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Vol. 55, Issue 6, 1000-1005, June 1999
Section of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland
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Summary |
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 Top
 Summary
 Introduction
Materials and Methods
Results
Discussion
References
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A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 µM pinacidil but only weakly by 100 µM diazoxide; in addition, they are blocked by 10 µM glibenclamide, but are insensitive to 500 µM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 µM diazoxide and 100 µM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level.
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Introduction |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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In
1986, Murry and coworkers (Murry et al., 1986
) described a paradoxical
form of cardioprotection whereby brief ischemic insults can reduce the
damage to the heart caused by subsequent, prolonged ischemia. This
phenomenon, known as ischemic preconditioning, appears to involve
ATP-sensitive potassium (KATP) channels as the
end effectors. Such a key role was first attributed to the classical
sarcolemmal KATP
(surfaceKATP) channels (Gross and Auchampach, 1992; Speechly-Dick et al., 1995; Liang, 1997; Kloner et al., 1998),
but recent evidence implicates the more nebulous mitochondrial KATP (mitoKATP) channels
(Inoue et al., 1991; Garlid et al., 1997; Liu et al., 1998; Sato et
al., 1998).
Native cardiac mitoKATP channels share some
pharmacological properties with their classical, sarcolemmal
counterparts. Both channels are activated by pinacidil and inhibited by
glibenclamide (Paucek et al., 1992; Garlid et al., 1997). Thus,
specific inhibitors and activators are in demand for the study of
cardiac mitoKATP channels; 5-hydroxydecanoate
(5HD) and diazoxide are candidates for such specific reagents (Garlid
et al., 1997; Liu et al., 1998; Sato et al., 1998). Nevertheless, there
is controversy as to whether 5HD blocks
surfaceKATP channels (Notsu et al., 1992; Garlid
et al., 1997; Liu et al., 1998). Likewise, the extent to which the activator diazoxide opens surfaceKATP channels is
a matter of some dispute (Inagaki et al., 1996; Garlid et al., 1997;
Liu et al., 1998).
The molecular identity of cardiac mitoKATP
channels remains unclear, although cardiac
surfaceKATP channels have been molecularly defined as an octameric complex of four pore-forming
Kir6.2 subunits and four SUR2A sulfonylurea
receptors (Inagaki et al., 1996; Clement et al., 1997). To distinguish
unambiguously between surfaceKATP and
mitoKATP channels, we compared native cardiac
mitoKATP channels to molecularly defined and
native surfaceKATP channels. Differences in
pharmacology and subcellular distribution reveal clear distinctions between cardiac mitoKATP and
surfaceKATP channels.
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Materials and Methods |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Functional Expression of Kir6.2 + SUR2A
Channels.
The transient transfection method in human embryonic
kidney (HEK)293 cells was modified from Hu et al. (1997). Briefly,
HEK293 cells were maintained in Dulbecco's minimal Eagle's medium
with glucose and L-glutamine, supplemented with 10% fetal
calf serum (GIBCO-BRL, Gaithersburg, MD) and 1% penicillin and
streptomycin (GIBCO-BRL). Cells were plated on 35-mm Petri dishes at a
density of 0.2 million cells/dish 1 day before transfection, and
maintained in a 37°C incubator. Cells were then transfected by
calcium phosphate precipitation (Graham and van der Eb, 1973; Calcium
Phosphate Transfection System, GIBCO-BRL) with 0.5 to 1 µg/dish of
equimolar plasmid DNAs of the rabbit cardiac
Kir6.2 subunit (Hu et al., 1999) and the
rat SUR2A sulfonylurea receptor (Inagaki et al., 1996), supplemented
with 0.2 µg/dish of mitochondrially targeted green fluorescent
protein, which enabled visual identification of transfected cells
(Marshall et al., 1995). The calcium phosphate-DNA mixture was left on
cells for 5 to 6 h before washing with PBS and the addition of
fresh media.
Electrophysiology of Expressed Kir6.2 + SUR2A
Channels.
For functionally expressed
surfaceKATP channels, electrophysiological
recordings were made 18 to 48 h after transfection. A coverslip
with cells was placed in a 0.3-ml perfusion chamber connected to a
gravity-driven perfusion system. Flow was maintained throughout the
experiment at rates of 2 to 3 ml/min. Membrane current was recorded
using the whole-cell patch configuration (Hamill et al., 1981), with
bath solution containing (in mM): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4 titrated with NaOH. The pipette solution contained: 120 mM K-glutamate, 5 mM K-EGTA, 25 mM KCl, 3 mM MgATP, and 10 mM HEPES, pH
7.2, titrated with KOH (21-22°C). Junction potential was corrected.
In voltage-clamp recordings, a ramp voltage command from 
100 mV to
+50 mV was applied over 100 ms every 10 s, from a holding
potential of 80 mV. Currents were recorded using a patch-clamp
amplifier (Axopatch 200, Axon Instruments, Inc.), and sampled at 10 kHz
after analog filtering at 2 to 5 kHz. Acquisition and analysis of the
data were performed with custom software. For data analysis, the
current at 0 mV was measured to assay KATP
channel activity. The holding current at 80 mV (near
EK) was also measured to verify the gigaseal stability. In the study of the effect of 5HD on membrane current, 1 min
of KATP currents before drug application were
averaged as the control, and the currents 5 to 6 min into drug
application were averaged for comparison with the control. The
effect of reagent diazoxide was assayed in a similar way, except that
steady-state currents (typically achieved within 5-10 min) were measured.
Confocal Imaging Green Fluorescent Protein
(GFP)-Kir6.2, Mitochondrial GFP, and
Mitochondria.
The murine Kir6.2 cDNA (Inagaki et al., 1995) was
amplified by the polymerase chain reaction and cloned into the vector
pEGFP-C3 (CLONTECH, Palto Alto, CA) to make pEGFP-Kir6.2. This
created a single open reading frame encoding a fusion protein between EGFP on the amino terminus and Kir6.2 on the carboxy terminus. Stable
cell lines were created by transfecting HEK293 cells with linearized
pEGFP-Kir6.2 using lipofectamine (Life Technologies, Gaithersburg, MD)
followed by selection with geneticin sulfate at 500 µg/ml. Cells
surviving selection were plated on glass coverslips and imaged.
Mitochondrially targeted GFP (Rizzuto et al., 1995) was transiently
transfected into HEK293 cells as described above, and the cells were imaged.
) by polymerase chain reaction
to allow for in-frame fusion with EGFP in the vector pEGFP-N3
(CLONTECH) to create pRabKir6.2GFP. The expression cassette was removed
and cloned into the multiple cloning site of modified adenovirus
shuttle vector pAdECd8I (Johns et al., 1999) to generate
pAdECd8Kir6.2GFP. The recombinant adenovirus containing the
RabKir6.2GFP fusion gene product was generated by Cre-lox recombination
as described previously (Johns et al., 1999). Isolated rabbit
ventricular myocytes were coinfected with AdRabKir6.2GFP and AdVgRXR
(Johns et al., 1999) and plated on to coverslips in M199 culture medium
with 5% fetal bovine serum at 37°C. Expression was induced by adding
5 µM ponasterone A for 72 h before imaging.
Confocal images were obtained using a 60× water immersion lens on a
Diaphot 300 inverted fluorescence microscope with a PCM-2000 confocal
scanning attachment (Nikon, Inc., Melville, NY). GFP was excited by the
488-nm line of an argon laser and the emission at 505 to 535 nm was
imaged. Mitochondria were stained with tetramethylrhodamine ethyl ester
(TMRE; added to the bath at a concentration of 100 nM and allowed to
redistribute into the mitochondrial matrix for 15 min). TMRE was
excited with the 543-nm line of a helium-neon laser and the 589- to
621-nm emission was imaged.
Flavoprotein Fluorescence and Electrophysiology of Rabbit
Ventricular Myocytes.
Ventricular myocytes were isolated from
adult rabbit hearts by conventional enzymatic dissociation (Liu et al.,
1996). Cells were then cultured on laminin-coated coverslips in M199
culture medium with 5% fetal bovine serum at 37°C. Experiments were
performed over the next 2 days. Mitochondrial redox state was monitored by recording the fluorescence of flavin adenine nucleotide
(FAD)-linked enzymes in the mitochondria and served as an index
of mitoKATP activity (Liu et al., 1998). Myocytes
were superfused with external solution containing: 140 mM NaCl, 5 mM
KCl, 1 mM CaCl2, 1 mM
MgCl2, and 10 mM HEPES, pH 7.4, with NaOH
(21-22°C). Endogenous flavoprotein fluorescence was excited for 100 ms every 6 s using a xenon arc lamp with a band-pass filter
centered at 480 nm, and emitted fluorescence was recorded at 530 nm by
a photomultiplier tube and digitized (Digidata 1200, Axon Instruments,
Foster City, CA). By focusing on individual myocytes with a 40×
objective, fluorescence was monitored from one cell at a time. The
flavoprotein fluorescence signal was averaged during the excitation
window and expressed as a percentage of the 2,4-dinitrophenol
(DNP)-induced maximal oxidation.
80 mV by two consecutive steps to 40
mV (for 100 ms) and 0 mV (for 380 ms). Currents at 0 mV were measured
200 ms into the pulse.
Chemicals. Diazoxide and glibenclamide were purchased from Sigma Chemical Co. (St. Louis, MO). Pinacidil and 5HD were purchased from Research Biochemical International (Natick, MA). TMRE was obtained from Molecular Probes (Eugene, OR). To prepare stock solutions, 5HD and DNP were dissolved in water, and the other drugs were dissolved in dimethyl sulfoxide. Ponasterone A was purchased from Invitrogen (San Diego, CA) and used according to the manufacturer's instructions.
Statistical Analyses. Pooled data are presented as mean ± S.E.M. Statistical comparison was evaluated by the two-tailed paired or unpaired Student's t test where appropriate, with P <.05 considered significant.
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Results |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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5HD Has Little Effect on Expressed Cardiac SurfaceKATP
Channels.
Figure 1 shows the effect
of 5HD on currents elicited by 100 µM pinacidil in a HEK293 cell
expressing Kir6.2 + SUR2A, which forms
cardiac-type surfaceKATP channels (Inagaki et
al., 1996). Figure 1A shows a time course of pharmacological responses
in a representative experiment quantified from membrane currents elicited by ramp pulses (as exemplified in Fig. 1B); these data illustrate that 500 µM 5HD did not convincingly inhibit the current, but subsequent application of glibenclamide (10 µM) did so
completely. Fig. 1C shows the pooled results in a box graph, presented
as the ratio of the current in the presence of both 5HD and pinacidil, to that originally induced by pinacidil alone. Overall, 5HD imposed no
significant inhibitory effect (3 ± 3%, n = 5).
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Diazoxide Weakly Activates Expressed Cardiac
SurfaceKATP Currents.
Diazoxide weakly activated
expressed KATP currents, as illustrated in Fig.
2. Upon washout of diazoxide, a
subsequent application of 100 µM pinacidil activated much more
current. Overall, 100 µM diazoxide elicited only 4 ± 1%
(n = 26, P <.03) of the current activated by 100 µM pinacidil, which is a nearly saturating
concentration in this expression system (Hu et al., 1998).
Diazoxide-induced currents were completely suppressed by glibenclamide
(n = 3, not shown).
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; Liu et al., 1998), we questioned
whether the minor activation of the expressed channels might have
actually reflected cross talk between the mitochondrial and the
sarcolemmal channels. We thus examined the effect of 5HD on
diazoxide-induced currents in the expression system. Figure 2 shows the
effect of 100 µM diazoxide and 500 µM 5HD on cells expressing
Kir6.2 + SUR2A channels. Diazoxide reversibly
activated a small outward current at 0 mV. In the presence of 5HD, a
second challenge with diazoxide induced a current comparable with the
first one. After washing out diazoxide and 5HD, pinacidil activated a
robust current, verifying that the channels were richly expressed. The
inset shows pooled data of the effect of 5HD, presented as the ratio of
the second current (diazoxide + 5HD) versus the first one (diazoxide
only). Overall, 5HD had no effect on diazoxide-induced currents. Thus,
diazoxide weakly activates surfaceKATP
channels encoded by Kir6.2 and SUR2A.
Surface Membrane KATP Currents in Heart Cells.
The
pattern of pharmacologic responsiveness observed for heterologously
expressed channels encoded by Kir6.2 and SUR2A
also applies to the native channels in rabbit cardiac myocytes. We have
previously shown that such channels are resistant to diazoxide but
sensitive to pinacidil (Liu et al., 1998; Sato et al., 1998). Figure
3 shows that pinacidil readily elicits
outward membrane current in a rabbit ventricular cell, an effect that
cannot be suppressed by 5HD. The pooled data (Fig. 3B) extend our
previous observations (Sato et al., 1998); taken together, the data in Figs. 1-3 verify that the molecularly defined
surfaceKATP channels mimic the behavior of native
cardiac surfaceKATP channels.
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Kir6.2 Does Not Target Mitochondrial Membranes.
To
address whether expressed Kir6.2 subunits are
targeted to mitochondrial membranes, we imaged HEK293 cells stably
expressing the fusion construct GFP-Kir6.2.
Figure 4A-C shows the distribution of
expressed Kir6.2 subunits (Fig. 4A, green), the
mitochondria of the cells (Fig. 4B, labeled red by TMRE), and the
overlay image (Fig. 4C). Green signals are distributed in both the
surface and internal membranes of cells, but this distribution does not
overlap that of the red mitochondria. As a positive control, we imaged HEK293 cells transiently expressing mitochondrially targeted GFP in the
same manner. The GFP shows up in the same organelles (Fig. 4D, green)
that are labeled red by TMRE (Fig. 4E), making them yellow in the
overlay image (Fig. 4F).
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5HD Inhibits mitoKATP in Intact Rabbit
Ventricular Myocytes.
Having studied the effects of diazoxide and
5HD on expressed surfaceKATP channels, we then
examined their effects on the native mitoKATP in
rabbit cardiomyocytes. We previously established single-cell methodologies to assay surfaceKATP and
mitoKATP channel activities by measuring membrane
current and flavoprotein fluorescence simultaneously (Liu et al., 1998;
Sato et al., 1998); in the present study, we verified the effects of
the reagents on mitoKATP channels in intact rabbit ventricular myocytes not invaded by patch pipettes.
). 5HD inhibited the effect of diazoxide to 7 ± 2% of
the DNP level (n = 5, P <.005 versus
diazoxide alone). These results indicate that diazoxide activates, and
5HD inhibits, mitoKATP channels in intact rabbit ventricular myocytes.
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Glibenclamide Independently Activates Flavoprotein Fluorescence in
Intact Rabbit Cardiomyocytes.
Glibenclamide, a well-known
sulfonylurea KATP channel blocker, has also been
reported to be an uncoupler of mitochondria (Szewczyk et al., 1997). On
uncoupling respiration from ATP synthesis, the mitochondrial redox
potential is oxidized like that induced by DNP. We therefore examined
the effect of glibenclamide on flavoprotein fluorescence. Figure
7 illustrates one such experiment.
Glibenclamide (100 µM) induced reversible oxidation of the
flavoprotein. This effect was concentration dependent. As summarized in
Fig. 7B, 10 and 100 µM glibenclamide increased mitochondrial
oxidation to 15 ± 4% (n = 4) and 27 ± 4%
(n = 5), respectively. Thus, glibenclamide is not a
useful probe of mitoKATP-induced mitochondrial
oxidation.
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Discussion |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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We have shown that expressed cardiac KATP channels (Kir6.2 + SUR2A) are activated strongly by pinacidil but only weakly by diazoxide, while being blocked by glibenclamide and essentially insensitive to 5HD. This pharmacology contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation in intact rabbit ventricular myocytes. MitoKATP channels are activated equally by diazoxide and pinacidil, and 5HD is an effective blocker of mitoKATP channels. These differences establish major pharmacological distinctions between the two channels at a functional level. In addition, confocal imaging reveals that expressed Kir6.2 subunits target sarcolemmal but not mitochondrial membranes. These key distinctions indicate that mitoKATP channels differ importantly from surfaceKATP channels at the molecular level.
In this study, we provide the first direct evidence that glibenclamide
alone (10 µM or 100 µM) causes mitochondrial oxidation. This effect
of glibenclamide precludes investigation in intact cells of its
reported inhibition of mitoKATP channels in
isolated mitochondria or reconstituted liposomes (Inoue et al., 1991;
Paucek et al., 1992); nevertheless, the observations are consistent
with the reported uncoupling effect of glibenclamide on mitochondria (Szewczyk et al., 1997). In addition, glibenclamide effectively blocks
both native and expressed cardiac surfaceKATP
channels (Edwards and Weston, 1993; Inagaki et al., 1996; Hu et al.,
1999). Thus, glibenclamide is a less-than-optimal probe in the study of
mitoKATP channels.
The results with glibenclamide illustrate one limitation of the
methodology that we have used to detect mitoKATP
activity. The method relies upon flavoprotein fluorescence
to report the redox potential of the mitochondrial matrix.
Although opening of inner membrane potassium channels will tend to
dissipate the mitochondrial potential and lead to net oxidation of the
matrix, so will uncouplers such as glibenclamide and DNP (Liu et al., 1998). The only other approaches devised so far to detect
mitoKATP activity use isolated mitochondria
(Inoue et al., 1991; Garlid et al., 1997), which necessarily involves
removing the organelles from their physiological surroundings. Thus,
the two types of approaches have complementary strengths and
limitations. It is therefore reassuring that similar conclusions
regarding the pharmacology of mitoKATP channels
have been derived from studies of flavoprotein fluorescence in intact
myocytes and potassium accumulation in isolated mitochondria (Garlid et
al., 1997; Liu et al., 1998).
Identifying a specific mitoKATP channel blocker
is very important, especially given that mitoKATP
channels have been pinpointed as likely effectors of ischemic
preconditioning (Garlid et al., 1997; Liu et al., 1998). Our data
identify 5HD as a specific blocker of mitoKATP
channels. At 500 µM, 5HD did not inhibit pinacidil-induced currents
through expressed or native cardiac surfaceKATP
channels, nor did it suppress diazoxide-induced
surfaceKATP currents. In native cardiomyocytes,
5HD dramatically suppresses mitoKATP channel activity, yet it does not affect surfaceKATP
currents (McCullough et al., 1991; Garlid et al., 1997; Liu et al.,
1998; Sato et al., 1998), with the exception of one report that 5HD
blocks surfaceKATP channels in excised patches
from guinea pig cardiomyocytes (Notsu et al., 1992). The reason for
this contradictory observation remains unclear. In animal studies, 5HD
has long been recognized to abolish cardioprotection (Auchampach et
al., 1992; Hide and Thiemermann, 1996; Schultz et al., 1997). The
establishment of its specificity further implicates
mitoKATP channels as key players in the mechanism of ischemic preconditioning.
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Acknowledgments |
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We thank Ms. Maria Janecki for technical assistance, Dr. Susumu Seino for providing SUR2A, and Dr. Yoshihisa Kurachi for providing mouse Kir6.2.
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Footnotes |
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Received August 18, 1998; Accepted March 7, 1999
1 These authors contributed equally to the work.
2 Current address: Osiris Therapeutics, Inc., 2001 Aliceanna St., Baltimore, MD 21231.
3 Current address: Maryland Research Laboratories, Otsuka America Pharmaceutical Inc., Rockville, MD 20850
This work was supported by National Insitutes of Health Grants R01 HL52768 and R01 HL44065 (to E.M.), and T32 HL07227 (to H.H.), an American Heart Association-Maryland Fellowship (to H.H.), and a Banyu Fellowship in Lipid Metabolism and Atherosclerosis (to T.S.).
Send reprint requests to: Eduardo Marbán, M.D., Ph.D., 844 Ross Bldg., The Johns Hopkins University School of Medicine, Baltimore, MD 21205. E-mail: marban{at}jhmi.edu
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Abbreviations |
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5HD, 5-hydroxydecanoate; DNP, 2,4-dinitrophenol; GFP, green fluorescent protein; EGFP, enhanced GFP; HEK, human embryonic kidney; Kir, inwardly rectifying potassium channels; KATP channels, ATP-sensitive potassium channels; mitoKATP, inner mitochondrial membrane KATP, SUR, Sulfonylurea receptor; surfaceKATP, sarcolemmal (surface membrane) KATP..
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References |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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)-opioid receptor in the intact rat heart.
J Mol Cell Cardiol
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B. O'Rourke Myocardial KATP Channels in Preconditioning Circ. Res., November 10, 2000; 87(10): 845 - 855. [Abstract] [Full Text] [PDF]
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W. Tang, M. H. Weil, S. Sun, A. Pernat, and E. Mason KATP channel activation reduces the severity of postresuscitation myocardial dysfunction Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1609 - H1615. [Abstract] [Full Text] [PDF]
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) in the presence
or absence of 100 µM pinacidil (Pin), 500 µM 5HD, or 10 µM
glibenclamide (Glib). The holding current at 
). B, representative ramp currents recorded under drug-free
conditions (a), when pinacidil was applied (b), when 5HD was added (c),
and when glibenclamide was added (d), at the times indicated in A. Pinacidil was present (b-d). C, pooled data from five experiments of
the relative current I5HD+Pin versus IPin,
i.e., the current at time c versus that at time b as indicated in A.
