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Am J Physiol Heart Circ Physiol 273: H1637-H1643, 1997;
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Vol. 273, Issue 4, H1637-H1643, October 1997

Priming effect of adenosine on KATP currents in intact ventricular myocytes: implications for preconditioning

Yongge Liu, Wei Dong Gao, Brian O'Rourke, and Eduardo Marban

Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21205

    ABSTRACT
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Abstract
Introduction
Materials
Results
Discussion
References

Activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA) has been shown to shorten the time to turn on ATP-sensitive potassium currents (IK,ATP) during metabolic inhibition (MI) but only when adenosine (Ado) is included. In the present study we tested whether pretreatment with Ado could mimic the effect of PMA in isolated rabbit ventricular myocytes. When IK,ATP was measured by conventional whole cell clamp, pretreatment with 100 µM Ado did not alter the time to IK,ATP activation: 13.5 ± 2.1 vs. 12.4 ± 1.9 min with Ado during MI. We repeated the experiment using the perforated patch technique. Consistent with the previous results in conventional whole cell patch recordings, the time to turn on IK,ATP during MI (with Ado included) was shortened from 27.1 ± 2.2 to 12.6 ± 2.4 min (P < 0.01) when cells were pretreated with PMA and Ado was included during MI. In contrast to conventional whole cell recordings, Ado pretreatment also abbreviated the time for IK,ATP activation during MI (with Ado included) to 16.4 ± 1.8 min. This effect was partially eliminated by simultaneous administration of an Ado receptor blocker or a PKC inhibitor during Ado pretreatment, suggesting that pretreatment with Ado stimulates PKC by activating Ado receptors. Our results demonstrate that Ado can prime IK,ATP during subsequent MI in the presence of Ado. This priming effect appears to be mediated by PKC upregulation of the channel. These results support the notion that Ado plays a dual role to initiate and to mediate ischemic preconditioning and links the roles of Ado receptors, PKC, and IK,ATP in ischemic preconditioning.

adenosine 5'-triphosphate-sensitive potassium currents; protein kinase C; patch clamp; amphotericin B

    INTRODUCTION
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Abstract
Introduction
Materials
Results
Discussion
References

ISCHEMIC PRECONDITIONING describes the ability of the heart to adapt itself during brief ischemia, becoming resistant to subsequent lethal ischemia (21). Such endogenous cardioprotection has been demonstrated in all species examined, including the human (3). Although a great deal of effort has been applied to identify the mechanisms underlying preconditioning, these remain poorly understood. Various lines of evidence implicate the activation of adenosine (Ado) receptors, activation of protein kinase C (PKC) (7), and opening of ATP-sensitive potassium (KATP) channels (10). These three mechanisms may be interrelated as parts of a common signal transduction cascade. One popular hypothesis proposes that Ado receptor activation during the preconditioning ischemia activates PKC, which then phosphorylates KATP channels. The phosphorylation alters the sensitivity of KATP channels in such a way that these channels open earlier and/or more intensely during the prolonged ischemia. Opening of KATP channels during ischemia shortens the action potential duration, reduces contractility, and thus decreases Ca2+ influx and conserves energy.

By means of conventional whole cell patch clamp, we have previously shown in rabbit ventricular myocytes that PKC activation by phorbol 12-myristate 13-acetate (PMA) increases the sensitivity of KATP channels to pinacidil, a KATP channel opener (18). We have also shown that PMA pretreatment combined with Ado during metabolic inhibition (MI) accelerates the activation of KATP current (IK,ATP) and action potential shortening during MI. The requirement for Ado during MI is consistent with the idea that Ado receptor activation is needed during the lethal ischemia for preconditioning (28). Two other studies have investigated the effect of PKC on sarcolemmal KATP channels. Hu et al. (12) showed that PKC activates whole cell IK,ATP in rabbit and human ventricular myocytes by reducing channel sensitivity to intracellular ATP. Using the excised patch technique, Light et al. (17) demonstrated that PKC activation reduces the Hill coefficient from 2.2 to 1.2 without changing the inhibition constant for ATP and thus increases IK,ATP at 1 mM intracellular ATP. These results are in the same direction as in our previous study (18) and further link the PKC and KATP channel hypotheses for preconditioning.

Several studies have suggested that Ado receptor activation initiates preconditioning by upregulating PKC (6, 19, 24). We rationalized that if Ado receptor stimulation indeed activates PKC, Ado pretreatment should have a similar priming effect on IK,ATP as PMA. Therefore, we investigated the effect of Ado pretreatment on the time to turn on IK,ATP during MI in this study. We also tested whether the effect of Ado pretreatment is mediated by PKC activation.

    METHODS AND MATERIALS
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Abstract
Introduction
Materials
Results
Discussion
References

Chemicals

Collagenase (type II) was purchased from Worthington (Freehold, NJ). 8-(p-Sulfophenyl)-theophylline (SPT) and chelerythrine (Che) were obtained from Research Biochemicals (Natick, MA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). SPT, Ado, and Che were directly added into the experimental solutions. PMA was dissolved in dimethyl sulfoxide (DMSO). Amphotericin B was freshly made on the day of the experiments as a stock solution of 50 mg/ml dissolved in DMSO. This stock solution was then added into the pipette solution at a final concentration of 240 µg/ml with brief sonication (1).

Preparation of Rabbit Myocytes

Isolated rabbit ventricular myocytes were obtained from rabbit hearts by conventional enzymatic dissociation methods (18). In brief, hearts were quickly excised from anesthetized (30 mg/kg iv pentobarbital) rabbits (New Zealand White) weighing 1-2 kg, and the aortas were cannulated for coronary perfusion on a Langendorff apparatus. The heart was perfused with modified Krebs-Henseleit-bicarbonate buffered solution composed of (in mM) 119 NaCl, 5 KCl, 1 MgSO4, 25 NaHCO3, 1 KH2PO4, 1 CaCl2, and 10 glucose. The perfusate was bubbled with 95% O2-5% CO2 and maintained at 37°C. After 5-min perfusion, hearts were perfused without Ca2+ for another 5 min, after which the perfusion solution was switched to one containing collagenase (0.8 mg/ml, Worthington type II). The perfusion pressure was monitored, and the flow rate was adjusted to maintain perfusion pressure at about 75 mmHg. After 10-15 min of collagenase perfusion, hearts were removed from the perfusion apparatus and the atria were trimmed away. The ventricles were minced and incubated in a shaking bath for another 5 min in collagenase-containing solution. Cells were then filtered through nylon mesh and stored at room temperature in a high-potassium solution containing (in mM) 120 K-glutamate, 25 KCl, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 glucose. Before each experiment, cells were washed in a modified Tyrode solution containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). This isolation procedure yielded more than 50% of Ca2+-tolerant, crisply striated ventricular myocytes. All experiments were performed at room temperature (21-22°C) within 7 h after isolation.

Membrane Current Measurements

Patch pipettes were pulled (model P-87, Sutter Instrument, San Rafael, CA) from filamented borosilicate glass (1.5 mm OD) and fire-polished before being used. When filled with intracellular solution, pipettes had 2-4 MOmega resistance. Conventional whole cell and perforated patch currents were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). After a gigaohm seal was formed, the conventional whole cell configuration was achieved by breaking the membrane with gentle suction. The internal solution for conventional whole cell patch recordings contained (in mM) 120 K-glutamate, 25 KCl, 0.5 MgCl2, 10 K-ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, and 1 MgATP (pH 7.2 with KOH). For the perforated patch configuration, adequate access to the cell was obtained in about 2-5 min after gigaohm seal formation (1). Pipette solution contained (in mM) 120 K-glutamate, 25 KCl, 0.5 MgCl2, and 1 CaCl2. If the membrane was inadvertently broken through, cells would crumple and die quickly because of the 1 mM Ca2+ in the pipette solution. The time course of change in IK,ATP was recorded by applying voltage ramps of 400 ms duration from 50 to -100 mV. Currents at 0 and -100 mV measured at the end of the pulses were shown vs. time. As previously described (18), the time to activate IK,ATP was determined by the time required to induce a clearly measurable outward current (>0.1 nA at 0 mV).

Protocols

Conventional whole cell IK,ATP during MI. PRIMING EFFECT OF ADO. We have previously shown that PMA can prime KATP channels so that during MI the time to turn on IK,ATP is abbreviated but only in the presence of Ado (18). We sought to determine whether Ado pretreatment could mimic the effect of PMA. MI was achieved by adding 2 mM sodium cyanide (CN) and omitting glucose from the Tyrode solution. The pH of the solution was adjusted to 7.4 with HCl. Three groups of cells were studied. For the controls (MI), after the conventional whole cell patch was established, cells were simply equilibrated for 20 min before exposure to MI. The second group of cells (MI + Ado) was exposed to MI with 100 µM Ado included in the perfusion solution. In the third (Ado + MI + Ado) group, cells were treated in the same way as the MI + Ado group, except these cells were pretreated with 100 µM Ado for 10 min. Ado was then omitted in the perfusion solution for 5 min before the MI.

Perforated patch IK,ATP during MI. PRIMING EFFECT OF PMA. To confirm the synergistic modulation of IK,ATP by PMA and Ado that we reported in conventional whole cell patch-clamp studies (18), we performed a similar protocol in the perforated patch configuration. Three groups of cells were studied. The first group was the control (MI). Five minutes after a gigaohm seal was established, recording was started. Cells were then exposed to MI including 10 mM 2-deoxyglucose, 2 mM CN, and zero glucose. The second group of cells (PMA + MI) was treated with 100 nM PMA for 10 min followed by 5-min Tyrode solution superperfusion before switching to MI. The third group of cells (PMA + MI + Ado) was treated the same as the PMA + MI group except that 100 µM Ado was added during the MI.

PRIMING EFFECT OF ADO. The priming effect of Ado was investigated in four additional groups. Cells in the MI + Ado group were exposed to MI with 100 µM Ado included. The Ado + MI + Ado group of cells was treated with 100 µM Ado for 10 min followed by 5-min Tyrode solution superperfusion before switching to MI with 100 µM Ado included. Cells in the Ado + Che + MI + Ado and Ado + SPT + MI + Ado groups were treated the same as the Ado + MI + Ado group, except that 10 µM Che, a PKC inhibitor, and 100 µM SPT, an Ado receptor antagonist, were simultaneously perfused, respectively, with 10-min Ado pretreatment before MI.

Data Analysis

All values are expressed as means ± SE. Analysis of variance combined with a Newman-Keuls post hoc test was used to test for differences among groups. Probability of null hypothesis, P < 0.05, was considered significant.

    RESULTS
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Abstract
Introduction
Materials
Results
Discussion
References

Conventional Whole Cell IK,ATP During MI

Priming effect of Ado. Figure 1 shows representative time courses of currents measured at 0 and -100 mV in each group. Figure 1A shows representative membrane currents elicited by ramp voltage commands obtained at the times indicated. After about 16 min of initiation of MI, current at 0 mV (IK,ATP) started to turn on and there was no change or a slight increase in currents at -100 mV in the control group. Inclusion of Ado during MI (MI + Ado, Fig. 1B) or pretreatment with Ado before MI plus Ado (Ado + MI + Ado, Fig. 1C) slightly shortened the time to activate IK,ATP, although the effect did not reach significance.


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  		Fig. 1.   Time courses of membrane current development during metabolic inhibition (MI) in conventional whole cell ATP-sensitive potassium current (IK,ATP) recordings. A: MI. Vc, voltage-clamp ramps from 50 to -100 mV; Im, currents activated by the ramps at the corresponding time points (a, b, and c) as shown in bottom panel. Time courses of currents measured at 0 and -100 mV from control cell are shown in bottom panel. B and C: time courses of currents of a cell from MI + adenosine (Ado) group and a cell from Ado + MI + Ado group, respectively. MI was induced by exposing the cell to cyanide (CN). See text for group names.

Figure 2 summarizes the times required for IK,ATP activation. In the control group (MI, n = 7), IK,ATP started to turn on at 16.5 ± 2.6 min during MI. When 100 µM Ado was included during MI (Ado + MI group), this time did not alter (13.8 ± 1.3 min, n = 7). When cells were pretreated with 100 µM Ado and had Ado included during MI (Ado + MI + Ado), this time was not significantly shortened either (12.3 ± 1.7 min, n = 7). This result suggests that Ado pretreatment does not mimic the effect of PMA to prime KATP channels in the conventional whole cell patch-clamp configuration.


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  		Fig. 2.   Pooled data for time required to activate IK,ATP during MI in conventional whole cell recordings. Ado does not prime ATP-sensitive potassium (KATP) channels.

Perforated Patch IK,ATP During MI

Priming effect of PMA. One of the reasons for the lack of a priming effect from Ado could be due to the intracellular dialysis that is known to occur with conventional whole cell patch clamp (20). We used the perforated patch configuration to avoid intracellular dialysis and then first tested whether we could reproduce the synergistic effect of PMA and Ado on IK,ATP as seen with the conventional whole cell configuration (18). Figure 3 shows the time course of membrane currents (measured at -100 and 0 mV) during MI from a representative cell in each group. When exposed to MI, the currents measured at -100 mV in the MI group did not change significantly over time. Nevertheless, currents at 0 mV (IK,ATP) started to increase after a delay of ~26 min (Fig. 3A). Currents activated by MI reversed at predicted equilibrium potential of potassium (-80 mV) under our experimental conditions. Prior activation of PKC did not alter this response; when cells were treated with 100 nM PMA before MI (PMA + MI group), the current changes were similar (Fig. 3B). However, if PMA-treated cells were subjected to MI with Ado included (PMA + MI + Ado group), the time required to increase the current at 0 mV was markedly abbreviated (Fig. 3D).


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  		Fig. 3.   Time courses of membrane current development during MI from representative cells from each of the 5 groups in perforated patch IK,ATP recordings: A: MI; B: phorbol 12-myristate 13-acetate (PMA) + MI; C: MI + Ado; D: PMA + MI + Ado; E: Ado + MI + Ado. PMA and Ado both prime KATP channels and accelerate the opening of KATP channels during MI in the presence of Ado (D and E). Currents were measured at -100 and 0 mV. MI was induced by perfusing the cell with CN and 2-deoxyglucose.

Figure 4 summarizes the pooled data for the times required for IK,ATP activation. This latency was 27.1 ± 2.2 min (n = 6) in the MI group and was not significantly altered in the PMA + MI group (22.4 ± 2.9 min, n = 5). However, the latency to develop IK,ATP in the PMA + MI + Ado group decreased to only 12.6 ± 2.4 min (n = 6, P < 0.01 vs. the other 2 groups). This synergistic effect of PMA and Ado seen here is consistent with the results we previously reported in conventional ruptured patch studies (18).


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  		Fig. 4.   Pooled data for priming effect of PMA in perforated patch recordings. PMA pretreatment abbreviates the time to turn on IK,ATP during MI in the presence of Ado. * P < 0.01.

We tested the effect of glibenclamide on IK,ATP activated by MI. Glibenclamide (100 µM) partially reduced IK,ATP in three of six cells tested. There was no blockade in the other three cells (data not shown). This is consistent with the previous finding from our laboratory as well as from other groups that glibenclamide loses its ability to inhibit KATP channels during MI (8, 18). To verify further that the current activated by MI is indeed IK,ATP, we have checked the characteristics of single channels in our previous study and showed that they matched the properties of KATP channels in cardiac myocytes (18).

Priming effect of Ado. Including Ado only during MI did not shorten the time to turn on IK,ATP (Fig. 3C). Figure 3E shows traces from a representative perforated patch recording in which we tested whether Ado could prime KATP channels to open earlier during MI. Similar to the effect of PMA, if the cells were exposed to Ado before MI with Ado included, IK,ATP indeed turned on much earlier.

Figure 5 summarizes the pooled data. The time to turn on IK,ATP during MI was significantly abbreviated to 16.4 ± 1.8 min in the Ado + MI + Ado group (n = 5, P < 0.05 vs. MI or MI + Ado group). This effect was almost completely blocked by simultaneous perfusion with either an Ado receptor blocker (SPT) or a PKC inhibitor (Che). The average times to turn on IK,ATP in the Ado + SPT + MI + Ado and Ado + Che + MI + Ado groups were 22.3 ± 1.8 min [n = 3, P not significant (NS) vs. MI or Ado + MI + Ado group] and 21 ± 1.8 min (n = 5, P = NS vs. MI or Ado + MI + Ado group).

    DISCUSSION
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Abstract
Introduction
Materials
Results
Discussion
References

The purpose of this study was to test whether Ado pretreatment can prime KATP channels and thus abbreviate the time to turn on IK,ATP during MI. Our results show that the priming effect of Ado can be observed in the perforated patch configuration but not in the conventional whole cell configuration. This suggests that an intact cell is required for the Ado priming effect. Our results also indicate that this priming effect from Ado is mediated by activation of Ado receptors and subsequent PKC activation.

Although the mechanism underlying preconditioning is not fully understood, several lines of evidence implicate Ado receptors, PKC, and KATP channels, at least in rabbit hearts (7, 30). A proposed scheme to link these three mechanisms is as follows: Ado receptor activation during the brief preconditioning ischemia upregulates PKC. PKC then phosphorylates KATP channels, priming the channels in such a way that they open earlier and/or more frequently during the lethal ischemia. Evidence also suggests that Ado receptor activation is required during the lethal ischemia to realize the protection (28).


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  		Fig. 5.   Pooled data for priming effect of Ado in perforated patch recordings. Pretreatment with Ado abbreviates time to turn on IK,ATP when Ado is included during MI. Che, chelerythrine; SPT, 8-(p-sulfophenyl)-theophylline. * P < 0.05.

The scheme linking Ado to PKC and PKC to KATP channels has been demonstrated in several preconditioning studies. First, Sakamoto et al. (24) were able to show that a PKC inhibitor blocks protection from an Ado receptor agonist in rabbit hearts. Second, a KATP channel blocker, glibenclamide, blocks protection from PKC activators (13, 33) in rabbit hearts as well as in human atrial trabeculae (26). Glibenclamide has also been shown to block protection from Ado receptor agonists (29, 32) and preconditioning (30) in rabbit hearts and human atrial trabeculae (26). Although the mechanisms outlined previously are based on data generated in rabbit and human myocardium, mechanisms may be different in other species. Studies have shown that PKC seems not to be involved in dog (22) and pig (31) hearts, although KATP channels have been consistently shown to play an important role in these two species as well (10, 25).

To investigate directly whether PKC phosphorylation alters KATP channels, we studied IK,ATP in rabbit ventricular myocytes activated by MI. Using conventional whole cell patch clamp, we have shown that activation of PKC by PMA greatly abbreviates the time to turn on IK,ATP in rabbit ventricular myocytes during MI in the presence of Ado (18). That PKC phosphorylation increases KATP channel activity has also been shown in two other studies in rabbit ventricular myocytes (12, 17). In addition to cardiac myocytes, PKC stimulation increases KATP channel activity in insulin-secreting cells (23). However, it is important to point out that PKC regulation of KATP channels may be tissue-type dependent because it has been shown that PKC inhibits KATP channels in smooth cells (4, 5).

In this study we further investigated whether Ado treatment can activate PKC and thus prime the KATP channels. Our results suggest that Ado receptor activation is able to stimulate PKC and then mimics the effect of PMA in intact cells. Dialyzing the cell with our pipette solution disrupts such a priming effect from Ado. We presently do not know which important element is altered by dialysis and thus causes the failure to activate PKC by Ado receptors. Possible candidates include Ca2+ because intracellular Ca2+ was buffered by 10 mM EGTA in the conventional whole cell configuration or the cytoskeleton, which may also be altered by intracellular dialysis. The cytoskeleton has been shown to modulate KATP channels (9, 27).

For preconditioning, one of the important links between Ado and PKC is that Ado receptor activation presumably stimulates PKC. Direct evidence for such a link is scarce. Henry et al. (11) were able to show that in rat myocytes Ado transiently stimulates PKC isoform. In contrast, Armstrong et al. (2) did not see a significant translocation of various PKC isoforms from cytosol to particulate fractions after preconditioning or Ado receptor stimulation in rabbit myocytes. Because of the complexity of PKC isoforms and their activation, more detailed studies are needed. Another possibility is that one of the intermediate messengers between receptors and PKC is upregulated. Indeed, Cohen et al. (6) have shown that both brief preconditioning and an Ado receptor agonist stimulate phospholipase D. Activation of phospholipase D stimulates diacylglycerol production and subsequently activates PKC. Further studies are required to dissect the link between Ado receptors and PKC activation.

Results from our previous study (18) and this one indicate that Ado receptor activation is required during MI to shorten the time to activate IK,ATP. This finding has important implications for the role played by Ado in preconditioning. The prevailing concept holds that Ado receptor activation plays a dual role: it initiates as well as mediates the protection (7, 28). Ado receptors have to be activated during both the preconditioning and the lethal ischemia to provide protection. In our experimental settings, cells were continually superperfused and Ado was not able to accumulate. Ado has to be added exogenously to mimic the Ado buildup during ischemia. Several studies have shown that Ado activates KATP channels in excised ventricular myocyte patches but only at nonphysiological submillimolar cytoplasmic ATP levels (15, 16). With 1 mM ATP in the pipette or using perforated patch and superfusion with normal Tyrode, we did not observe any IK,ATP even with concentrations of Ado as high as 10 mM in the solution (unpublished data). In addition, 100 µM Ado alone did not significantly shorten the latency for IK,ATP activation during MI (Fig. 2C). Thus two periods of Ado receptor stimulation are required. KATP channels have to be primed by Ado (or PMA) before MI or ischemia. It is not known how KATP channels are primed, but channel phosphorylation or PKC translocation may be involved. Ado receptors then have to be stimulated again during MI or ischemia. The involvement of Ado receptors during MI was demonstrated in our previous study (18). The mechanism for the second stimulation of Ado receptors is not clear. One possibility is that KATP channels become gated by Ado receptors when channels are phosphorylated by PKC. Such phosphorylation may occur during the preconditioning ischemia or Ado pretreatment. Most recently, Yang et al. (34) stringently tested this hypothesis by using the protein kinase inhibitor staurosporine. Their results suggest that phosphorylation is not required during the preconditioning period but is only critical during lethal ischemia for the protection. This seems contradictory to our study, because Che blocked the priming effect of Ado during pretreatment. However, one of proposed mechanisms for preconditioning is upregulation of PKC by translocation (19). Preconditioning merely translocates PKC from cytosol to membrane, and the critical phosphorylation occurs during the lethal ischemia. Staurosporine blocks phosphorylation but does not interfere with PKC translocation (14). Che is a specific PKC inhibitor, but it is not known whether it also affects translocation. One possible explanation for the discrepancy between this study and that of Yang et al. (34) is that Che also interferes with PKC translocation and thus blocks PKC upregulation. The priming effect of Ado is through translocation of PKC rather than through phosphorylation of KATP channels.

In summary, we have provided evidence that Ado treatment primes IK,ATP in intact rabbit ventricular myocytes, and the primed IK,ATP turns on earlier during MI. The priming effect is apparently mediated by Ado receptor activation and subsequent PKC upregulation of the channel. These results provide further evidence to link Ado receptors, PKC, and KATP channels in preconditioning.

    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-44065 and RO1-HL-54598.

    FOOTNOTES

Address for reprint requests: E. Marban, Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University, Ross 844, 720 Rutland Ave., Baltimore, MD 21205.

Received 11 April 1997; accepted in final form 10 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials
Results
Discussion
References

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AJP Heart Circ Physiol 273(4):H1637-H1643
0363-6135/97 $5.00 Copyright © 1997 the American Physiological Society


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