to provide charge compensation and measured from changes in PBFI fluorescence (12).
Chemicals and Reagents-- PBFI was obtained from Molecular Probes, Inc.; HEPES was obtained from Calbiochem; 5-HD was obtained from Research Biochemicals Inc.; and other drugs and chemicals were obtained from Sigma. The Tris salt of ATP was titrated to pH 7.2 with Tris base.
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RESULTS |
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Specific and Nonspecific Cation Flux in Respiring Heart Mitochondria-- Respiring mitochondria take up K+ by nonspecific and specific mechanisms, i.e. via diffusion and via mitoKATP. We used TEA+ to distinguish between these two parallel mechanisms, as demonstrated by the four traces in Fig. 1A. K+ flux (trace a) is greater than TEA+ flux (trace b). Addition of ATP inhibits K+ flux (trace c) but not TEA+ flux (trace d). These results, which are routinely observed in rat heart (Fig. 1) and liver (5) mitochondria, permit the following conclusions: (i) TEA+ is transported solely by diffusive leak (16) and is not transported by mitoKATP; and (ii) in the presence of ATP, K+ is transported solely by diffusion, and its flux equals that of TEA+. Thus, TEA+ flux may be used as a control for the component of K+ flux due to leak.
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Nonspecific Effects of Glyburide on K+ Flux in
Mitochondria--
In massive doses, glyburide does inhibit
K+ flux, as previously reported by us (5) and by Belyaeva
et al. (17) and Szewczyk et al. (18).
We concluded, however, that this effect was secondary to inhibition of
respiration (5). Cation uptake into respiring mitochondria is
exquisitely sensitive to (16), and it is essential to
differentiate between nonspecific inhibition due to reduced driving
force and specific inhibition of mitoKATP. Accordingly, we
examined the effect of glyburide on uncoupled respiration, on
respiration-driven K+ and TEA+ uptake, and on
respiration-driven K+ uptake in the presence of
valinomycin.
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Nonspecific Effects of 5-HD on K+ Flux in Mitochondria-- An identical series of experiments was carried out using 5-HD. 5-HD had no effect on K+ flux under these conditions. In contrast to glyburide, 5-HD did not inhibit uncoupled respiration or cation uptake in either rat liver or heart mitochondria up to 500 µM (data not shown).
Open States in which Glyburide and 5-HD Specifically Inhibit Rat Heart MitoKATP-- Under the conditions of Fig. 1, mitoKATP is open because no inhibitory ligands are present. In vivo, mitoKATP would be opened by pharmacological agents (KCO), such as diazoxide or cromakalim (15), or physiological ligands, such as GTP (12). Moreover, ATP and Mg2+ would also be present in vivo. When mitochondria were studied under these more physiological conditions, glyburide and 5-HD were potent, specific inhibitors of K+ flux, as illustrated by the traces in Fig. 4. Control K+ flux (Fig. 4, trace a) was inhibited in the presence of ATP and Mg2+ (Fig. 4, trace b) and then restored to control values by diazoxide (Fig. 4, trace c). This pharmacologically induced K+ flux was strongly inhibited by 10 µM glyburide to the level associated with ATP inhibition (Fig. 4, trace d). Note that this glyburide dose had no effect on respiration in ascorbate/TMPD and no effect on TEA+ flux (see Fig. 3A). Therefore, the effect is specific for K+ flux through mitoKATP.
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Open States in Which Glyburide and 5-HD Specifically Inhibit Rat Liver MitoKATP-- An identical series of experiments was carried out with rat liver mitochondria, and the results were qualitatively identical to the results with rat heart mitochondria. We were also able to compare the effect of substrates on glyburide potency in liver mitochondria because the margin of safety for nonspecific inhibition during respiration on succinate is much wider in liver than in heart mitochondria (compare Fig. 3, A and B). The results are summarized in Table I. The K1/2 for glyburide inhibition was 3.4 µM when either succinate or ascorbate/TMPD was the respiratory substrate. 5-HD also inhibited cromakalim-opened mitoKATP in liver mitochondria respiring on either succinate or ascorbate/TMPD with equal potency (K1/2 = 73 µM).
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Specific Inhibition of Reconstituted MitoKATP by Glyburide and 5-HD-- Table I also summarizes the effects of glyburide and 5-HD on reconstituted mitoKATP purified from rat liver mitochondria. In the presence of Mg2+, ATP, and cromakalim, both glyburide and 5-HD inhibited mitoKATP. Glyburide inhibited with K1/2 = 90 nM, and 5-HD inhibited with K1/2 = 85 µM. In the absence of ATP, 5-HD had no effect, but glyburide inhibited K+ flux with K1/2 = 250 nM. The latter result is consistent with our previous results on reconstituted mitoKATP in the presence of Mg2+ (19). Glyburide was also a potent inhibitor (K1/2 = 80 nM) of the reconstituted mitoKATP in the presence of Mg2+, ATP, and GTP.
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DISCUSSION |
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There is growing evidence for the hypothesis that mitoKATP is the receptor for the cardioprotective actions of K+ channel openers and the cardio-damaging actions of glyburide and 5-HD (4, 9). Glyburide is a prototypical sulfonylurea inhibitor that acts on all KATP channels and blocks the protective effects of both KCO and cardiac preconditioning (20, 21). 5-HD, which is structurally unrelated to glyburide, has been characterized as an ischemia-selective inhibitor of KATP channels (22, 23). Like glyburide, 5-HD selectively blocks the protective effects of both KCO (22) and cardiac preconditioning (24, 25). There have been no previous studies of the effects of 5-HD on mitochondria. The hypothesis that mitoKATP is involved in cardiac protection has been clouded by the lack of a crucial piece of evidence: a convincing demonstration that glyburide and 5-HD inhibit ATP-dependent K+ uptake in intact mitochondria.
MitoKATP activity can readily be elicited in respiring
mitochondria, as demonstrated by the data in Fig. 1. We showed
previously that glyburide inhibits K+ flux under these
conditions; however, inhibition required high doses that inhibit
respiration in the presence of succinate, and we concluded that this
inhibition was nonspecific (5). Belyaeva et al. (17)
concluded to the contrary that mitoKATP was specifically inhibited by 150 µM glyburide. To resolve this
disagreement, we carried out a thorough study of the nonspecific
effects of glyburide, as reported under "Results." We found that
glyburide is a potent inhibitor of uncoupled respiration in both liver
and heart mitochondria when succinate was used as the respiratory
substrate (Fig. 2). Over the same dose range, glyburide also inhibited
diffusive K+ and TEA+ fluxes (Fig. 3).
TEA+ is transported solely by leak pathways; accordingly,
glyburide inhibited K+ and TEA+ fluxes by
depression of and not by inhibition of mitoKATP. Thus, glyburide inhibition of K+ flux under these
conditions is nonspecific.
Respiration using ascorbate/TMPD is relatively insensitive to inhibition by glyburide and 5-HD, and the flux experiments using these substrates (Fig. 3) permit a further conclusion: glyburide and 5-HD, at any dose, are completely ineffective under the conditions that have routinely been used to study inhibition of K+ flux through mitoKATP, namely respiration on succinate with rotenone.
These findings presented a serious obstacle to studies of the pharmacological regulation of mitoKATP in mitochondria. We finally recognized, however, that the conditions routinely used, namely, in the absence of other ligands of mitoKATP, are far from those present in vivo. In a living cell, the open channel would never be exposed to glyburide under such conditions. Rather, the channel would be exposed to ATP and Mg2+ and then opened by GTP or a K+ channel opener (12, 15). Indeed, when the in vitro experiments were adjusted to mimic these in vivo conditions, we found glyburide and 5-HD to be potent, specific blockers of K+ flux in the open states induced by physiological or pharmacological ligands (Figs. 4 and 5 and Table I). We emphasize that three components, Mg2+, ATP, and a physiological or pharmacological opener, were required to achieve inhibition by either of these drugs. No single component nor any combination of two components was sufficient. This phenomenon was observed in both heart and liver mitochondria.
Results with reconstituted mitoKATP qualitatively reflect results in intact mitochondria (Table I). With glyburide, the K1/2 value was reduced when studied in the pharmacological open state. With 5-HD, no inhibition was observed in the absence of ATP, but 5-HD inhibited in the pharmacological open state, when ATP and a KCO were present.
Some aspects of these findings raise new scientific questions. (i) What renders mitoKATP susceptible to these inhibitors in one open state and not the other? In view of the fact that K+ fluxes are identical in all open states, we conclude that the protein must be in a different state. Thus, we infer that Mg2+, ATP, and an opener induce a conformation in mitoSUR that renders it susceptible to glyburide and 5-HD. (ii) Why is glyburide a potent inhibitor of reconstituted mitoKATP under conditions in which it is ineffective in intact mitochondria? The logical inference is that mitochondria are regulated by a factor that is lost during reconstitution. These hypotheses are under investigation.
In conclusion, our results are consistent with the hypothesis that mitoKATP is the essential drug receptor involved in ischemic cardioprotection. They also remove the previous deterrent to pharmacological studies on mitoKATP in intact mitochondria.
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ACKNOWLEDGEMENTS |
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We thank Yuliya Yarova-Yarovaya and Craig Semrad for excellent technical assistance.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grant GM55324 (to K. D. G.) from the National Institutes of General Medical Sciences and by a National Scientist Development Grant 9630004N (to P. P.) from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was in partial fulfillment of requirements for the Ph.D.
degree.
§ To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, P. O. Box 91000, Portland, OR 97291-1000. Tel.: 503-690-1680; Fax: 503-690-1464; E-mail: garlid{at}bmb.ogi.edu.
1 The abbreviations used are: mitoKATP, mitochondrial ATP-sensitive potassium channel; KCO, potassium channel opener(s); 5-HD, 5-hydroxydecanoate; PBFI, potassium-binding benzofuran isophthalate; TEA+, tetraethylammonium cation; TES, N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid; TMPD, N,N,N,N'-tetramethyl-p-phenylenediamine; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; CCCP, carbonyl cyanide p-chlorophenylhydrazone; mitoSUR, mitochondrial sulfonylurea receptor.
2 A preliminary account of these findings was reported in abstract form (26).
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REFERENCES |
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