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1
Department of Pharmacology, University of Alberta, Edmonton, Alberta; and
* Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada
1Correspondence: 9–58 Medical Sciences, Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada. T6G 2H7. E-mail: peter.light{at}ualberta.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ischemic preconditioning • protein kinase C • adenosine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
). However, the precise signal transduction pathways involved in the triggering and maintenance of protection are the subject of intense research and remain to be determined conclusively. Many candidate molecules for the triggering, transduction, and initiation of IPC have now been identified (5
, 6)
. In particular, adenosine release and activation of protein kinase C (PKC) and ATP-sensitive potassium (KATP) channels are common elements in the majority of IPC models tested (1
, 6)
. There are two separate populations of KATP channels within the myocardium: the mitochondrial (mito) and the sarcolemmal (sarc) KATP channel (7)
. KATP channels possess the unique ability to couple plasma membrane potential and/or mitochondrial redox potential/free radical release to the metabolic status of the cell (8
9
10)
. However, the roles of sarcKATP and mitoKATP channels in the preconditioning pathway are the source of recent debate and controversy (7
, 10
, 11)
. Current opinion favors a predominant role for the mitoKATP channel in IPC (10
, 12
13
14)
, although several lines of evidence demonstrate that sarcKATP channels are important mediators of protection during the reoxygenation phase of injury (15
, 16)
. Determination of the respective roles of both sarc and mito KATP channels during hypoxia/reoxygenation injury with respect to PKC activation and adenosine is important in understanding the cellular events that occur during IPC. Therefore, in this study we set out to determine the relative contributions of the sarcKATP channel and mitoKATP channel populations in response to activation by PKC and adenosine. A cellular model of calcium overload during hypoxia/reoxygenation was used in combination with cell viability assays, allowing us to resolve the respective time courses of sarc and mito KATP channel contribution to myoprotection. Our data indicate that both sarc and mito KATP channels reduce calcium loading and protect myocytes from hypoxia/reoxygenation injury, but their mechanism and timing of action are distinct from one another. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. tsA201 cells (an SV40 transformed variant of the HEK293 human embryonic kidney cell line) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10 mmol/l glucose, 2 mmol/l L-glutamine, 10% fetal calf serum, and 0.1% penicillin/streptomycin at 37°C (10% CO2). Cells were plated at 30–40% confluency on 35 mm culture dishes 4 h before transfection. The KATP channel subunit clones were generously provided by Dr. S. Seino (mouse Kir6.2 and rat SUR2A; 18
, 19
). Clones were inserted into mammalian expression vectors (pCDNA3 or pCMV6) and transfected into tsA201 cells using the calcium phosphate precipitation technique. Transfected cells were identified using fluorescence optics in combination with coexpression of the KATP channel subunit clones with the blue fluorescent variant of the green fluorescent protein (Living Colors, Clontech, Palo Alto, CA). Recordings were made from cells 48–72 h after transfection. Expression of the mammalian expression vector pCDNA3 without any channel constructs inserted did not produce any measurable whole-cell current.
Single-channel recording
Standard patch-clamp techniques were used to record single-channel currents in the inside-out patch configuration. The internal faces of the patches were directly exposed to test solutions via a multi-input perfusion pipette (time to change solution at the tip of the recording pipette was less than 2 s). Single-channel currents were recorded at fixed holding potentials, amplified (Axopatch 200, Axon Instruments, Foster City, CA), digitized (Neuro-corder DR-384, Neuro Data Instruments Corp., New York, NY), then stored on videotape. Data were sampled at 500 Hz and filtered at 200 Hz.
The pipette solution used for all patch recordings contained the following (in mmol/l): KCl 140, HEPES 10, MgCl2 1.4, EGTA 1, glucose 10. The pH of the solution was adjusted to 7.4 with KOH. This solution was also used in the recording chamber to superfuse the cells/patches for experiments using symmetrical [K+].
Myocyte viability assays
Freshly isolated populations of single myocytes from rat right ventricle were used for this study and stored in the experimental solution containing (in mmol/l): NaCl 136.5, KCl 5.4, CaCl2 1.8, MgCl2 0.53, glucose 5.5, HEPES-NaOH 5.5, pH 7.4 at 20–22°C. Myocyte viability was assessed in each group after a 5 min exposure to chemically induced hypoxia (CIH) and then 3 min reoxygenation at 37°C. For induction of CIH, 2 mmol/l NaCN and 5 mmol/l 2-deoxyglucose were added to the experimental solution. Myocytes were gently pelleted in 1.5 ml tubes using a Microfuge for 30 s at 2000 rpm and solutions were then changed. All normoxic controls underwent the same handling procedure to minimize experimental artifacts. A modified Tyrode solution contained (in mmol/l) KCl 2.68, CaCl2 1.8, NaH2PO4 0.42, NaHCO3 11.9, MgSO4 0.83, glucose 5.55, and amylobarbitone 3.0, 5 µg/ml glutaraldehyde; 5 µg/ml trypan blue was applied before assessing myocyte viability. Under these conditions, dead cells stain blue and viable cells do not stain. Approximately 200 myocytes per sample were counted in <10 min from each sample; > 1000 myocytes were sampled in each experimental group. From these data the percentage of dead cells was calculated and compared among experimental groups. All results were normalized to control data. Experimental protocols of this nature have been used previously to assess cardiac myocyte viability (20
, 21)
.
Calcium overload measurements
The sustained increase in intracellular calcium has been proposed to be a good correlate of cellular viability during ischemia/reperfusion injury (for a review, see ref 22
). Therefore, the tracking of intracellular calcium loading has recently been used as an assay to measure calcium handling in an isolated model of chemically induced hypoxia/reoxygenation (23
, 24)
. Right ventricular myocytes from rat were superfused with the experimental solution. Cells were loaded for 15 min with the esterified form of the Ca2+-sensitive fluorescent probe FURA-2-AM (5 µmol/l, dissolved in a 60% dimethyl sulfoxide and 40% v/v Pluronic acid mixture; Molecular Probes, Eugene, OR). Once loaded, cells were washed and placed on a coverslip for observation at 200x under an inverted microscope (Zeiss Axiovert 100). A Photon Technology International (PTI, Lawrenceville, NJ) imaging system with PTI Imagemaster software was used for data acquisition and analysis. FURA-2 was excited with alternating 340 and 380 nm wavelengths of light, and the emitted light intensity at 520 nm was digitized and stored. An estimate of intracellular calcium was obtained from the calculated 340/380 nm ratio [experimental range was 1.07–1.24 under normoxic (control) conditions]. In experiments on recombinant sarcKATP channels, the calcium-sensitive dye FLUO-3-AM (Molecular Probes) was used in conjunction with expression of the blue fluorescent protein (BFP) marker. We used BFP instead of GFP to avoid overlap of the absorption/emission spectra of the fluorescent moieties. Intracellular calcium concentration was measured by the fluorescence intensity emitted at 520 nm light during excitation with 488 nm light. Normalized values were expressed as the ratio F(test)/F(control), where F(test) = fluorescence intensity at a given time point and F(control) is the mean fluorescence intensity measured during the stable 1 min pre-CIH normoxic period.
Experimental compounds
ATP (as MgATP; Sigma, St. Louis, MO) was added as required from a 10 mmol/l stock prepared immediately before use. 5-Hydroxydecanoate (5-HD; Sigma/RBI) and HMR 1098 (sodium salt of HMR 1883; Aventis, Frankfurt, Germany) were dissolved as stock solutions in distilled water. 2-Chloro-N6-cyclopentyladenosine (CCPA, Sigma/RBI) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, Sigma/RBI) were made up as stock solutions in dimethyl sulfoxide. The phorbol esters 4-ß-PMA (Sigma) and 4-
-PDD (Calbiochem, San Diego, CA) were stored in ethanol at a concentration of 100 µmol/l at -20°C. Chelerythrine chloride (Calbiochem) was stored at 5 mmol/l in distilled H2O at -20°C. Stock solutions were diluted to the required concentration immediately before use. Statistical significance was evaluated using Student’s t test and paired t test, as appropriate. Differences with values of probability (P<0.05) were considered to be significant. All values in the text are mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, to our knowledge, no studies have been conducted directly at the single-channel level to establish sarcKATP channel inhibitory concentration-response relations for HMR 1098. Therefore, we first performed a pharmacological assay to determine concentrations of HMR 1098 suitable for this study. Single-channel inside-out patch clamp studies were performed on native KATP channels from rat right ventricular myocytes and the sarcKATP channel inhibitory effects of 0.1, 0.5, 1, 5, 10, 50, and 1000 µmol/l HMR 1098 were measured. HMR 1098 was an effective inhibitor of sarcKATP channels in a dose-dependent manner (see Fig. 1
A.). Fitting of the HMR 1098 dose-response relationship determined the IC50 and Hill coefficient to be 0.88 µmol/l and 1.27, respectively. Accordingly, an HMR 1098 concentration of 10 µmol/l was used to block >90% of the sarcKATP channel current in subsequent experiments.
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5-Hydroxydecanoate has no effect on sarcKATP channels
In addition to the HMR 1098 experiments, it was necessary to determine whether the mitoKATP channel inhibitor 5-HD had any effect on sarcKATP channels. Application of 5-HD at concentrations of 10, 50, 100 and 500 µmol/l did not significantly inhibit the activity of sarcKATP channels in excised inside-out ventricular membrane patches (Fig. 1B
). Normalized NPo at 10, 50, 100, and 500 µmol/l 5-HD were 104.9±5.9, 100.4±8.3, 93.9±6.7, and 102.9±6.4% (P>0.05 for all concentrations).
PKC, KATP channels, and Ca2+ overload
To assess the relative importance of sarc and mito KATP channels in the protection observed by PKC activation during and after hypoxic insults, a real-time assessment of intracellular calcium ([Ca2+]i) was used. Myocyte calcium overload is a well-known correlate of ischemia/reperfusion injury (22)
and has been used to assess the protective effects of recombinant KATP channel activation (15)
. In myocytes loaded with the calcium-sensitive dye FURA-2AM, no change in intracellular calcium was observed during normoxic steady-state conditions. However, superfusion with the CIH solution caused a steady-rise in [Ca2+]i and reached a value of 9.1 ± 0.75% after 3 min. The rate of rise accelerated on reoxygenation with normoxic solution; after 2 min, the increase reached 37.6 ± 4.6% (P<0.001; see Fig.2A
). Pretreatment with the PKC-activating 4-ß-phorbol 12-myristate 13-acetate (PMA, 100 nmol/l) completely abolished the increase in [Ca]int induced by the CIH (2.1±1.0%) and reoxygenation (3.0±1.2%; see Fig. 2A
.). Similarly, addition of levcromakalim (20 µmol/l), a nonspecific KATP channel opener, prevented Ca2+ loading during CIH and reoxygenation (5.3±1.0%). Neither the inactive phorbol ester PDD added alone nor PMA added in the presence of the PKC inhibitor chelerythrine (10 µmol/l) inhibited the calcium loading observed during CIH/reoxygenation (see Fig. 2B
.).
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To investigate roles of mito and sarc KATP channels in the calcium overload lowering effects of PKC activation, the following experiments were performed. Continuous treatment of myocytes with the specific mitoKATP channel blocker 5-HD (100 µmol/l) relieved some of the protection afforded by PMA during the CIH period (see Fig. 2C
.) but did not hinder the PMA-induced protection from Ca2+ loading during reoxygenation (2.5±0.9%). Conversely, exposure to HMR 1098 (10 µmol/l) had little effect on the protection afforded by PMA during CIH but resulted in the reappearance of calcium overload during reoxygenation (21.3±2.7%, P<0.001; see Fig. 2C
). The addition of both 5-HD and HMR 1098 in the presence of PMA restored calcium overload during the CIH (12.1±1.6%) and reoxygenation period (38±4.6%, P<0.001) to levels even higher than those observed during CIH/reoxygenation in control conditions (see Fig. 3
A.). Taken together, these data show that PKC-mediated activation of sarc and mitoKATP channels differentially moderate calcium overload during distinct phases of hypoxia and reoxygenation.
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KATP channels and myocyte viability
To assess the roles of both KATP channel populations on myocyte survival, a series of experiments was performed in which cell viability was assessed using the trypan blue exclusion assay. After a 5 min CIH and 3 min reoxygenation period, myocyte survival was reduced to 34.3 ± 2.2% (P<0.001, normalized to normoxic control; see Fig. 3B
.). In contrast, treatment with PMA (100 nmol/l) or the KATP channel opener levcromakalim (20 µmol/l) increased cell survival to values of 87.6 ± 3.1% and 81.4 ± 4.1%, respectively (vs. control).
The protection afforded by PMA was significantly reduced by the addition of HMR 1098 (10 µmol/l), with only 38.3 ± 1.4% of cells surviving (P<0.001, compared with PMA alone). The addition of 5-HD reduced the protection afforded by PMA (71.2±2.3%, P<0.05 vs. PMA alone), though not to the same extent as HMR 1098. Simultaneous administration of both HMR 1098 (10 µmol/l) and 5-HD (100 µmol/l) abolished the protective effects of PMA (25.4±1.1%, P<0.001 from PMA alone). This value is also significantly lower than control (P<0.01), suggesting that basal activity of KATP channels may contribute to cell survival during CIH/reoxygenation in the absence of exogenous PKC activators.
Adenosine A1 agonists, calcium overload, and myocyte viability
Strong evidence indicates that adenosine, acting via the A1 receptor, is a major physiological trigger of IPC in the mammalian heart (6
, 26)
. The signaling pathways downstream of A1 receptor stimulation likely involve the activation and translocation of several PKC isoforms as well as activation of KATP channels (1
, 6)
. However, the respective contributions of the sarc and mito KATP channel populations as mediators of adenosine-induced protection remain to be determined. In the next series of experiments, we set out to determine whether both KATP channel populations are involved in the protection afforded by adenosine.
Treatment of cells with 1 µmol/l of the A1 receptor-selective agonist CCPA (27)
induced protection from calcium overload to an extent similar to that observed with PKC activation after CIH (2.9±0.3%) or during reoxygenation (4.1±0.3%, Fig. 4
A). The CCPA-mediated protection from Ca2+ loading was significantly reduced by the addition of HMR 1098 (10 µmol/l) to CCPA (22.4±3.3% increase after reoxygenation; P<0.001 compared with CCPA alone). When similar experiments were conducted with 5-HD alone (100 µmol/l), CCPA-mediated protection was maintained (3.2±0.4%, n=15, Fig. 4A
). The addition of DPCPX (10 µmol/l), a selective A1 antagonist (28)
, prevented the CCPA Ca2+ overload protection, and calcium loading during CIH (9.3±2%) and reoxygenation (22.6±2%) were observed (P<0.001 from CCPA). The selective PKC blocker chelerythrine (100 µmol/l) also prevented CCPA-induced protection from calcium overload (8.2±1.3% during CIH and 15.4±0.5% during reoxygenation; P<0.01 from CCPA). These data suggest that the effects of A1 receptor stimulation are mediated through the activation of PKC and sarcKATP channels (see Fig. 4B
.).
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Treatment of myocytes with CCPA (1 µmol/l) during 5 min hypoxia and 3 min reoxygenation significantly increased cell survival to 79.6 ± 2.2% (from 40.4±1.4% in control, P<0.001; Fig 4C
). The CCPA-mediated protection was abolished in the presence of either DPCPX (10 µmol/l) or chelerythrine (1 µmol/l) resulting in cell survival of 32.6 ± 4.0% and 34.3 ± 5.1%, respectively. HMR 1098 also prevented the protection afforded by CCPA, resulting in only 26.3 ± 3.1% of cells surviving (P<0.01 from control). 5-HD (100 µmol/l) also reduced the number of cells surviving in the presence of CCPA, but not as effectively as HMR 1098 (55.6±2.2%; P<0.001 from CCPA, P<0.01 from CIH).
A single residue substitution in the sarcKATP channel prevents PKC-mediated reduction in calcium overload after reoxygenation
The recent cloning of the sarcKATP channel subunits has provided a useful tool with which to probe the involvement of KATP channels in mediating the protective effects of PKC at the molecular level. Our previous studies demonstrated that PKC activates the sarcKATP channel at physiological levels of ATP by altering the Hill coeffient for ATP binding (29)
. We have determined that a single threonine residue at position 180 in the pore-forming Kir6.2 subunit is the target for the functional PKC-mediated phosphorylation of the sarcKATP channel complex. Substitution of this T180 residue with an alanine (T180A) prevents PMA-induced activation of the sarcKATP channel (30)
. Therefore, in the next series of experiments we wanted to determine whether the PKC-mediated reduction in calcium overload observed during reoxygenation can be prevented by this single residue substitution in a model of hypoxia/reoxygenation utilizing recombinant KATP channels.
In tsA201 cells transiently expressing BFP alone, a biphasic calcium overload was observed during CIH/reoxygenation (using fluo-3 fluorescence) similar to that observed with native ventricular myocytes with Fura-2. The calcium increase persisted after pretreatment with PMA (100 nmol/l) in cells expressing only BFP (119.0±8.9% increase in fluo-3 fluorescence; see Fig. 5A.
). It has been shown that pharmacological activation of expressed recombinant KATP channels is required to elicit protection from CIH/reoxygenation-induced calcium overload (15
, 23)
. In accordance with these findings, we show that without exogenous activation, transient expression of the recombinant sarcKATP channel subunits has little effect on the level of calcium overload observed (fluorescence increase, 102.1±20.3% vs. BFP alone). However, in cells expressing the sarcKATP channel subunits, treatment with PMA (100 nmol/l) significantly reduced the level of calcium overload during the reoxygenation period (fluorescence increase, 27.4±5.0%, P<0.001, vs. results in the absence of PMA; see Fig. 5A
). Moreover, expression of the SUR2A and mutant Kir6.2, T180A subunits significantly attenuated the PMA-mediated reduction in calcium overload (65.3±5.0%, P<0.01; see Fig. 5B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several previous studies have suggested that pharmacological activation of sarcKATP channels can reduce the calcium overload observed during reoxygenation (15
, 23)
. Our data are consistent with these observations and demonstrate that PKC stimulation is also able to mediate this effect via activation of the sarcKATP channel population. It is now possible to take advantage of the distinct pharmacological properties of sarcKATP and mitoKATP channel isoforms and use isoform-specific inhibitors. Specific blockade of sarcKATP channels with the novel sulfonylurea HMR 1098 has little effect on calcium handling during the hypoxic period; however, PKC-mediated reduction in calcium overload is lost during reoxygenation. In contrast, blockade of mitoKATP channels with 5-HD did not cause any change in calcium overload during reoxygenation in response to PKC activation, but resulted in a slight increase in calcium loading during the hypoxic period. The reasons for this increase are unclear but may reflect the ability of activated mitoKATP channels to protect the mitochondria from calcium overload (31)
.
The cell viability data demonstrate that the PKC-induced increase in cell survival after hypoxia/reoxygenation is mediated by both sarcKATP and mitoKATP channel populations. In a recent whole-heart study, it was suggested that the mitoKATP channels act to regulate infarct size whereas functional recovery of the surviving tissue is modulated by activation of sarcKATP channels (16)
. Our data indicate that PKC-induced activation of sarcKATP channels may be an important pathway by which myocytes can regulate fluctuations in intracellular calcium during reperfusion. The exact mechanism(s) whereby sarcKATP channel activation elicits these protective effects remains to be determined. Protection by PKC via the mitoKATP channel population is unlikely to involve the ability of myocytes to handle cytosolic calcium during the reoxygenation period. Recent data suggests that the activation of mitoKATP channels results in a transient increase in free radical species, which in turn mediates the protection observed during IPC (9
, 14)
, probably via activation of a MAP kinase signaling cascade (5
, 6)
.
There is much evidence to suggest that adenosine is a major physiological trigger of IPC and that the signal transduction pathway downstream of adenosine A1 receptor activation likely involves PKC translocation and activation of KATP channels (6)
. Links have been proposed between adenosine/PKC activation and sarcKATP channels (32)
as well as mitoKATP channels (33)
further implicating these signaling pathways in the processes underlying IPC. We recently proposed that sarcKATP and mitoKATP channels may be activated by common signaling pathways, consistent with data suggesting that the regulatory sites of mitoKATP channels face the cytoplasm (34)
. Our present study demonstrates that A1 receptor stimulation inhibits calcium overload during the hypoxia/reoxygenation period and that this effect is mediated largely via PKC and sarcKATP channel activation during reoxygenation. The myocyte viability results indicate that A1 receptor activation prevents cell death via a PKC-dependent mechanism that involves both sarc and mitoKATP channel activation. The differences between A1 receptor agonists and phorbol esters and the relative importance of sarcKATP and mitoKATP channels in mediating protection may be due to the isoforms of PKC stimulated by these respective pathways. Although the cellular model used in our study allowed us to temporally resolve the contributions of sarcKATP and mitoKATP channels, there are limitations to extrapolation of findings from isolated cells to the events occurring in the whole heart in vivo. Nevertheless, our data agree with recent whole-heart studies that implicate physiological stimuli such as adenosine and indicate in a role for both types of KATP channel: mitoKATP channel activation reduces cell death whereas sarcKATP channel activation reduces reperfusion induced stunning, possibly through a calcium-dependent mechanism (16
, 37)
.
The molecular sites of action of signal transduction events in IPC are important to our understanding of the IPC process. Our recent identification of the PKC phosphorylation site on the sarcKATP channel complex provided an opportunity to functionally uncouple channel activation from PKC stimulation (30)
. Our present results indicate that substitution of the phospho-acceptor threonine residue in the pore-forming subunit of the KATP channel (Kir6.2, T180A) prevents PKC-mediated reduction in calcium overload in a recombinant model of hypoxia/reoxygenation. These findings demonstrate, for the first time, a functional coupling of PKC directly to the protective effects of the sarcKATP channel complex.
The importance of KATP channels in the response of the heart to acute ischemia/reperfusion injury, and ischemic preconditioning, has been studied extensively, and the respective contributions of the sarc and mito KATP channel populations remain under debate (7
, 10
, 11)
. Some recent evidence favors the activation of mitoKATP channels, rather than sarcKATP channels, as the key link in the process of IPC (10
, 12
13
14)
. These conclusions are based on selective pharmacology of the different KATP channel opening drugs, even though the KATP channel isoform selectivity of KATP channel openers such as diazoxide may be intrinsically linked to the metabolic status of the cell (35)
, which is altered during simulated ischemia/reperfusion protocols. Nevertheless, much evidence suggests that mitoKATP channels are intimately involved in the transduction of the preconditioning event leading to cellular protection, and our results support the specific conclusion that mitoKATP channels contribute to PKC-mediated protection during acute hypoxia.
Most striking, however, our data also demonstrate that activation of the sarcKATP channel is an important myoprotective mechanism acting via a PKC- and adenosine-dependent pathway and prevents intracellular calcium overload during the posthypoxic reoxygenation period. The ability of sarcKATP channel activation to reduce calcium overload has direct implications for understanding the reperfusion-induced stunning observed in many studies, which is thought to be mediated in part by excess intracellular calcium entry (22
, 36)
. Furthermore, adenosine-induced IPC has been shown to induce anti-infarct and anti-stunning components of protection (37)
. Whether these protective components are mediated via separate activation of the sarc and mito KATP channel populations, respectively, remains to be determined, although our results and those of others indicate that this may be the case (16
, 37
, 38)
.
In conclusion, we propose that the sarc and mitoKATP channel populations have distinct but complementary roles to play in the transduction of the cardioprotective effects of IPC mediated by adenosine and PKC. The apparent occurrence of similar processes in the central nervous system (39)
suggests they are of wide significance, as does our own observation of protection in the tsA201 cell line.
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ACKNOWLEDGMENTS |
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Note added in proof: Most of the results reported here were described in a thesis by Hussein D. Kanji presented to the University of Calgary in partial fulfillment of requirements for an M.Sc. degree.
Received for publication May 9, 2001.
Revision received August 16, 2001.
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REFERENCES |
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H. J. Ranki, G. R. Budas, R. M. Crawford, A. M. Davies, and A. Jovanovic 17{beta}-Estradiol regulates expression of KATP channels in heart-derived H9c2 cells J. Am. Coll. Cardiol., July 17, 2002; 40(2): 367 - 374. [Abstract] [Full Text] [PDF]
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