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Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca²⁺ homeostasis under metabolic stress

NENAD JOVANOVI, SOFIJA JOVANOVI, ALEKSANDAR JOVANOVI and ANDRE TERZIC¹

Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA

¹Correspondence: Guggenheim-7F, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905, USA. E-mail: terzic.andre{at}mayo.edu

	ABSTRACT

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Metabolic injury is a complex process affecting various tissues, with intracellular Ca²⁺ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP-sensitive K⁺ (K_ATP) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca²⁺ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode K_ATP channel subunits, into a somatic cell line lacking native K_ATP channels. Hypoxia-reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca²⁺ loading was induced by Ca²⁺ influx during hypoxia and release of Ca²⁺ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the K_ATP channel activator pinacidil, prevented intracellular Ca²⁺ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of K_ATP channel, 5-hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca²⁺ homeostasis under hypoxia-reoxygenation irrespective of the stage of the metabolic insult.—Jovanovi, N., Jovanovi, S., Jovanovi, A., Terzic, A. Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca²⁺ homeostasis under metabolic stress.

Key Words: K_ATP channels • ischemia • gene therapy • potassium channel opener

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A COMMON FORM of insult that affects metabolically active cells is reoxygenation that follows hypoxia. Such metabolic stress initiates a series of cellular reactions leading to cell injury (1) . Although the mechanism of hypoxia-reoxygenation injury is complex, an increase in the intracellular levels of Ca²⁺ has been recognized as a trigger of events that ultimately lead to cell death (1, 2) . Thus, successful handling of intracellular Ca²⁺ homeostasis is essential to protect cells against hypoxia-reoxygenation injury (2, 3) . However, conventional approaches developed so far have shown only limited efficacy, and further identification of novel mechanisms of cytoprotection is in order.

The ATP-sensitive K⁺ (K_ATP)² channel is a heteromultimer composed of a pore-forming K⁺ channel core, Kir6.2, and a regulating ATP binding cassette protein, SUR (4, 5) . Recombinant Kir6.2 physically associates with SUR to reconstitute the properties of native K_ATP channels, including sensing of the cellular metabolic state (5 6 7 8 9 10 11 12) . Opening of native or recombinant K_ATP channels under metabolic stress has been implicated in promoting cellular survival (13 14 15) . More recently, we have observed that cells overexpressing recombinant K_ATP channel subunits can acquire increased resistance against metabolic stress (16) . On the other hand, cells from K_ATP channel-deficient transgenic animals, in which the Kir6.2 subunit has been disrupted, lose their ability to regulate intracellular Ca²⁺ levels, suggesting a critical role of the channel complex in the maintenance of cellular Ca²⁺ homeostasis (17) . This raises the possibility that delivery of K_ATP channel genes could promote the ability of a cell to preserve Ca²⁺ homeostasis under stress conditions.

Therefore, we here delivered genes encoding K_ATP channel subunits and examined whether intracellular Ca²⁺ can be controlled by turning on the activity of recombinant channel proteins. We report that K_ATP channel gene delivery, in conjunction with pharmacological activation, has the potential to regulate Ca²⁺ homeostasis during hypoxia-reoxygenation.

	MATERIALS AND METHODS

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Gene delivery into COS-7 cells
The mammalian somatic COS-7 cell line (ATCC, Manassas, Va.) was cultured in a tissue flask (at 5% CO₂) containing Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM glutamine. Cells were trypsinized (5 min, 37°C), and plated (2–6·10⁶) on 60·15 mm culture dish, containing 25- mm glass coverslips. At 40–60% confluence, cells were transfected, using 24 µl lipofectamine (GIBCO, Brooklyn, N.Y.), with 6 µg of total plasmid DNA, i.e., full-length Kir6.2 (5) and SUR2A (7, 18) cDNAs subcloned into mammalian expression vectors pcDNA3.1⁺ and pcDNA3, respectively, and with 0.6 µg of the reporter green fluorescent protein (GFP) gene (GIBCO), as described previously (10, 11, 15) .

Digital epifluorescent imaging
Untransfected or COS-7 cells transfected with Kir6.2/SUR2A were superfused with Tyrode solution (in mM: 136.5 NaCl; 5.4 KCl; 1.8 CaCl₂; 0.53 MgCl₂; 5.5 glucose; 5.5 HEPES-NaOH; pH 7.4). Cells were loaded (for 30 min) with the esterified form of the Ca²⁺-sensitive fluorescent probe, Fluo-3AM (5 µM dissolved in dimethyl sulfoxide plus pluronic acid; Molecular Probes, Eugene, Oreg.). Before loading, transfected cells were preselected based on GFP fluorescence, and the GFP-emitted fluorescence was digitally subtracted (15) . COS-7 cells were imaged using a digital epifluorescence imaging system coupled to an inverted microscope (Zeiss Axiovert-135 TV) with a x40 (numerical aperture 1·3) oil-immersion objective lens. A 100W mercury lamp served as a source of light to excite Fluo-3 at 488 nm. An excitation dichroic mirror with a cutoff of 510 nm and a long pass emission filter with a cutoff of 520 nm were used to detect Fluo-3 fluorescence using an intensified charge-coupled device camera. Detected fluorescence was digitized with the aid of an imaging software (Attoflor RatioVision, Atto Instruments, Rockville, Md.). An estimate of the cytosolic Ca²⁺ concentration, as a function of Fluo-3 fluorescence, was calculated according to the equation: [Ca²⁺]=K_d(F-F_min/F_max-F), where F_min and F_max are minimal and maximal fluorescence intensity, K_d is the dissociation constant of the Fluo-3-Ca²⁺ complex (422 nM), and F is intensity of fluorescence. To obtain F_min and F_max values, cells were exposed to 100 µM ionomycin either in the absence of Ca²⁺ (extracellular Ca²⁺ was removed and 3 mM EGTA added to the extracellular solution) or in the presence of saturating concentrations of Ca²⁺ (10 mM CaCl₂), respectively (15, 19 20 21) .

Experimental protocol
COS-7 cells superfused with Tyrode solution were exposed to 2,4-dinitrophenol (DNP; Sigma, St. Louis, Mo.), a metabolic poison that inhibits mitochondrial oxidative phosphorylation (22) . After treatment for 3 min, DNP was removed and cells were reexposed to Tyrode solution (15, 23) . When indicated, Ca²⁺ was removed from the Tyrode solution by omitting Ca²⁺ and adding the Ca²⁺ chelator EGTA (1 mM). This chemically induced hypoxia-reoxygenation protocol was conducted in the absence or presence of the potassium channel opener pinacidil (RBI, Natick, Mass.) (24) , with and without the potassium channel blocker, 5-hydroxydecanoate (5-HD; RBI) (25) . In a separate series of experiments, COS-7 cells were pretreated (10 min) with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase (26) , ryanodine, which depletes endoplasmic Ca²⁺ stores (27) , and m-iodobenzylguanidine (MIBG, Sigma), an inhibitor of Ca²⁺ release from mitochondria (28, 29) . All drugs except 5-HD were dissolved in dimethyl sulfoxide, which did not exceed 0.1% in its final concentration. At this concentration, DMSO did not affect Ca²⁺ levels or channel activity (15) . 5-HD was dissolved in distilled water.

	RESULTS

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Chemical hypoxia-reoxygenation and intracellular Ca²⁺
The cytosolic Ca²⁺ concentration in COS-7 cells was 103 ± 9 nM (n=14; Fig. 1 ). A 3 min exposure to chemical hypoxia, induced by the mitochondrial poison DNP (2 mM), did not significantly change the intracellular concentration of Ca²⁺ (86 ± 11 nM, n=14, P>0.05; Fig. 1 ). However, reoxygenation evoked by removal of DNP produced rapid and significant increase in cytosolic Ca²⁺ levels (256 ± 29 nM, n=14; P<0.01; Fig. 1 ). Thus, short exposure to hypoxia does not alter Ca²⁺ levels in COS-7 cells, whereas reoxygenation induces intracellular Ca²⁺ loading.

View larger version (85K): [in this window] [in a new window]   Figure 1. The effect of hypoxia and reoxygenation on Ca2+ concentration in COS-7 cells. A) Epifluorescent digital images of Fluo-3 loaded cells under control conditions after chemical hypoxia (induced by 2 mM DNP) and reoxygenation (removal of DNP). White bar corresponds to 50 µM. Color bar represents the relative intensity of fluorescence. A1) Time course of Fluo-3 fluorescence in cells presented in panel A. AU: arbitrary units. A2) Average concentration of intracellular Ca2+ under conditions as indicated. Bars represent mean ± standard error of the mean (n=14); *P<0.01, when compared with the control.

Source of Ca²⁺ loading
This hypoxia-reoxygenation protocol did not induce intracellular Ca²⁺ loading when cells were in a Ca²⁺-free solution containing EGTA (control: 111 ± 16 nM; hypoxia: 99 ± 15 nM; reoxygenation: 94 ± 11 nM, P>0.05, n=6; Fig. 2 A). Removal of Ca²⁺ from the extracellular solution during hypoxia prevented Ca²⁺ loading after reoxygenation (control: 104 ± 16 nM; hypoxia: 97 ± 15 nM; reoxygenation: 96 ± 11 nM, P>0.05, n=6) despite the presence of extracellular Ca²⁺ during the reoxygenation stage (Fig. 2B ). In contrast, removal of Ca²⁺ from the extracellular solution only in the phase of reoxygenation did not prevent hypoxia-reoxygenation-induced Ca²⁺ loading (control: 103 ± 12 nM; hypoxia: 97 ± 17 nM; reoxygenation: 297 ± 22 nM, P<0.01, n=6; Fig. 2C ). Thus, in addition to a role of extracellular Ca²⁺ in hypoxia, another source of Ca²⁺ appears to be critical for reoxygenation-induced Ca²⁺ loading. In cells treated with a mixture that inhibits intracellular Ca²⁺ stores (10 µM thapsigargin, 5 µM ryanodine, and 100 µM MIBG), hypoxia-reoxygenation could no longer induce intracellular Ca²⁺ loading (control: 147 ± 19 nM; hypoxia: 145 ± 25 nM; reoxygenation: 144 ± 19 nM, P>0.05, n=7; Fig. 2D ). Moreover, combined use of thapsigargin and ryanodine prevented the majority of Ca²⁺ overload (Fig. 2E, F ). Thus, Ca²⁺ loading under hypoxia-reoxygenation is apparently due to Ca²⁺ influx during hypoxia, and release of Ca²⁺ from intracellular stores during reoxygenation.

View larger version (44K): [in this window] [in a new window]   Figure 2. The effect of extracellular and intracellular Ca2+ on hypoxia-reoxygenation induced Ca2+ loading in COS-7 cells. A–F) Average concentration of intracellular Ca2+ in cells after hypoxia (DNP) and reoxygenation (washout) when Ca2+ was removed from the extracellular solution throughout the experiment (A), only during hypoxia (B), only during reoxygenation (C), only during reoxygenation in cells pretreated with MIBG (100 µM), ryanodine (5 µM), and thapsigargin (10 µM) in combination (D) or separately (E, F). Bars represent mean ± standard error of the mean (n=6–7); *P<0.01, when compared with the control.

Pinacidil, Kir6.2/SUR2A genes, and Ca²⁺ loading
The K_ATP channel opener, pinacidil (100 µM), did not affect intracellular concentration of Ca²⁺ in untransfected COS-7 cells exposed to chemical hypoxia-reoxygenation protocol (control: 88 ± 10 nM; hypoxia: 86 ± 11 nM; reoxygenation: 228 ± 19 nM, P<0.01, n=6; Fig. 3 A). In cells cotransfected with Kir6.2/SUR2A genes, hypoxia-reoxygenation induced cytosolic Ca²⁺ loading (control: 95 ± 10 nM; hypoxia: 99 ± 11 nM; reoxygenation: 187 ± 19 nM, P<0.01, n=6; Fig. 3B ). However, in cells cotransfected with Kir6.2/SUR2A, treatment with pinacidil (100 µM), an opener of recombinant K_ATP channels (15, 17) , prevented reoxygenation-induced Ca²⁺ loading (control: 99 ± 12 nM; hypoxia: 96 ± 11 nM; reoxygenation: 97 ± 11 nM, P>0.05, n=6; Fig. 4 A). Pinacidil (100 µM), when added only during hypoxia, prevented hypoxia-reoxygenation induced Ca²⁺ loading (control: 101 ± 15 nM; hypoxia: 95 ± 14 nM; reoxygenation: 98 ± 12 nM, P>0.05, n=6; Fig. 4B ). Also, when pinacidil (100 µM) was added only during reoxygenation, hypoxia-reoxygenation did not significantly change intracellular Ca²⁺ (control: 103 ± 15 nM; hypoxia: 97 ± 14 nM; reoxygenation: 90 ± 10 nM, P>0.05, n=4; Fig. 4C ). Thus, delivery of Kir6.2/SUR2A genes into COS-7 cells, in conjunction with pinacidil, can control intracellular Ca²⁺ under hypoxia-reoxygenation throughout the different phases of injury.

View larger version (52K): [in this window] [in a new window]   Figure 3. The effect of pinacidil or delivery of Kir6.2/SUR2A genes on hypoxia-reoxygenation induced Ca2+ loading in COS-7 cells. Time course of Fluo-3 fluorescence (A, B) and average concentration of intracellular Ca2+ (A1, B1) in cells exposed to hypoxia (DNP) and reoxygenation (washout) in the presence of 100 µM pinacidil (A, A1) or after delivery of Kir6.2/SUR2A genes (B, B1). AU: arbitrary units. (A1) Bars represent mean ± standard error of the mean (n=6); *P<0.01.

View larger version (50K): [in this window] [in a new window]   Figure 4. Pinacidil in hypoxia or reoxygenation on Ca2+ loading in COS-7 cells cotransfected with Kir6.2/SUR2A. A) Time course of Fluo-3 fluorescence (A) and average concentration of intracellular Ca2+ (A1) in cells cotransfected with Kir6.2/SUR2A and exposed to hypoxia (DNP) and reoxygenation (washout) in the presence of 100 µM pinacidil (A1). AU: arbitrary units. A1) Bars represent mean ± standard error of the mean (n=6). B, C) Average concentration of intracellular Ca2+ in cells cotransfected with Kir6.2/SUR2A under conditions as indicated with pinacidil (100 µM) present only during hypoxia (B) or reoxygenation (C). Same protocol repeated in the presence of 100 µM 5-HD (D, E). Bars represent mean ± standard error of the mean (n=6); *P<0.01.

When pinacidil (100 µM), together with the selective antagonist of K_ATP channels, 5-HD (100 µM), was added during the stage of hypoxia, reoxygenation induced significant Ca²⁺ loading in cells cotransfected with Kir6.2/SUR2A (control: 81 ± 8 nM; hypoxia: 75 ± 8 nM; reoxygenation: 141 ± 13 nM, P<0.01, n=6; Fig. 4D ). Similarly, when pinacidil (100 µM) was added with 5-HD (100 µM) during reoxygenation, a significant Ca²⁺ loading also occurred (control: 73 ± 8 nM; hypoxia: 70 ± 7 nM; reoxygenation: 128 ± 11 nM, P<0.01, n=6; Fig. 4E ). 5-HD (100 µM) by itself did not modify intracellular Ca²⁺ concentration in cotransfected COS-7 cells exposed to chemical hypoxia and reoxygenation (control: 93 ± 12 nM; hypoxia: 91 ± 9 nM; reoxygenation: 208 ± 29 nM, P<0.01, n=6). Thus, the control of intracellular Ca²⁺ by pinacidil, under hypoxia-reoxygenation, in cells expressing Kir6.2/SUR2A is sensitive to 5-HD.

	DISCUSSION

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In the present study, we show that delivery of K_ATP channel genes, in conjunction with a pharmacological switch in channel activity, tightly regulates intracellular Ca²⁺ during various stages of hypoxia-reoxygenation. Specifically, this report demonstrates that turning on recombinant K_ATP channels, either during hypoxia and/or reoxygenation, could prevent Ca²⁺ overload in mammalian cells natively lacking K_ATP channels. Such findings indicate that K_ATP channel-based strategy has the potential to be effective in regulating Ca²⁺ homeostasis under metabolic stress.

It is well established that intracellular Ca²⁺ loading is potentially lethal for cells exposed to hypoxia-reoxygenation (1, 2) . During the hypoxic stage, intracellular Ca²⁺ may remain low, but rapidly increases after reoxygenation (1, 2) , as observed here. The mechanism responsible for Ca²⁺ overload in hypoxia-reoxygenation remains largely controversial (1 2 3) . In the present study, we provide evidence that both extracellular Ca²⁺, as well as Ca²⁺ stored in subcellular compartments, contribute to Ca²⁺ overload under hypoxia-reoxygenation. First, we show that removal of extracellular Ca²⁺ throughout hypoxia-reoxygenation can abolish Ca²⁺ loading. It was the transient removal of extracellular Ca²⁺ during hypoxia, and not during reoxygenation, that effectively prevented Ca²⁺ loading. Second, treatment with agents that deplete or inhibit intracellular Ca²⁺ stores also abolished Ca²⁺ loading. Thus, Ca²⁺ loading after hypoxia-reoxygenation of mammalian COS-7 cells may have occurred through influx of Ca²⁺ during the hypoxic stage and release of Ca²⁺ from intracellular stores during the reoxygenation stage.

Influx of Ca²⁺ into COS-7 cells during hypoxia did not manifest as significant increase in intracellular Ca²⁺. This is probably due to an avid uptake of entering Ca²⁺ by mitochondria and/or the endoplasmic reticulum, as described previously to occur under hypoxic stress (30) . In support of this is our finding that concomitant inhibition of intracellular Ca²⁺ stores attenuated hypoxia-reoxygenation induced Ca²⁺ loading. The significance of both extracellular and intracellular Ca²⁺ stores in hypoxia-reoxygenation injury is further underscored by the absence of protection afforded by calcium channel blockers when applied during reoxygenation (30, 31) and by the efficacy of pre- and postischemic administration of Na/H exchange inhibitors to diminish cytosolic Ca²⁺ overload (32) . Thus, an effective mean to maintain Ca²⁺ homeostasis under different stages of hypoxia-reoxygenation should be able to target both sources of Ca²⁺.

In recent years, attempts have been made to implement gene therapy as a more efficient strategy to treat or prevent injury induced by hypoxia-reoxygenation. So far, such approaches have yielded variable success, in part due to difficulties in controlling the function of recombinant proteins after delivery into somatic cells (33 34 35) . Previously, we have shown that the activity of recombinant K_ATP channels expressed in a cell line can be switched on and off by pharmacological means (15) . Since disruption of K_ATP channels induces loss of intracellular Ca²⁺ control (17, 36) whereas activation of K_ATP channels has been associated with a cytoprotective outcome (37 38 39 40 41) , use of genes that encode the two channel subunits may generate a unique approach to limit cell injury. We provide evidence favoring such a concept, since delivery of K_ATP channel genes, in conjunction with pharmacological activation of channel subunits, efficiently prevented intracellular Ca²⁺ loading.

The prototype potassium channel opener, pinacidil, maintained low levels of intracellular Ca²⁺ in cells cotransfected with K_ATP channel subunit isoforms, Kir6.2 and SUR2A, irrespective of whether the opener was applied throughout the duration of hypoxia-reoxygenation or transiently during either the hypoxic or reoxygenation stage. These actions of pinacidil are likely mediated through K_ATP channel proteins, since this opener has been shown to have no effect in untransfected COS-7 cells (see also ref 15 ) and the effect on preventing Ca²⁺ loading in Kir6.2/SUR2A cotransfected cells was shown to be antagonized by the selective K_ATP channel blocker, 5-HD (25) . Under present experimental conditions, the K_ATP channel opener could maintain low intracellular Ca²⁺ by impeding Ca²⁺ influx during hypoxia and/or by limiting Ca²⁺ release from intracellular stores during reoxygenation. Previously, we have demonstrated, at the single channel level, that recombinant K_ATP channels expressed in the plasma membrane of COS-7 cells are vigorously activated by pinacidil (15) . Such action may account for keeping the membrane potential close to the value of the reversal potential for K⁺ and away from the more positive membrane potential of COS-7 cells (42) . This would limit Ca²⁺ influx associated with membrane depolarization, which accompanies hypoxia (43) . In addition, the action of potassium channel openers in a number of cell types has been associated with regulation of intracellular Ca²⁺ stores (44, 45) .

In summary, the present study demonstrates that combined use of gene delivery and pharmacological targeting of recombinant proteins can be used to efficiently control intracellular Ca²⁺ homeostasis under hypoxia-reoxygenation. The property of recombinant Kir6.2/SUR2A, in conjunction with a K_ATP channel opener, to limit Ca²⁺ loading irrespective of the stage of metabolic stress may provide a basis for future therapies of hypoxia-reoxygenation injury.

	ACKNOWLEDGMENTS

 
The authors acknowledge the kind gifts of Kir6.2 and SUR2A cDNAs received from Dr. S. Seino (Chiba University, Chiba, Japan) and Dr. Y. Kurachi (Osaka University, Osaka, Japan), respectively. This work was supported by the American Heart Association, Merck Company Foundation, Miami Heart Research Institute, and the Bruce and Ruth Rappaport Program in Vascular Biology and Gene Delivery.

	FOOTNOTES

 
² Abbreviations: DNP, 2,4-dinitrophenol; GFP, green fluorescent protein; 5-HD, 5-hydroxydecanoate; K_ATP, ATP-sensitive K⁺; MIBG, m-iodobenzylguanidine.

Received for publication October 28, 1998. Revision received December 21, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Granger, D. N., Korthius, R. J. (1995) Physiologic mechanisms of postischemic tissue injury. Annu. Rev. Physiol. 57,311-332[Medline]
2. Kristian, T., Siesjo, B. K. (1998) Calcium in ischemic cell death. Stroke 29,705-718[Abstract/Free Full Text]
3. Maxwell, S. R. J., Lip, G. Y. H. (1997) Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int. J. Cardiol. 58,95-117[Medline]
4. Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E., Gonzales, G., Hererra-Sosa, H., Nguy, K., Bryan, J., Nelson, D. A. (1995) Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268,423-426[Abstract/Free Full Text]
5. Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzales, G., Aguilar-Bryan, L., Seino, S., Bryan, J. (1995) Reconstitution of I_KATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270,1166-1170[Abstract/Free Full Text]
6. Inagaki, N., Gonoi, T., Clement, J. P., Wang, C. Z., Aguilar-Bryan, L., Bryan, J., Seino, S. (1996) A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K⁺ channels. Neuron 16,1011-1017[Medline]
7. Isomoto, S., Kondo, C., Yamada, M., Matsumoto, S., Higashiguchi, O., Horio, Y., Matsuzawa, Y., Kurachi, Y. (1996) A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K⁺ channel. J. Biol. Chem. 271,24321-24324[Abstract/Free Full Text]
8. Nichols, C. G., Shyng, S. L., Nestorowicz, A., Glaser, B., Clement, J. P., Gonzales, G., Aguilar-Bryan, L., Permutt, M. A., Bryan, J. (1996) Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 272,1785-1787[Abstract]
9. Clement, J. P., Kunjilwar, K., Gonzales, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L., Bryan, J. (1997) Association and stoichiometry of K_ATP channel subunits. Neuron 18,827-838[Medline]
10. Alekseev, A. E., Kennedy, M. E., Navarro, B., Terzic, A. (1997) Burst kinetics of co-expressed Kir6.2/SUR1 clones: comparison of recombinant with native ATP-sensitive K⁺ channel behavior. J. Membr. Biol. 159,161-168[Medline]
11. Lorenz, E., Alekseev, A. E., Krapivinsky, G. B., Carrasco, A. J., Clapham, D. E., Terzic, A. (1998) Evidence for direct physical association between a K⁺ channel (Kir6.2) and in ABC protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K⁺ channel. Mol. Cell. Biol. 18,1652-1659[Abstract/Free Full Text]
12. Ashcroft, F. M., Gribble, F. M. (1998) Correlating structure and function in ATP-sensitive K⁺ channels. Trends Neurosci 21,288-294[Medline]
13. Lauritzen, I., De Weille, J. R., Lazdunski, M. (1997) The potassium channel opener (-) cromakalim prevents glutamate-induced cell death in hippocampal neurons. J. Neurochem. 69,1570-1579[Medline]
14. Liu, Y. G., Sato, T., Orourke, B., Marban, E. (1998) Mitochondrial ATP-dependent potassium channels—novel effectors of cardioprotection. Circulation 97,2463-2469[Abstract/Free Full Text]
15. Jovanovi, A., Jovanovi, S., Lorenz, E., Terzic, A. (1998) Recombinant cardiac ATP-sensitive K⁺ channel subunits confer resistance towards chemical hypoxia-reoxygenation injury. Circulation 98,1548-1555[Abstract/Free Full Text]
16. Jovanovi, A., Jovanovi, S., Carrasco, A. J., Terzic, A. (1998) Acquired resistance of a mammalian cell line to hypoxia-reoxygenation through co-transfection of Kir6.2 and SUR1 clones. Lab. Invest. 78,1101-1107[Medline]
17. Miki, T., Nagashima, K., Tashiro, F., Kotake, K., Yoshitami, H., Tamamoto, A., Gonoi, T., Iwanaga, T., Miyazaki, J., Seino, S. (1998) Defective insulin secretion and enhanced insulin action in K_ATP channel-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 95,10402-10406[Abstract/Free Full Text]
18. Okuyama, Y., Yamada, M., Kondo, C., Satoh, E., Isomoto, S., Shindo, T., Horio, Y., Kitakaze, M., Hori, M., Kurachi, Y. (1998) The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K⁺ channel expressed in a mammalian cell line, HEK293T cells. Pfluegers Arch 435,595-603[Medline]
19. Lopez, J. R., Jovanovic, A., Terzic, A. (1995) Spontaneous calcium waves without contraction in cardiac myocytes. Biochem. Biophys. Res. Commun. 214,781-787[Medline]
20. Jovanovic, A., Lopez, J. R., Terzic, A. (1996) Cytosolic Ca²⁺ domain-dependent protective action of adenosine in a cardiomyocytes. Eur. J. Pharmacol. 298,63-69[Medline]
21. Jovanovic, A., Alekseev, A. E., Lopez, J. R., Shen, W. K., Terzic, A. (1997) Adenosine prevents hyperkalemia-induced calcium loading in cardiac cells: relevance for cardioplegia. Ann. Thorac. Surg. 63,153-161[Abstract/Free Full Text]
22. Brady, P. A., Zhang, S., Lopez, J. R., Jovanovi, A., Aleeksev, A. E., Terzic, A. (1996) Dual effect of glyburide, an antagonist of K_ATP channels, on metabolic inhibition-induced Ca²⁺ loading in cardiomyocytes. Eur. J. Pharmacol. 308,343-349[Medline]
23. Terzic, A., Jahangir, A., Kurachi, Y. (1995) Cardiac ATP-sensitive K⁺ channels regulation by intracellular nucleotides and K⁺ channel opening drugs. Am. J. Physiol. 269,C525-C545[Abstract/Free Full Text]
24. Notsu, T., Tanaka, I., Takano, M., Noma, A. (1992) Blockade of the ATP-sensitive K⁺ channel by 5-hydroxydecanoate in guinea pig ventricular myocytes. J. Pharmacol. Exp. Ther. 260,702-708[Abstract/Free Full Text]
25. Kirby, M. S., Sagara, Y., Gaa, S., Inesi, G., Lederer, W. J., Rogers, T. B. (1992) Thapsigargin inhibits contraction and Ca²⁺ transient in cardiac cells by specific inhibition of the sarcoplasmatic reticulum Ca²⁺ pump. J. Biol. Chem. 267,12545-12551[Abstract/Free Full Text]
26. Hansford, R. G., Lakatta, E. G. (1987) Ryanodine releases calcium from sarcoplasmatic reticulum in calcium-tolerant rat cardiac myocytes. J. Physiol. (London) 390,453-467[Abstract/Free Full Text]
27. Juedes, M. J., Kaas, G. E. N., Orrenius, S. (1992) M-Iodobenzylguanidine increases the mitochondrial Ca²⁺ pool in the isolated hepatocytes. FEBS Lett 313,39-42[Medline]
28. Kim, J. S., Southard, J. H. (1998) Alterations in cellular calcium and mitochondrial functions in the rat liver during cold preservation. Transplantation 65,369-375[Medline]
29. Miller, T. W., Tormey, J. McD. (1995) Subcellular calcium pools of ischaemic and reperfused myocardium characterised by electron probe. Cardiovasc. Res. 29,85-94[Medline]
30. Watts, J. A. (1986) Protection of ischemic hearts by Ca²⁺ antagonists. J. Mol. Cell. Cardiol. 18(Suppl. 4), 71–75
31. Kusuoka, H., Correti, M. C., Koretsune, Y., Marban, E. (1998) Mechanism for the cardioprotective effects of the calcium channel blocker clentiazem during ischemia and reperfusion. Jpn. Circ. J. 62,611-616[Medline]
32. Levitsky, J., Gurell, D., Frishman, W. H. (1998) Sodium ion hydrogen ion exchange inhibition—a new pharmacologic approach to myocardial ischemia and reperfusion injury. J. Clin. Pharmacol. 38,887-897[Abstract]
33. Zwack, R. M., Zhou, W., Zhang, Y., Darby, C. J., Dudus, L., Halldorson, J., Oberley, L., Engelhardt, J. F. (1998) Redox gene therapy for ischemia/reperfusion injury of the liver reduces AP1 and NF-B activation. Nature Med 4,698-704[Medline]
34. Daniel, C., Erzurum, S. C., Prayssac, P., Eissa, N. T., Crystal, R. G., Herve, P., Baudet, B., Mazmanian, M., Lemerchand, P. (1998) Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury. Hum. Gene Ther. 9,1487-1496[Medline]
35. French, BA (1998) Gene therapy and cardiovascular diseases. Curr. Opin. Cardiol. 13,205-213[Medline]
36. Miki, T., Tashiro, F., Iwanaga, T., Nagashima, K., Yoshitomi, H., Aihara, H., Nitta, Y., Gonoi, T., Inagaki, N., Miyazaki, J., Seino, S. (1997) Abnormalities of pancreatic islets by targeted expression of a dominant-negative K_ATP channel. Proc. Natl. Acad. Sci. U. S. A. 94,11969-11973[Abstract/Free Full Text]
37. Gross, G. J. (1995) ATP-sensitive potassium channels and myocardial preconditioning. Basic Res. Cardiol. 90,85-88[Medline]
38. Hearse, D. J. (1995) Activation of ATP-sensitive potassium channels: a novel pharmacological approach to myocardial protection. Cardiovasc. Res. 30,1-17[Medline]
39. Lopez, J. R., Jahangir, R., Jahangir, A., Shen, W. K., Terzic, A. (1996) Potassium channel openers prevent potassium-induced calcium loading of cardiac cells: possible implications in cardioplegia. J. Thorac. Cardiovasc. Surg. 112,820-831[Abstract/Free Full Text]
40. Grover, G. J. (1997) Pharmacology of ATP-sensitive potassium channel (K_ATP) openers in models of myocardial ischemia and reperfusion. Can. J. Physiol. Pharmacol. 75,309-315[Medline]
41. Behling, R. W., Malone, H. J. (1995) K_ATP-channel openers protect against increased cytosolic calcium during ischemia and reperfusion. J. Mol. Cell. Cardiol. 27,1809-1817[Medline]
42. Ishii, T., Hashimoto, T., Ohmori, H. (1996) Hypotonic stimulation induced Ca²⁺ release from IP3-sensitive internal stores in a green monkey kidney cell line. J. Physiol. (London) 493,371-384[Medline]
43. Balastrino, M. (1995) Pathophysiology of anoxic depolarization: new findings and working hypothesis. J. Neurosci. Methods 59,99-103[Medline]
44. Yanagisawa, T., Teshigawara, T., Taira, N. (1990) Cytoplasmic calcium and the relaxation of canine coronary arterial smooth muscle produced by cromakalim, pinacidil and nicorandil. Br. J. Pharmacol. 101,157-165[Medline]
45. Holmuhamedov, E., Jovanovic, S., Dzeja, P. P., Jovanovic, A., Terzic, A. (1998) Mitochondrial ATP-sensitive K⁺ channels modulate cardiac mitochondrial function. Am. J. Physiol. 275,H1567-H1576[Abstract/Free Full Text]

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