AJP - Heart AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 283: H1545-H1554, 2002. First published May 16, 2002; doi:10.1152/ajpheart.01052.2001
0363-6135/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/4/H1545    most recent
01052.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (49)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, J.
Right arrow Articles by Earm, Y. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, J.
Right arrow Articles by Earm, Y. E.
Vol. 283, Issue 4, H1545-H1554, October 2002

ATP-sensitive K+ channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes

Jin Han1, Nari Kim1, Hyun Joo2, Euiyong Kim1, and Yung E. Earm3

1 Department of Physiology and Biophysics, College of Medicine, Inje University, Busan 614-735; 2 Department of Molecular Science and Technology/Life Science, Ajou University, Suwon 442-749; and 3 National Research Laboratory for Cellular Signaling and Department of Physiology, College of Medicine, Seoul National University, Seoul 110-799, Korea


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K+ (KATP) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated KATP channel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 µM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 µM) increased KATP channel activity in cell-attached patches. PKG (5 U/µl) added together with ATP and cGMP (100 µM each) directly to the intracellular surface increased the channel activity. Activation of KATP channels was abolished by the replacement of ATP with ATPgamma S. Rp-pCPT-cGMP (100 µM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the KATP channels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated KATP channel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of KATP channels in rabbit ventricular myocytes.

phosphorylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A BRIEF PERIOD OF ischemic preconditioning has been shown to protect the heart against subsequent prolonged ischemia in all species examined to date, including humans (61). In general, ischemic preconditioning appears to provide electrical stability and tissue resistance to ischemia via some underlying mechanisms whereby metabolic conservation occurs (54).

ATP-sensitive K+ (KATP) channels are thought to play a role in the phenomenon of ischemic preconditioning in the heart, and the activation of these channels may improve recovery of regional contractile function of stunned myocardium by shortening action potential duration and attenuating membrane depolarization, thus decreasing contractility and preserving energy during ischemia. These effects in turn reduce the duration of Ca2+ influx through L-type Ca2+ channels and increase the time for the Na+/Ca2+ exchanger to extrude Ca2+ from the cell, both of which will prevent intracellular Ca2+ overload and myocardial tissue injury (3, 11, 13).

The activation of cardiac muscarinic receptors due to vagal stimulation (35, 60), the release of myocardial nitric oxide (NO) (23, 38), and generation of bradykinin (41, 55) during ischemia have been suggested to play roles in ischemic preconditioning. A common mechanism of these findings is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute to the cardioprotective effect against ischemia-reperfusion injury in various species (7, 40, 58).

NO has been known to activate guanylate cyclase and to generate cGMP (1). A recent study (5) showed that cardiac myocytes express NO synthase, not only the inducible isoform (6) but also the constitutive isoform. In fact, recent studies (38) have shown that the NO level increases dramatically in the ischemic heart to reduce both coronary vascular tone and the extent of the ischemia. Furthermore, NO can protect the heart against ischemia-induced reperfusion injury (22). Thus one would predict that NO modulates the KATP channel during ischemia and reperfusion injury.

Thus it seems reasonable that cGMP-mediated intracellular signal transduction plays an important role in the mechanism of ischemic preconditioning. cGMP is a second messenger that mediates a considerable part of its effects by protein kinase G (PKG) (15, 20, 27, 31, 32, 52, 53). PKG is a serine-threonine protein kinase and has been shown to play a role in the mechanism of cardioprotection during ischemia (39, 41). The KATP channel is activated by phosphorylation of the serine-threonine residue in rat cardiac myocytes, as shown in a recent study (29). Indeed, there are potential phosphorylation sites, including serine-threonine residues in the cloned KATP channels (4, 25, 34).

Previous findings raise the intriguing possibility that the myocardial protection afforded by KATP channel activation may involve phosphorylation of KATP channels by PKG during ischemia. Accordingly, in this study, we tested the hypothesis that NO-cGMP-PKG-mediated phosphorylation of KATP channels is involved in the activation of KATP channels in rabbit ventricular myocytes. We observed that sodium nitroprusside (SNP), a potent stimulator of cGMP formation, and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a potent stimulator of PKG, potentiated the pinacidil-induced KATP channel activity. Furthermore, we observed evidence that PKG activates KATP channels in the presence of cGMP and ATP and that the PKG-mediated KATP channel activity is inhibited by 8-pCPT-cGMP Rp isomer (Rp-pCPT-cGMP), a specific blocker of PKG, and protein phosphatase 2A (PP2A). These results suggest that PKG is involved in the phosphorylation of the KATP channel or an associated protein. Such results may be important in understanding the mechanism by which the NO-cGMP-PKG signaling pathway acts as a link in receptor-mediated increases in KATP channel activity during ischemic preconditioning.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. Single ventricular myocytes were isolated from rabbit hearts by an enzymatic dissociation procedure, as discussed previously (18, 19). Briefly, rabbits weighing 150-200 g were anesthetized by injection of pentobarbital sodium (50 mg/ml, 1 ml/kg body wt) and heparin (300 IU/ml) into the marginal ear vein. Hearts were rapidly removed via thoracotomy with artificial ventilation and the aorta was cannulated. A dissected heart was mounted on a Langendorff apparatus and perfused retrogradely with oxygenated normal Tyrode solution for 5-6 min until all signs of blood were removed with gentle squeezing of the heart. The hearts were then perfused with a normal Ca2+-free Tyrode solution for 5 min, followed by perfusion with Ca2+-free Tyrode solution containing 0.01% collagenase (5 mg/50 ml, Yakult). After 15-25 min of enzymatic treatment, Kraftbrühe (KB) solution was perfused. After being perfused with KB solution for 5 min, the hearts were removed from the cannula, the atria were discarded, and the ventricular walls and septum were cut vertically into 4-6 pieces. They were gently agitated in a small beaker with KB solution to obtain single cells. Isolated ventricular cells were stored in a KB solution at 4°C and used within 12 h. Langendorff column was kept at 37°C during all previous steps.

Electrophysiological methods. Single channel currents were measured in the cell-attached, inside-out, and outside-out patch configurations of the patch-clamp technique (17). Channel activity was measured using a patch-clamp amplifier (Axopatch-1D, Axon Instruments; Foster City, CA). Pipettes of 5- to 10-MOmega resistance were pulled from borosilicate glass capillaries (Clark Electrochemical; Pangbourne, UK) using a vertical puller (model PP-83, Narishige; Tokyo, Japan). Their tips were coated with Sylgard and fire polished. Membrane currents were digitized at a sampling rate of 20 kHz and stored in digitized format on digital audiotapes with the use of a recorder (model DTR-1200, Biologic; Grenoble, France). For the analysis of single channel activity, the data were transferred to a personal computer (Pentium III 450, IBM; Busan, Korea) with pCLAMP software (version 6.3; Axon Instruments, Union City, CA) through an analog-to-digital converter interface (Digidata-1200, Axon Instruments).

Data analysis and quantification of channel activity. The threshold for judging the open state was set at one-half of the single channel amplitude (12). Open probability (Po) was calculated by using the formula
P<SUB>o</SUB><IT>=</IT><FENCE><LIM><OP>∑</OP><LL><IT>j=</IT>1</LL><UL><IT>N</IT></UL></LIM><IT>t<SUB>j</SUB> · j</IT></FENCE><IT>/</IT>(<IT>T</IT><SUB>d</SUB><IT> · N</IT>)
where tj is the time spent at current levels corresponding to j = 0, 1, 2, ... N channels in the open state, Td is the duration of the recording, and N is the number of channels active in the patch. The number of channels in a patch was estimated by dividing the maximum current observed during an extended period at zero ATP by the mean unitary current amplitude. When the dose-response relationship between pinacidil and KATP channel activation was examined at a dose range of 0.1-1,000 µM, we found that the concentration at which KATP channel activity was maximal for pinacidil was estimated to be 600-800 µM. On the basis of the data, the 1,000 µM pinacidil dose was used for the estimation of the maximum number of KATP channels in the cell-attached and outside-out patch experiments. Po was calculated over 30-s records.

Rundown of KATP channels. KATP channel activity in rabbit ventricular myocytes decreases slowly with time after patches are excised into ATP-free solution. This phenomenon is known as "rundown." At the time of excision, patches were continuously exposed to ATP (100 µM) except for a brief exposure to zero ATP at the beginning and end of experiments to estimate the number of channels in a patch and the degree of rundown. The data from patches exhibiting >50% rundown were discarded. In experiments designed to test the effects of PKG activation on KATP channel activity, patches were continuously exposed to 100 µM ATP, unless otherwise stated. This concentration of ATP was chosen to represent a half-maximal inhibition level of ATP while giving a Po to allow single channel events to be observed.

Solutions and drugs. Normal Tyrode solution contained (in mM) 143 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 5.5 glucose, and 5 HEPES (pH 7.4) with NaOH. In the cell-attached and inside-out patch clamp experiments, the pipette solution contained (in mM) 140 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4) with KOH, whereas the bath solution contained (in mM) 127 KCl, 13 KOH, 1 MgCl2, 5 EGTA, 10 glucose, and 10 HEPES (pH 7.4) with KOH. In outside-out patch-clamp experiments, the pipette and bath solution were opposite to those used in the cell-attached and inside-out patch experiments. The modified KB solution had the following composition (in mM): 25 KCl, 10 KH2PO4, 16 KOH, 80 glutamic acid, 10 taurine, 14 oxalic acid, 10 HEPES, and 11 glucose at pH 7.4 adjusted with KOH.

ATP was added to the intracellular solution and glibenclamide was added to the extracellular solutions according to the experimental protocols described in the text. Glibenclamide was dissolved as a 0.2 mM stock solution in 2% dimethyl sulfoxide (DMSO) and diluted into the test solution appropriately before study. The final concentration of DMSO contained in the test solution was <0.01%. We confirmed that DMSO at this concentration had no effect on KATP channel activity. After drugs were added to the test solution, the pH was readjusted to 7.4 with KOH. Pinacidil (RBI; Natick, MA) was freshly prepared before experiments and diluted into the test solution to obtain the final concentrations indicated in the text. Okadaic acid (OA) was purchased from RBI. OA was stored at a stock concentration of 100 µM in ethanol at 4°C and used at a final concentration of 5 nM. PP2A was purchased from UBI (Lake Placid, NY). PKG was obtained from Promega (Madison, WI). 8-CPT-cGMP and Rp-pCPT-cGMP were obtained from Biolog Life Science Institute (Bremen, Germany). Unless noted otherwise, the agents were obtained from Sigma (St. Louis, MO). The experiments were performed at a room temperature of 25 ± 2°C.

Solution exchange system. In most experiments, we used a superfusion system (DAD-12, Adams and List; New York, NY) to change the bath solution and drugs. The system was designed to simplify the application of various concentrations of drugs and solutions to cells. The system takes advantage of the "sewer pipe effect." When pointed at the cell to be studied, if the cell remains within the stream of the solution, the cell was essentially immersed completely in the solution that was applied. We could stop and start the flow quickly and change it with up to 12 variables by using the system.

Statistics. Data are presented as means ± SE when appropriate. Student's unpaired t-test was used to calculate statistical significance. P <=  0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of SNP on KATP channel activity of rabbit ventricular myocytes. To test the hypothesis that KATP channels are involved in the action of NO, the effects of the NO donor on KATP channels in cell-attached patches were investigated (Fig. 1, A and B). NO is well known as a stimulator of soluble guanylate cyclase and produces its effects by increasing intracellular cGMP concentrations, leading to an activation of PKG (1). We used sodium nitroprusside (SNP), a potent stimulator of cGMP formation, which has been known to be a NO donor (49). After gigaseal formation, the bath solution was switched from normal Tyrode solution to a high-K+ solution. At the pipette potential of -40 mV, unitary currents through the inward rectifier K+ channel were recorded, which was identified from its current-voltage (I-V) relations showing a slope conductance of ~33 pS for the inward current. Under these conditions, KATP channels were inactive even in the presence of 1 mM SNP, but subsequent bath application of pinacidil (50 µM) opened KATP channels (Fig. 1A). The I-V relations for such a channel showed a mean single channel conductance of about 76 pS and a reversal potential of near 0 mV in cell-attached patches. Potentiation of the pinacidil-induced KATP channel activity by the addition of SNP (1 mM) was observed in 12 patches, in which activity (measured as Po) of KATP channels increased from 0.112 ± 0.053 to 0.287 ± 0.083 mM (P < 0.05, n = 12 patches) (Fig. 1B). This potentiation was reversed by washout of SNP. SNP had no effect on single channel current amplitude. Mean single channel currents before and after the addition of SNP were 2.9 ± 0.1 and 2.9 ± 0.3 pA, respectively (n = 12 patches).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1.   The effect of sodium nitroprusside (SNP) on the ATP-sensitive K+ (KATP) channel activity in rabbit ventricular myocytes. SNP (1 mM), pinacidil (50 µM), ATP (1 mM), and glibenclamide (30 µM) were added to the bath solution for the periods indicated by the thin bars. A: effect of SNP on the activation of KATP channels in the absence of pinacidil in cell-attached patches. B: reversible activating effect of SNP on the pinacidil-induced KATP channel activity in cell-attached patches. The pipette potential was held at -40 mV in the cell-attached patch experiments. C: effect of SNP on the KATP channel activity in inside-out patches held at -40 mV. D: effect of SNP on the KATP channel activity in outside-out patches held at -40 mV. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level.

The effects of SNP on KATP channel activity were examined in either inside-out or outside-out patches to minimize interference from cellular metabolic changes and regulatory enzymes. Figure 1C shows a representative result obtained in an inside-out patch. As soon as a membrane patch was excised in ATP-free internal solution (vertical arrow above current trace), spontaneous KATP channel openings appeared. Application of SNP (1 mM) to the bath failed to enhance the channel activity at -40 mV. In six patches, the average Po was 0.184 ± 0.055 mM before and 0.176 ± 0.071 mM during the addition of SNP (P > 0.05, n = 6 patches). Application of SNP (1 mM) to the extracellular surface of outside-out patch failed to enhance the channel activity (Fig. 1C): the average Po was 0.135 ± 0.081 mM before and 0.141 ± 0.069 mM during the addition of SNP (P > 0.05, n = 3 patches). Addition of ATP (1 mM, Fig. 1B) and glibenclamide (30 µM, Fig. 1D) immediately suppressed this channel activity confirming that observed openings were due to K+ flowing through KATP channels.

To investigate whether SNP facilitates the pinacidil-induced KATP channel activity via a cGMP/PKG-dependent mechanism, we applied Rp-pCPT-cGMP, a selective and membrane-permeant inhibitor of PKG in cell-attached patches (Fig. 2). The potentiating effect of SNP on KATP channel activity was suppressed by Rp-pCPT-cGMP (100 µM) in a reversible manner in all of the cells tested (Fig. 2A); the average Po was 0.237 ± 0.049 before and 0.092 ± 0.053 during the addition of Rp-pCPT-cGMP (P < 0.05, n = 5 patches, Fig. 2B).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   The effect of a selective inhibitor of protein kinase G (PKG) on the SNP-induced KATP channel activity. A: current recording from cell-attached patches (see inset) held at -40 mV. The solution exchange protocol for pinacidil, SNP, and Rp isomer 8-(4-chlorophenylthio)-cGMP (Rp-pCPT-cGMP) is shown above the current trace. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level. Arrows indicate channel activities of the records at specific times. B: histogram showing the pooled data (means ± SE) for open probability (Po) for the following conditions: 50 µM pinacidil alone (a), 50 µM pinacidil + 1 mM SNP in the bath solution (b), additional application of 100 µM Rp-pCPT-cGMP (c), and washout of 100 µM Rp-pCPT-cGMP (d). * P < 0.05, significant difference from control (before application of Rp-pCPT-cGMP) value.

Effects of membrane-permeable cGMP analog on KATP channels. To evaluate more directly whether the cGMP/PKG-dependent mechanism produces effects similar to those of the application of SNP, we examined effects of the potent PKG activator 8-pCPT-cGMP on the pinacidil-induced single channel activity in cell-attached patches. 8-pCPT-cGMP has the following advantages over other membrane-permeable analogs of cGMP: 1) it has higher lipophilicity so that it should permeate the cell membrane at higher rates, 2) it has a high degree of specificity of the PKG, 3) it has little effect on cGMP-regulated phosphodiesterases, and 4) it is resistant to degradation by phosphodiesterases. In the experiment shown in Fig. 3A, the pinacidil-induced KATP channel activity was reversibly facilitated by the addition of 8-pCPT-cGMP (100 µM). In six patches, the average Po increased 2.13 ± 0.12 times by 8-pCPT-cGMP (Po = 0.170 ± 0.049) when compared with the Po (0.079 ± 0.028) recorded before the addition of 8-pCPT-cGMP (P < 0.05, n = 6). The pinacidil-induced single channel activity was inhibited by subsequent application of 30 µM glibenclamide (Po = 0.007 ± 0.005). These data are summarized in Fig. 3B.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3.   The effect of a potent activator of PKG on pinacidil-induced single channel activity. The pipette potential was held at -40 mV in cell-attached patches (see inset). A: representative record of the effect of 100 µM 8-pCPT-cGMP, followed by 30 µM glibenclamide, on the pinacidil-induced single channel activity. Glibenclamide applied extracellularly caused a marked inhibition of pinacidil-induced single channel activity. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level. Arrows indicate channel activities of the records at specific times. B: histogram showing the pooled data (means ± SE) for Po for the following conditions: pinacidil alone (a), pinacidil + 8-pCPT-cGMP (b), and subsequent application of glibenclamide (c). * P < 0.05 relative to the pinacidil alone group.

Effect of PKG activation on KATP channel in excised inside-out patches. Because SNP and 8-pCPT-cGMP enhance the KATP channel activity in the cell-attached patches, as described above, these data support the idea that a cGMP-PKG-dependent mechanism can activate KATP channels. To evaluate more directly the involvement of a cGMP-PKG-dependent mechanism in activation of the KATP channel, we applied PKG to the intracellular side of the KATP channel in excised inside-out patches. In the experiments shown in Fig. 4A, after excision of the patch, ATP (100 µM) inhibited spontaneous KATP channel openings, reducing Po from 0.148 to 0.003. In the continuous presence of ATP, PKG (5 U/µl) with cGMP (100 µM), added to the intracellular surface, enhanced the channel activity (Po = 0.239). Such an increase in channel activity by PKG with cGMP in the presence of ATP was observed in six patches, in which the average Po of KATP channels increased from 0.008 ± 0.005 to 0.250 ± 0.083 (P < 0.05, n = 6 patches). In the experiments described in Fig. 4, B and C, a maximal response to PKG activation was observed with 100 µM ATP. As the intracellular ATP concentration increased, the responses were progressively smaller. In Fig. 4D, the channel activity for the ATP concentration used was normalized using the equation y = (Po - Po,min)/(Po,max - Po,min), where y is the relative Po, Po,max is the Po at a given concentration of 100 µM ATP, and Po,min is the Po at 1,000 µM ATP. The continuous line in the graph is the curve fitted to the Hill equation using the least-squares method Po = (Po,max - Po,min)K<UP><SUB>d</SUB><SUP><IT>n</IT></SUP></UP>/(K<UP><SUB>d</SUB><SUP><IT>n</IT></SUP></UP> + [ATP]n) + Po,min, where [ATP] is each ATP concentration, Kd is the concentration of ATP at the half-maximal inhibition of the channel, and n is the Hill coefficient. The Kd value for this inhibitory effect was 384 ± 29 µM (n = 7 patches). cGMP alone (100 µM; P > 0.05, n = 3 patches), cGMP and ATP together (100 µM each; P > 0.05, n = 3 patches), PKG alone (5 U/µl; P > 0.05, n = 3 patches), and PKG and cGMP together (5 U/µl and 100 µM each; P > 0.05, n = 3 patches) had no effect on the channel activity (data not shown).


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.   The effect of PKG on the KATP channel activity. A: change of channel activity in response to PKG activation. Addition of 100 µM ATP to the internal side of patch (see inset) reduced activity of KATP channels present. Subsequent application of PKG together with cGMP enhanced the channel activity. B: effect of intracellular concentrations of ATP on PKG activation-induced increase in KATP channel activity. C: concentration-response relationship for the effects of ATP on KATP channel activity from a series of experiments similar to that shown in B. The Po for each ATP concentration was normalized by referring to the value in the presence of 100 µM ATP. D: PKG activation-induced increase in KATP channel activity for ATP was normalized using the equation in RESULTS. The solid line was drawn from calculations that are described in RESULTS. E: effect of the replacing ATP with ATPgamma S on the channel activity under the same experimental condition as in A. Note that PKG together with cGMP had no effects on ATPgamma S-inhibited channels. Current recording from an inside-out patch configuration (inset) held at -40 mV. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level.

The above results would indicate that PKG is effective only in the presence of cGMP and ATP, which in turn suggests that PKG acts through phosphorylation of the KATP channel or an associated protein. To verify this hypothesis, we replaced ATP with ATPgamma S, a nonhydrolyzable analog of ATP, under experimental conditions similar to that shown in Fig. 4A. In the record shown in Fig. 4E, KATP channels were inhibited by ATPgamma S (100 µM), and Po was reduced from 0.094 to 0.002. However, the subsequent addition of PKG (5 U/µl) along with cGMP (100 µM) failed to enhance the channel activity (Po = 0.003). Similar effects were observed in three other patches.

To confirm the specificity of the PKG in stimulating the channel activity, we repeated the same experiments with heat-inactivated PKG. A stock solution containing only PKG was incubated in a hot water bath (100°C) for 20 min, and this heated PKG was used for the internal solution. Figure 5A shows a representative result obtained in an inside-out patch exposed to ATP (100 µM) at the intracellular surface. In the continuous presence of 100 µM ATP at the intracellular surface (Po = 0.0016), application of heated PKG (5 U/µl) along with cGMP (100 µM) to the intracellular surface failed to enhance the channel activity (Po = 0.0018). Similar results were observed in four other patches; the average Po was 0.003 ± 0.002 µM before and 0.004 ± 0.003 µM during the addition of heated PKG (5 U/µl) and cGMP (100 µM) (P > 0.05, n = 5 patches).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 5.   The specificity of the PKG in stimulating the KATP channel activity. A: effect of heated PKG on the KATP channel activity in the presence of intracellular ATP and cGMP. Note that stimulatory effect of PKG on KATP channel activity results from its enzymatic activity. B: effect of a PKG inhibitor Rp-pCPT-cGMP on PKG activation-induced KATP channel activity. Addition of ATP to the internal side of the patch (see inset) reduced activity of the KATP channel present. PKG increased the channel activity in the presence of ATP and cGMP together. In the same patch, Rp-pCPT-cGMP was added to the bath solution. Note that Rp-pCPT-cGMP reversed the stimulatory effects of PKG on KATP channel activity. Current recording from an inside-out patch configuration held at -40 mV. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level.

To reaffirm the specificity of the PKG in stimulating the channel activity, another experiment was performed with Rp-pCPT-cGMP, a selective and membrane-permeant inhibitor of PKG. Figure 5B illustrates the effects of applying Rp-pCPT-cGMP (100 µM) in the presence of PKG activation in an excised inside-out patch. PKG (5 U/µl) caused an increase in KATP channel activity in the presence of 100 µM ATP and 100 µM cGMP. Addition of Rp-pCPT-cGMP (100 µM) resulted in a reversal of the PKG-mediated KATP channel activation, reducing the Po from 0.090 to 0.005. Similar results were obtained in five of the six patches examined; the average Po was 0.133 ± 0.032 µM before and 0.006 ± 0.002 µM during the addition of Rp-pCPT-cGMP (100 µM) (P < 0.05, n = 5 patches).

PKG-induced activation of KATP channel is reversed by protein phosphatase. In the present study, we observed that the activation of the KATP channel by PKG occurred in the presence of cGMP and ATP. The result suggests that the cGMP stimulates PKG, which in turn activates the channel by phosphorylation. We sought to investigate whether exogenous PP2A, applied intracellularly during PKG activation, could impair the PKG-induced KATP channel activation. Figure 6A illustrates the effects of applying PP2A (1 U/ml) in the presence of PKG activation in an excised inside-out patch. After the patch was excised, 100 µM ATP inhibited spontaneous KATP channel openings and Po was reduced from 0.163 to 0.012. In the continuous presence of ATP, PKG (5 U/µl) and cGMP (100 µM), added to the intracellular surface of the patch, enhanced the channel activity (Po = 0.122). Application of exogenous PP2A inhibited the PKG-mediated KATP channel activity (Po = 0.032). Such a decrease in channel activity by PP2A was observed in five other patches (Fig. 6B).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of exogenous protein phosphatase 2A (PP2A) on the KATP channel activity stimulated by PKG in rabbit ventricular myocytes. A: current recording from an inside-out patch (inset) held at -40 mV. In the presence of ATP, cGMP, and PKG caused an increase in the channel activity. In the same patch, PP2A was added to the bath solution. PP2A caused an inhibition of PKG activation-induced channel activity. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level. Arrows indicate channel activities of the records at specific times. B: change of channel activity in response to PP2A in inside-out patches. Histogram shows the pooled data (mean ± SE) for Po for the following conditions: ATP alone (a), PKG activation (b), and additional application of PP2A (c). * P < 0.05, significant difference from control (before application of PP2A) value.

To investigate whether the PKG-mediated phosphorylation of the KATP channel underlies the PKG-induced activation of KATP channels, further experiments were performed with OA, a potent inhibitor of type 1 protein phosphatase and PP2A (Fig. 7A). OA was used at a low concentration (5 nM) to specifically block the activity of PP2A in excised inside-out patches (21). Application of PKG in the presence of ATP and cGMP caused an increase in the channel activity (Po = 0.268). When ATP, cGMP, and PKG were then washed out and OA was applied to the patches, KATP channel activity remained unchanged (Po = 0.275). In the presence of OA, PP2A did not alter the channel activity (Po = 0.274). The results were observed in six of seven patches examined (Fig. 7B).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of exogenous PP2A on the KATP channel activity stimulated by PKG activation in the presence of okadaic acid (OA), a potent inhibitor of PP2A. A: current recording from an inside-out patch (see inset) held at -40 mV. PKG activation increased the channel activity. On removal of ATP, cGMP, and PKG and exposure to OA, activation of KATP channel persisted. PP2A was ineffective in the presence of OA. Data were sampled at 20 kHz and filtered at 1 kHz. Dashed line indicates the zero current level. Arrows indicate channel activities of the records at specific times. B: change of channel activity in response to PP2A in the presence of OA. Histogram shows the pooled data (mean ± SE) for Po for the following conditions: PKG activation (a) after application of OA on removal of ATP + cGMP + PKG (b) and additional application of PP2A in the presence of OA (c). NS indicates that the differences are not statistically significant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous local agents or systemic factors that elevate myocardial cGMP have been reported to release during myocardial ischemia and play important roles in ischemic preconditioning via cGMP-related signal transduction mechanism like PKG (15, 39). Therefore, it is particularly important to know whether there is any interaction between the cGMP-PKG signaling pathway and KATP channels in the heart. The present study showed, for the first time to our knowledge, that KATP channels can be opened through the cGMP-PKG signaling pathway in rabbit ventricular myocytes.

Our study provided evidence that the activation of KATP channels in rabbit ventricular myocytes can occur through a signal transduction pathway involving stimulation of guanylate cyclase, increased production and accumulation of cGMP, and activation of PKG, which phosphorylates and activates the KATP channel. First, SNP facilitated the pinacidil-induced KATP channel activity in cell-attached patches (Fig. 1). The potentiating effect of SNP was probably mediated by NO, as it is known to be a potent NO donor (48). The role of NO in modulating the KATP channel is further supported by the study by Shinbo and Iijima (47), who demonstrated that NO could potentiate the effects of K+ channel opener in cardiac cells, although they could not explain the underlying mechanism for the potentiation of NO. It seems unlikely that NO directly activates KATP channels because in the present study SNP (NO) failed to facilitate the KATP channel activity in inside-out and outside-out patches. Second, SNP-induced KATP channel activity was reduced by Rp-pCPT-cGMP, a selective inhibitor of PKG (Fig. 2). This finding provided direct evidence that the potentiating effect of SNP (NO) is due to an activation of PKG. This excludes the possibility that the effect of SNP (NO) is mediated via the activation of PKA by cross activation (26) or by cGMP-inhibited phosphodiesterases. Third, 8-pCPT-cGMP, a potent stimulator of PKG, potentiated the pinacidil-induced KATP channel activity (Fig. 3). Fourth, PKG, added together with cGMP and ATP directly to the intracellular surface of inside-out patches, also increased the channel activity (Fig. 4). Finally, the effect of PKG was prevented by Rp-pCPT-cGMP, and heat-inactivated PKG had little effect on the channel activity (Fig. 5).

Such results would predict that PKG acts through phosphorylation of KATP channels or some associated protein. Our present data support this notion in the several ways. First, the PKG-mediated activation of KATP channels required both cGMP and ATP. This was further confirmed by the fact that under similar experimental conditions, replacement of ATP by ATPgamma S, a nonhydrolyzable analog of ATP, abolished the effect of PKG (Fig. 4). Second, application of exogenous PP2A reversed the PKG-induced activation of KATP channels, and OA, a potent inhibitor of type 1 protein phosphatase and PP2A, abolished the effects of PP2A (Figs. 6 and 7). Finally, OA prevented the spontaneous reversal of the PKG-induced activation of KATP channels (Fig. 7). The results also suggested that an endogenous membrane-associated PP2A is responsible for the reversal of PKG-mediated activation of KATP channels. We therefore speculate that KATP channels are under the control of both PKG and PP2A in rabbit ventricular myocytes. The processes of phosphorylation and dephosphorylation thus may regulate the KATP channel activity in rabbit ventricular myocytes, proving a mechanism by which cellular excitability can be reversibly controlled.

The present results obtained from rabbit ventricular myocytes are compatible with others, demonstrating the regulation of KATP channels by a cGMP-related signaling mechanism in vascular smooth muscle cells and follicle-enclosed Xenopus oocytes. Kubo et al. (28) have demonstrated that both atrial natriuretic factor (ANF) and isosorbide dinitrate (ISDN), activators of particulate and soluble guanylate cyclase, respectively, activate KATP channels via an increase of intracellular cGMP in the cultured rat thoracic aorta. They also showed that 8-bromo-cGMP (8-BrcGMP) activated KATP channels and had effects similar to those of ANF and ISDN, suggesting that the modulation of KATP channels by ANF and ISDN is mediated by cGMP. In addition, SNP has been found to hyperpolarize rabbit mesenteric arteries by activating KATP channel with accumulation of cGMP as an intermediate step (36). In follicle-enclosed Xenopus oocytes, ANF potentiated KATP currents via the activation of guanylate cyclase and consequent accumulation of cGMP (45) and similar potentiating effects of 8-BrcGMP were observed for the KATP channel activity-induced by K+ channel openers (46). These results suggest that activation of the KATP channel may occur through a cGMP-related signaling mechanism more likely due to the activation of PKG. In contrast to the preceding studies, 8-BrcGMP has been found to inhibit the KATP channel activity in guinea pig ventricular cells (47) and mouse pancreatic beta -cells (44). On the other hand, SNP has been reported to have no effect on the KATP currents in guinea pig coronary arterial smooth muscle (57). In addition, Tsuura et al. (49) have found that SNP does not affect the KATP channel activity in rat ventricular myocytes. Thus it is a likely that the effects of cGMP and PKG on KATP channel function are tissue specific and depend on the signaling pathway to which PKG activation is linked.

Protein phosphorylation has been invoked as a putative effector mechanism in the infarct size-limiting effect of ischemic preconditioning (43), a phenomenon whereby a brief period of ischemia and reperfusion can protect the heart against subsequent prolonged ischemia and reperfusion injury (37). Indeed, it has been shown that phosphorylation levels are decreased during ischemia, whereas they are enhanced during ischemic preconditioning (56). A number of cellular proteins have been proposed as potential phosphorylation targets in the ischemic preconditioned heart, including the KATP channel, stress proteins, and cytoskeletal proteins (2, 10, 16). To date, considerable evidence have been provided to suggest that the modulation of KATP channel activity by phosphorylation play an important role in the cardioprotective effects of ischemic preconditioning (50, 59). The majority of the studies on the contribution of phosphorylation to KATP channel activity have been centered on the role of PKC in the heart. It has been shown that PKC may act as a link in one or more receptor-mediated pathways to increase KATP channel activity by phosphorylation and lead to ischemic preconditioning (30).

It is of interest that other newly described kinases like PKG may also play a role in the mechanism of ischemic preconditioning (15, 39). Iliodromitis et al. (24) reported that cardiac tissue cGMP is higher in the preconditioned than the nonpreconditioned regions of the heart. A rationale exists for a protective role for cGMP-PKG signaling pathway against ischemia-reperfusion injury. PKG may act by the modulation of Ca2+ availability (32, 53) or myofilament sensitivity to Ca2+ (51). This together with the effect of the cGMP-PKG signaling pathway in activating KATP channels would reduce myocardial contractility and thus serve to reduce oxygen consumption, energy demand and the rate of Ca2+ loading into the cell, an important factor in ischemic damage. Furthermore, there is close relationship between KATP channels and intracellular phosphotransfer reaction through creatine kinase and adenylate kinase, promoting delivery of mitochondrial signals to the channel site (9, 42, 62). Such signal transduction cascades provide a novel pathway for integration of cellular energetics with membrane electrical events believed to be critical in ischemic preconditioning.

KATP channel activation has been shown to be involved in cardioprotection by a variety of stimuli, including brief ischemia in the heart or remote organs and nonischemic stimuli in the heart such as ventricular pacing, stretch, and heat stress. Moreover, pharmacological agents that open KATP channels also produce cardioprotection. Although the exact mechanism by which KATP channel activation protects is still incompletely understood, recent evidence suggests that mitochondrial KATP channels mediate cardioprotection in ischemic preconditioning (35). However, our results do not provide any experimental evidence to support this interesting possibility. It is not yet known whether the cardioprotection of ischemic preconditioning is due to mitochondrial KATP channels or sarcolemmal KATP channels or to a mixture of both. The mitochondrial KATP channel is regulated by every ligand that regulates sarcolemmal KATP channels. Our finding that the NO-cGMP-PKG signaling pathway can potentiate the KATP channel in rabbit ventricular myocytes raises the intriguing possibility that the NO-cGMP-PKG signaling pathway could modulate mitochondrial KATP channels in the heart. Such a hypothesis is supported by recent studies (8, 14) that addressed a possible role for NO in mediating late ischemic preconditioning.

In conclusion, our data show that rabbit ventricular myocytes may have a phosphorylation sites associated with the KATP channel that can be phosphorylated by a NO-cGMP-PKG signaling mechanism. These sites, when phosphorylated, increase the KATP channel activity, suggesting that this mechanism may contribute, at least in part, to the preconditioning-induced cardioprotection.


    ACKNOWLEDGEMENTS

The authors thank the following individuals for valuable input and comments: Prof. W. K. Ho (Department of Physiology, Seoul National University), Prof. S. H. Kim (Department of Physiology, Chonbuk National University), Prof. D. K. Kim and J. B. Park (Department of Medicine, Sungkyunkwan University), Prof. Y. J. Lee (Department of Life Science, Sejong University), Prof. D. H. Seog and S. Park (Department of Microbiology, Inje University), Prof. J. Y. Jung (Institute of Malaria, Inje University), and Prof. W. G. Park (Department of Anesthesiology, Yonsei University).


    FOOTNOTES

This study was supported by grant from the Korea Health Research and Development Project 21 and by Korean Ministry of Health and Welfare Grant 01-PJ1-PG1-01CH06-0003.

Addresses for reprint requests and other correspondence: J. Han, Dept. of Physiology and Biophysics, College of Medicine, Inje Univ., 633-165 Gaegeum-Dong, Busanjin-Ku, Busan, 614-735, Korea (E-mail: phyhanj{at}ijnc.inje.ac.kr) and Y. E. Earm, National Research Laboratory for Cellular Signaling and Dept. of Physiology, College of Medicine, Seoul National Univ., Seoul 110-799, Korea (E-mail: earmye{at}snu.ac.kr).

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.

May 16, 2002;10.1152/ajpheart.01052.2001

Received 3 December 2001; accepted in final form 6 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahlner, J, Andersson RGG, Torfgard K, and Axelsson XL. Organic nitrate esters: clinical use and mechanisms of actions. Pharmacol Rev 43: 351-423, 1991[Web of Science][Medline].

2.   Auchampach, JA, and Gross GJ. Adenosine A1-receptors, KATP channels, and ischemic preconditioning in dogs. Am J Physiol Heart Circ Physiol 264: H1327-H1336, 1993[Abstract/Free Full Text].

3.   Auchampach, JA, Maruyama M, Cavero I, and Gross GJ. Pharmacological evidence for a role of ATP-dependent potassium channels in myocardial stunning. Circulation 86: 311-319, 1992[Abstract/Free Full Text].

4.   Babenko, AP, Gonzalez G, Aguilar-Bryan L, and Bryan J. Reconstituted human cardiac KATP channels: functional identity with the native channels from the sarcolemma of human ventricular cells. Circ Res 83: 1132-1143, 1998[Abstract/Free Full Text].

5.   Balligand, JL, Kelly RA, Marsden PA, Smith TW, and Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci USA 90: 347-351, 1993[Abstract/Free Full Text].

6.   Balligand, JL, Ungureanu-longrois D, Simmons WW, Pimental D, Malinski TA, Kapturczak M, Taha Z, Lowenstein CJ, Davidoff AJ, Kelly RA, Smith TW, and Michel T. Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. J Biol Chem 269: 27580-27588, 1994[Abstract/Free Full Text].

7.   Billman, GE. Effect of carbachol and cyclic GMP on susceptibility to ventricular fibrillation. FASEB J 4: 1668-1673, 1990[Abstract].

8.   Bolli, R, Dawn B, Tang XL, Qiu Y, Ping P, Xuan YT, Jones WK, Takano H, Guo Y, and Zang J. The nitric oxide hypothesis of late preconditioning. Basic Res Cardiol 93: 325-338, 1998[Web of Science][Medline].

9.   Carrasco, AJ, Dzeja PP, Alekseev AE, Pucar D, Zingman LV, Abraham MR, Hodgson D, Bienengraeber M, Puceat M, Janssen E, Wieringa B, and Terzic A. Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels. Proc Natl Acad Sci USA 98: 7623-7628, 2001[Abstract/Free Full Text].

10.   Cohen, MV, and Downey JM. Ischemic preconditioning: can the protection be bottled? Lancet 342: 6, 1993[Web of Science][Medline].

11.   Cole, WC, McPherson CD, and Sontag D. ATP-regulated K+ channels protect the myocardium against ischemia/reperfusion damage. Circ Res 69: 571-581, 1991[Abstract/Free Full Text].

12.   Colquhoun, D, and Sigworth FJ. Fitting and statistical analysis of single channel records. In: Single-Channel Recording, edited by Sakmann B, and Neher E.. New York: Plenum, 1983, p. 191-263.

13.   D'Alonso, AJ, Darbenzio RB, Parham CS, and Grover GJ. Effects of intracoronary cromakalim on postischemic contractile function and action potential duration. Cardiovasc Res 26: 1046-1053, 1992[Abstract/Free Full Text].

14.   Dawn, B, Xuan YT, Qiu Y, Takano H, Tang XL, Ping P, Banerjee S, Hill M, and Bolli R. Bifunctional role of protein tyrosine kinases in late preconditioning against myocardial stunning in conscious rabbits. Circ Res 85: 1154-1163, 1999[Abstract/Free Full Text].

15.   De Jonge, HR. Cyclic GMP-dependent protein kinase in intestinal brush borders. Adv Cyclic Nucleotide Res 14: 315-333, 1981[Web of Science][Medline].

16.   Ganote, C, and Armstrong S. Ischemia and the myocyte cytoskeleton: review and speculation. Cardiovasc Res 27: 1387-1403, 1993[Free Full Text].

17.   Hamill, OP, Marty A, Nether E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[Web of Science][Medline].

18.   Han, J, Kim N, and Kim E. Trifluoroacetic acid activates ATP-sensitive K+ channels in rabbit ventricular myocytes. Biochem Biophys Res Commun 285: 1136-1142, 2001[Web of Science][Medline].

19.   Han, J, Kim E, Lee SH, Yoo S, Ho WK, and Earm YE. cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes. Pflügers Arch 435: 388-393, 1998[Web of Science][Medline].

20.   Han, J, Kim N, Kim E, Ho WK, Earm YE, and Kim H. Increase of L-type calcium current by cGMP-dependent protein kinase regulates in rabbit ventricular myocytes. Korean J Physiol Pharmacol 2: 733-742, 1998.

21.   Hardie, DG. Roles of protein kinases and phosphatases in signal transduction. Symp Soc Exp Biol 44: 241-255, 1990[Medline].

22.   Hoshida, S, Yamashita N, Igarashi J, Nishida M, Hori M, Kamada T, Kuzuya T, and Tada M. Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits. J Pharmacol Exp Ther 274: 413-418, 1995[Abstract/Free Full Text].

23.   Hoshida, S, Yamashita N, Igarashi J, Nishida M, Hori M, Kamada T, Kuzuya T, and Tada M. Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits. J Pharmacol Exp Ther 274: 413-418, 1995[Abstract/Free Full Text].

24.   Iliodromitis, EK, Papadopoulos CC, Markianos M, Paraskevaidis IA, Kyriakides ZS, and Kremastinos DT. Alterations in circulating cyclic guanosine monophosphate (c-GMP) during short and long ischemia in preconditioning. Basic Res Cardiol 91: 234-239, 1996[Web of Science][Medline].

25.   Inagaki, N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J, and Seino S. A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16: 1011-1017, 1996[Web of Science][Medline].

26.   Jiang, H, Shabb JB, and Corbin JD. Cross-activation: overriding camp/cGMP selectivities of protein kinases in tissues. Biochem Cell Biol 70: 1283-1289, 1992[Web of Science][Medline].

27.   Joyce, NC, Decamilli P, Lohmann SM, and Walter U. cGMP-dependent protein kinase is present in high concentrations in contractile cells of the kidney vasculature. J Cyclic Nucleotide Protein Phosphor Res 11: 191-198, 1986[Web of Science][Medline].

28.   Kubo, M, Nakaya Y, Matsuoka S, Saito K, and Kuroda Y. Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP-sensitive K+ channels in cultured vascular smooth muscle cells. Circ Res 74: 471-476, 1994[Abstract/Free Full Text].

29.   Kwak, YG, Park SK, Cho KP, and Chae SW. Reciprocal modulation of ATP-sensitive K+ channel activity in rat ventricular myocytes by phosphorylation of tyrosine and serine/threonine residues. Life Sci 58: 897-904, 1996[Web of Science][Medline].

30.   Light, PE, Sabir AA, Allen BG, Walsh MP, and French RJ. Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K+ channels: a possible mechanistic link to ischemic preconditioning. Circ Res 79: 399-406, 1996[Abstract/Free Full Text].

31.   Lincoln, TM, and Corbin JD. Characterization and biological role of the cGMP-dependent protein kinase. Adv Cyclic Nucleotide Res 15: 139-142, 1983[Web of Science].

32.   Lincoln, TM, and Keely SL. Regulation of cardiac cGMP-dependent protein kinase. Biochem Biophys Acta 676: 230-244, 1980.

33.   Liu, Y, Sato T, O'Rourke B, and Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation 97: 2463-2469, 1998[Abstract/Free Full Text].

34.   Lorenz, E, and Terzic A. Physical association between recombinant cardiac ATP-sensitive K+ channel subunits Kir6.2 and SUR2A. J Mol Cell Cardiol 31: 425-434, 1999[Web of Science][Medline].

35.   Miyazaki, T, and Zipes DP. Protection against autonomic denervation following acute myocardial infarction by preconditioning ischemia. Circ Res 64: 437-448, 1989[Abstract/Free Full Text].

36.   Murphy, ME, and Brayden JE. Nitric oxide hyperpolarizes rabbit mesenteric arteries via ATP-sensitive potassium channels. J Physiol 486: 47-58, 1995[Abstract/Free Full Text].

37.   Murry, CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124-1136, 1986[Abstract/Free Full Text].

38.   Node, K, Kitakaze M, Kosaka H, Komamura K, Minamino T, Tada M, Inoue M, Hori M, and Kamada T. Plasma nitric oxide end products are increased in the ischemic canine heart. Biochem Biophys Res Commun 211: 370-374, 1995[Web of Science][Medline].

39.   Parratt, JR. Protection of the heart by ischemic preconditioning: mechanisms and possibilities for pharmacological exploitation. Trends Pharmacol Sci 15: 19-25, 1994[Medline].

40.   Parratt, JR. Possibilities for the pharmacological exploitation of ischaemic preconditioning. J Mol Cell Cardiol 27: 991-1000, 1995[Web of Science][Medline].

41.   Parratt, JR, Vegh A, and Papp JG. Bradykinin as an endogenous myocardial protective substance with particular reference to ischemic preconditioning-a brief review of the evidence. Can J Physiol Pharmacol 73: 837-842, 1995[Web of Science][Medline].

42.   Pucar, D, Dzeja PP, Bast P, Juranic N, Macura S, and Terzic A. Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR. J Biol Chem 276: 44812-44819, 2001[Abstract/Free Full Text].

43.   Rapundalo, ST. Cardiac protein phosphorylation: functional and pathphysiological correlates. Cardiovasc Res 38: 559-588, 1998[Abstract/Free Full Text].

44.   Ropero, AB, Fuentes E, Rovira JM, Ripoll C, Soria B, and Nadal A. Non-genomic actions of 17-oestradiol in mouse pancreatic cells are mediated by a cGMP-dependent protein kinase. J Physiol 521: 397-407, 1999[Abstract/Free Full Text].

45.   Sakuta, H, Okamoto K, and Tandai M. Atrial natriuretic factor potentiates glibenclamide-sensitive K+ currents via the activation of receptor guanylate cyclase in follicle-enclosed Xenopus oocytes. Eur J Pharmacol 267: 281-287, 1994[Web of Science][Medline].

46.   Sakuta, H, Okamoto K, and Watanabe Y. Modification by cGMP of glibenclamide-sensitive K+ currents in Xenopus oocytes. Jpn J Pharmacol 61: 259-262, 1993[Medline].

47.   Shinbo, A, and Iijima T. Potentiation by nitric oxide of the ATP-sensitive K+ current induced by K+ channel openers in guinea-pig ventricular cells. Br J Pharmacol 120: 1568-1574, 1997[Web of Science][Medline].

48.   Southam, E, and Garthwaite J. Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices. Neurosci Lett 130: 107-111, 1991[Web of Science][Medline].

49.   Tsuura, Y, Ishida H, Hayashi S, Sakamoto K, Horie M, and Seino Y. Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibit glucose-induced insulin release in pancreatic beta  cells. J Gen Physiol 104: 1079-1099, 1994[Abstract/Free Full Text].

50.   Van Winkle, DM, Chien GL, Wolff RA, Soifer BE, Kuzume K, and Davis RF. Cardioprotection provided by adenosine receptor activation is abolished by blockade of the KATP channel. Am J Physiol Heart Circ Physiol 266: H829-H839, 1994[Abstract/Free Full Text].

51.   Vila-Petroff, MG, Younes A, Egan J, Lakatta EG, and Sollott SJ. Activation of distinct camp-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ Res 84: 1020-1031, 1999[Abstract/Free Full Text].

52.   Wahler, GM, and Dollinger SJ. Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. Am J Physiol Cell Physiol 268: C45-C54, 1995[Abstract/Free Full Text].

53.   Waldmann, R, Bauer S, Gobel C, Hofmann F, Jakobs KH, and Walter U. Demonstration of cGMP-dependent phosphorylation in cell-free extracts of platelets. Eur J Biochem 158: 203-210, 1986[Web of Science][Medline].

54.   Walker, DM, and Yellon DM. Ischaemic preconditioning: from mechanisms to exploitation. Cardiovasc Res 26: 734-739, 1992[Free Full Text].

55.   Wall, TM, Sheehy R, and Hartman JC. Role of bradykinin in myocardial preconditioning. J Pharmacol Exp Ther 270: 681-689, 1994[Abstract/Free Full Text].

56.   Weinbrenner, C, Liu GS, Cohen MV, and Downey JM. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J Mol Cell Cardiol 29: 2383-2391, 1997[Web of Science][Medline].

57.   Wellman, GC, Quayle JM, and Standen NB. ATP-sensitive K+ channel activation by calcitonin generelated peptide and protein kinase A in pig coronary arterial smooth muscle. J Physiol 507: 117-129, 1998[Abstract/Free Full Text].

58.   Yamaguchi, F, Nasa Y, Yabe K, Ohba S, Hashizume Y, Ohaku H, Furuhama K, and Takeo S. Activation of cardiac muscarinic receptor and ischemic preconditioning effects in in situ rat heart. Heart Vessels 12: 74-83, 1997[Web of Science][Medline].

59.   Yao, Z, and Gross GJ. A comparison of adenosine-induced cardioprotection and ischemic preconditioning in dogs: efficacy, time course, and role of KATP channels. Circulation 89: 1229-1236, 1994[Abstract/Free Full Text].

60.   Yao, Z, and Gross GJ. Acetylcholine mimics ischemic preconditioning via a glibenclamide-sensitive mechanism in dogs. Am J Physiol Heart Circ Physiol 264: H2221-H2225, 1993[Abstract/Free Full Text].

61.   Yellon, DM, Alkhulaifi AM, and Pugsley WB. Preconditioning the human myocardium. Lancet 342: 276-277, 1993[Web of Science][Medline].

62.   Zingman, LV, Alekseev AE, Bienengraeber M, Hodgson D, Karger AB, Dzeja PP, and Terzic A. Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance. Neuron 31: 233-245, 2001[Web of Science][Medline].

Am J Physiol Heart Circ Physiol 283(4):H1545-H1554
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Kuno, N. V. Solenkova, V. Solodushko, T. Dost, Y. Liu, X.-M. Yang, M. V. Cohen, and J. M. Downey
Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1288 - H1295.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
M. Miyamae, S. Sugioka, and V. M. Figueredo
Additive Cardioprotection by Ethanol and Sevoflurane: Are Sarcolemmal KATP Channels Also Involved?
Anesth. Analg., June 1, 2008; 106(6): 1926 - 1927.
[Full Text] [PDF]

Home page
 J. Physiol.Home page
K. E. Brack, V. H. Patel, J. H. Coote, and G. A. Ng
Nitric oxide mediates the vagal protective effect on ventricular fibrillation via effects on action potential duration restitution in the rabbit heart
J. Physiol., September 1, 2007; 583(2): 695 - 704.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. V. Cuong, M. Warda, N. Kim, W. S. Park, J. H. Ko, E. Kim, and J. Han
Dynamic changes in nitric oxide and mitochondrial oxidative stress with site-dependent differential tissue response during anoxic preconditioning in rat heart
Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1457 - H1465.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Y. Jang, H. Wang, J. Xi, R. A. Mueller, E. A. Norfleet, and Z. Xu
NO mobilizes intracellular Zn2+ via cGMP/PKG signaling pathway and prevents mitochondrial oxidant damage in cardiomyocytes
Cardiovasc Res, July 15, 2007; 75(2): 426 - 433.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. V. Cuong, N. Kim, J. B. Youm, H. Joo, M. Warda, J.-W. Lee, W. S. Park, T. Kim, S. Kang, H. Kim, et al.
Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K+ channels in rat hearts
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1808 - H1817.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Q. Qin, X.-M. Yang, L. Cui, S. D. Critz, M. V. Cohen, N. C. Browner, T. M. Lincoln, and J. M. Downey
Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism
Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H712 - H718.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Z. Xu, X. Ji, and P. G. Boysen
Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial KATP channels, and ERK
Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1433 - H1440.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
O. Oldenburg, Q. Qin, T. Krieg, X.-M. Yang, S. Philipp, S. D. Critz, M. V. Cohen, and J. M. Downey
Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection
Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H468 - H476.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
R. C Kukreja, R. Ockaili, F. Salloum, and L. Xi
Sildenafil-induced cardioprotection in rabbits
Cardiovasc Res, December 1, 2003; 60(3): 700 - 701.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. P. D'Souza, D. M. Yellon, C. Martin, R. Schulz, G. Heusch, A. Onody, P. Ferdinandy, and G. F. Baxter
B-type natriuretic peptide limits infarct size in rat isolated hearts via KATP channel opening
Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1592 - H1600.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/4/H1545    most recent
01052.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (49)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, J.
Right arrow Articles by Earm, Y. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, J.
Right arrow Articles by Earm, Y. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online