Ischemic preconditioning, by which brief episodes of myocardial ischemia reduce injury resulting from subsequent prolonged ischemia and reperfusion, produces an acute “memory” phase of approximately 1 h between the “preconditioning” and “test” ischemic periods (1). Anesthetic preconditioning also produces approximately 1 h of cardioprotection and may depend on similar signaling pathways involving activation of G-protein coupled receptors linked to phospholipases, membrane hydrolysis, and release of diacylglycerol (DAG) second messengers that activate protein kinase C (PKC) (2–8). This activation initiates translocation of specific PKC isoforms, including the novel PKC-δ and -ε isoforms, modulating infarct size (2,9), from the cytosol to bind with proteins having specific receptors for activated C-kinases located at various intracellular sites, including the sarcolemma and mitochondria (8–11). It is likely that the kinetics of activation, transport, phosphorylation, and dephosphorylation of targeted intracellular effector proteins is responsible for the characteristic duration of acute cardioprotection (12). The PKC-dependent pathway along with other kinase cascades are thought ultimately to facilitate the opening of sarcolemmal and mitochondrial adenosine triphosphate (ATP)-sensitive potassium channels (sarcKATP and mitoKATP channels, respectively), the latter a major effector of cardioprotection associated with generation of reactive oxygen species (3,13). Although the relative importance of sarc- and mitoKATP channels in acute cardioprotection is controversial (13–16), little is known about what anesthetic actions might underlie changes in cardiac ionic channel function that persist long after anesthetic removal.
Our studies in isolated guinea pig cardiomyocytes have shown that isoflurane facilitates the opening of sarcKATP channels during metabolic inhibition or by the KATP channel opener pinacidil at a reduced intracellular ATP concentration achieved by dialysis with a patch pipette (17–19). We also found that isoflurane did not facilitate the opening of sarcKATP channels in excised membrane patches, but isoflurane would facilitate activation of whole-cell sarcKATP current (IKATP) in a model of a test ischemia in which PKC was stimulated by a phorbol ester during the reduction of intracellular ATP by dialysis (18). Hu et al. (20) reported that stimulation of PKC alters the sensitivity of sarcKATP channels to ATP, increasing the threshold (half-maximal inhibitory concentration) for ATP-dependent opening to approximately 0.6 mM, a concentration that might occur in ischemia. In the present study, we tested the hypothesis that the induction of IKATP by isoflurane in a model of simultaneous stimulation of PKC, using phorbol 12-myristate-13 acetate (PMA) and cell dialysis to decrease intracellular ATP to the range of 0.5–1.0 mM, would be inhibited by the PKC antagonists chelerythrine (4) and bisindolylmaleimide (20). In addition, because DAGs may be involved in activating PKC during both the triggering and effector phases of cardioprotection (2,21), we determined how isoflurane pretreatment might alter the response of the sarcKATP channels to a synthetic DAG activator of PKC (di-octanoyl-glycerol [DOG]) (22) applied via the patch pipette during the reduction of intracellular ATP.
After approval by the Institutional Animal Care and Use Committee, isolated ventricular myocytes were obtained from adult guinea pig hearts, as previously reported (17). Guinea pigs were anesthetized with intraperitoneal pentobarbital sodium (50 mg). During anesthesia, the hearts were quickly excised and mounted via the aorta on a Langendorff-type apparatus. The hearts were perfused at 37°C with Joklik medium containing heparin at a pH value of 7.23 for 3–4 min and then perfused with a recirculating medium containing 0.25 mg/mL of collagenase (Invitrogen Type II, Invitrogen, San Diego, CA), 0.13 mg/mL of protease (Sigma Type XIV, Sigma, St. Louis, MO), and 1 mg/mL of bovine serum albumin at a pH value of 7.23. After 14 min, the hearts were cut into fragments and further incubated for approximately 8 min in a shaker bath. The cell suspension was filtered, centrifuged, washed, and stored in Tyrode solution, and cells were used within 12 h of isolation.
An aliquot of cells was placed on the stage of an inverted microscope at 22°C in 1 mL of Tyrode solution containing (in mM) NaCl 132, KCl 4.8, MgCl2 1.2, CaCl2 1, dextrose 5, and HEPES 10 at a pH value adjusted to 7.4 with NaOH. After establishing whole-cell voltage clamp using a List EPC-7 amplifier (Adams and List Associates, Westbury, NY), potassium channel currents were filtered at 3 kHz using an 8-pole Bessel filter and isolated by changing solutions at 2 mL/min to an external solution containing (mM) N-methyl-d-glucamine 132 (substituting for Na), KCl 5, MgCl2 1.2, CaCl2 1, and HEPES 10 with a pH value of 7.4 (with HCl). Sodium channel current was inactivated with a holding potential of −40 mV, and nisoldipine (200 nM; Miles-Pentex, West Haven, CT) was added to block l-type calcium channel current. The standard pipette solution contained (mM) K-glutamate 60, KCl 50, MgCl2 1, CaCl2 1, EGTA 11, and K2-ATP 0.5–1 (pH value of 7.4 with KOH). Resting membrane potentials under these conditions were between −65 and −75 mV. The PKC activators PMA and 1,2-dioctanoyl sn-glycerol (Calbiochem, La Jolla, CA) were diluted and added to the external and pipette solutions, respectively, from frozen aliquots of stock solution in dimethylsulfoxide (DMSO). The antagonists chelerythrine and bisindolylmaleimide (Calbiochem) were prepared as frozen stock solutions in DMSO and thawed for daily use. The final concentration of DMSO was <0.05% and by itself did not affect sarcKATP channel activity.
IKATP was monitored every 15 s at the end of a 100-ms test voltage pulse to 0 mV from a holding potential of −40 mV, beginning approximately 5 min after membrane rupture and solution changes. Current amplitudes, measured at the end of each pulse, are shown verses time in the figures, were normalized to cell capacitance, and are summarized in the text as the mean current density (pA/pF) ± sem. IKATP was identified by its sensitivity to glibenclamide (500 nM). Peak values of current density were measured after each intervention, and paired or unpaired t-tests were used to compare mean values within and between groups, respectively. P ≤ 0.05 was considered statistically significant.
To evaluate anesthetic actions after PKC stimulation, cells were continuously exposed to 0.2 μM of PMA externally. Isoflurane was applied by switching solutions after at least 30 min of cell dialysis to permit diffusional exchange of pipette ATP with the cell. Two ATP concentrations were used to evaluate the ATP dependence of peak IKATP induced by isoflurane. Isoflurane solutions (1 mM; approx. 2.1 vol%) were prepared by dissolving liquid anesthetic volumetrically in a 50-mL flask, adding other drugs as required, sonicating, and decanting into glass syringes. The concentration of isoflurane applied was determined at the end of each experiment by pumping solution through the bath, collecting duplicate 1-mL samples in metal-capped 2-mL glass vials, and analyzing the samples by gas chromatography.
To evaluate the role of PKC in “priming” or facilitating isoflurane-induced IKATP, chelerythrine (2 μM) or bisindolylmaleimide (200 nM) was applied concurrently with PMA from the start of cell dialysis and continued for 30 min before isoflurane exposure. In another series, PMA was first applied for 30 min, and the response of primed channels to a brief first period of isoflurane was measured. After activation of IKATP, isoflurane was washed out, and either chelerythrine or bisindolylmaleimide was applied for 5 min followed by a second application of isoflurane with PKC inhibition to evaluate the contribution of PKC to isoflurane-induced current.
The concentration-related effects of DAG stimulation of PKC were determined by adding 0.1, 0.5, or 1.0 μM of DOG to the pipette solution and monitoring IKATP during cell dialysis with an intermediate concentration (0.75 mM) of ATP. Cells that did not exhibit activation by DOG alone after at least 30 min were challenged with isoflurane in a manner analogous to the experiments with PMA. The effects of isoflurane preconditioning on IKATP were evaluated by exposing cells on the microscope stage to isoflurane in Tyrode solution for 7–10 min and washing out the anesthetic for 5–20 min before establishing whole-cell clamp by rupturing the membrane with suction. Thereafter, the cells were internally dialyzed with 0.5 μM of DOG and 0.75 mM of ATP in a protocol designed to simulate anesthetic preconditioning followed by a test period of PKC stimulation at reduced intracellular ATP concentration.
Figure 1A illustrates the stable time course of whole-cell currents elicited by test pulses from −40 mV to 0 mV during prolonged intracellular dialysis with 1.0 mM of ATP. The subsequent addition of 1 mM of isoflurane did not activate IKATP, as we previously reported in this model (17–19). However, as shown in Figure 1B, pretreatment with the PKC activator PMA (0.2 μM), which alone did not elicit sarcKATP channel activation, facilitated or primed the channels for activation of IKATP by later exposure to isoflurane. The current density elicited by 1 mM of isoflurane after PKC stimulation by PMA was 2 ± 1 pA/pF (n = 6 cells) with 1 mM of ATP in the pipette and was smaller (P ≤ 0.05; unpaired t-test) in magnitude than at 0.5 mM of ATP (10 ± 5 pA/pF; n = 5 cells). Figure 1C illustrates an experiment in which an inactive phorbol ester analog was substituted for PMA. Repeated trials of isoflurane failed to activate IKATP with a phorbol ester, which does not stimulate PKC.
To examine the role of PKC in altering the response of sarcKATP channels to subsequent isoflurane exposure, the PKC inhibitors chelerythrine or bisindolylmaleimide were superfused simultaneously with PMA from the start of the experiments. Figure 2 shows that neither inhibitor applied with PMA activated IKATP before isoflurane exposure. Figure 2A illustrates bisindolylmaleimide antagonism of PMA-associated facilitation of IKATP induction by isoflurane. Complete abolition of the PMA effect was observed in all trials in six cells. However, as shown in Figure 2B, chelerythrine (2 μM) did not consistently antagonize the actions of PMA facilitating IKATP activation by isoflurane in that activation occurred in three of six trials. Thus, bisindolylmaleimide was more effective than chelerythrine in inhibiting the priming effect of PKC stimulation on isoflurane responses.
Further experiments were performed using PMA and two brief exposures to isoflurane to determine if PKC inhibition just before the second anesthetic exposure would prevent activation of sarcKATP channels previously primed or sensitized to isoflurane. As shown in Figure 3A, the first application of isoflurane after PMA elicited the expected IKATP response of primed channels. Although chelerythrine after isoflurane (Fig. 3A) did not activate IKATP, chelerythrine enhanced activation of sarcKATP channels by a second application of isoflurane, increasing the current magnitude (P ≤ 0.05; paired t-test) in four cells to 1.7 ± 0.5 nA from 0.3 ± 0.2 nA in response to the first isoflurane exposure. In contrast (Fig. 3B), the application of bisindolylmaleimide, just before the second anesthetic exposure, abolished the induction of IKATP by isoflurane, indicating that the actions of isoflurane opening sarcKATP channels previously primed by PMA were also dependent on PKC.
To better characterize the effects of PKC stimulation on sarcKATP channel function, we examined the concentration-related actions of the DAG DOG applied intracellularly via the patch pipette to activate PKC (22). Using DOG and 0.75 mM of ATP in the pipette, none of the four cells exhibited IKATP with 0.1 μM of DOG, whereas three of nine cells and five of five cells exhibited IKATP with 0.5 and 1.0 μM of DOG, respectively, within 20–35 min after establishing the whole-cell patch configuration. Figure 4A illustrates sarcKATP channel activation with 0.5 μM of DOG in the pipette. The magnitudes of the peak IKATP density were: none (0) at 0.1 μM of DOG (n = 4 cells) and 5 ± 3 pA/pF (n = 9) at 0.5 μM of DOG, whereas 1.0 μM of DOG induced a larger (P ≤ 0.05; unpaired t-test) current density of 63 ± 8 pA/pF (n = 5) compared to that with 0.5 μM of DOG. Thus, an intracellularly applied DAG by itself facilitated sarcKATP channel opening in a concentration-related manner.
Figure 4B illustrates the results of experiments performed with DOG, analogous to those with PMA (Fig. 1B), to examine the sarcKATP channel response to isoflurane in cells in which previous stimulation of PKC by 0.1–0.5 μM of DOG was insufficient to activate IKATP (n = 7 cells). Unlike PMA, which alone did not activate IKATP but primed the channels for activation by isoflurane, DOG applied from the start of cellular dialysis (Fig. 4B) did not facilitate activation by subsequent exposure to isoflurane in all seven trials in cells in which DOG alone failed to elicit IKATP.
The persistence of effects of isoflurane on sarcKATP channel activity was tested by initially exposing cells on the microscope stage to isoflurane. The anesthetic was then washed out with Tyrode solution, and whole-cell clamp was established in 10 cells using 0.5 μM of DOG and 0.75 mM of ATP in the pipette. The average duration of the isoflurane preconditioning period was 9 min and the washout period 12 min before PKC stimulation with DOG in the pipette. As illustrated in Figure 4C, 7 (7) of 10 cells pretreated with isoflurane exhibited robust IKATP activation with peak current densities of 40 ± 9 pA/pF (n = 10) and onset, when present, averaging 48 ± 2 min after the end of the isoflurane preconditioning period. This magnitude of IKATP (40 ± 9 pA/pF; n = 10) in cells pretreated with isoflurane and dialyzed with 0.5 μM of DOG was larger (P ≤ 0.01; unpaired t-test) than that found with 0.5 μM of DOG alone (Fig. 4A), which averaged 5 ± 3 pA/pF in nine cells not pretreated with isoflurane and was qualitatively larger than that typically observed with isoflurane after priming the channels with PMA. Thus, preconditioning with isoflurane facilitated or primed sarcKATP channels to subsequent stimulation of PKC by an intracellularly applied DAG. However, intracellular DOG did not facilitate activation of IKATP by a subsequent brief exposure to isoflurane as did the phorbol ester PMA.
This study demonstrates that isoflurane facilitates, in a PKC-dependent manner, the opening of sarcKATP channels of guinea pig myocytes during cellular dialysis to intracellular ATP levels (0.5–1.0 mM) that might occur during myocardial ischemia. Pretreatment with the phorbol ester PMA, which itself did not elicit IKATP, altered channel function to facilitate IKATP activation during later isoflurane exposure, although isoflurane alone (without stimulation of PKC) does not activate the channels (17–19). The role of PKC in these actions regulating IKATP was confirmed by findings that the PKC antagonist bisindolylmaleimide prevented the actions of PMA, facilitating isoflurane-induced IKATP, as well as preventing activation of IKATP when superfused just before a second isoflurane challenge. Our findings are consistent with other reports indicating that phorbols alter the sensitivity of sarcKATP channels to ATP by activating PKC and not by nonspecific membrane effects (20). In addition, we (23) recently reported that specific PKC-isoform selective peptide agonists (PKC-ε) and antagonists (PKC-ε more so than PKC-δ) similarly modulate the induction of IKATP by isoflurane in this model. However, there are clearly important differences in how different PKC agonists (PMA verses an intracellular DAG) and antagonists (chelerythrine verses bisindolylmaleimide) influence the subsequent induction of IKATP by isoflurane that are not readily explained. In particular, chelerythrine did not consistently inhibit the priming effect of PMA on isoflurane-induced IKATP and by itself enhanced IKATP induced by a second isoflurane exposure. Thus, the actions of chelerythrine are probably not limited to effects on PKC, and use of this antagonist in studies of anesthetic actions may be problematical.
We found that a synthetic DAG activator of PKC produces concentration-related activation of IKATP in a manner consistent with studies showing that increasing numbers of brief ischemic preconditioning stimuli enhance PKC translocation and cardioprotection (2). A graded response of IKATP to PKC stimulation without hypoxic challenge is consistent with the idea that some preconditioning drugs may act “upstream” of PKC, at the level of the G-protein coupled receptors, PI3-kinase, or phospholipases (5–7,19), to produce graded increases of endogenous DAG and thereby graded priming the sarcKATP channels via PKC-dependent phosphorylation. The findings do not exclude a role for additional signaling elements “downstream” of PKC, such as at the level of the mitochondria, that may “feedback” to facilitate sarcKATP channel activation via the tightly coupled creatinine kinase cascade (23,24) or indirectly by altering the translocation of different PKC isoforms (2,3,16). Further studies examining cellular DAG levels in response to isoflurane and IKATP responses to isoflurane in the presence of free-radical scavengers and selective mitoKATP channel antagonists would be helpful to discriminate between these possible sites of anesthetic action. Although both phorbol esters and DAG bind to the same molecular region of PKC to produce activation, DOG did not prime the sarcKATP channels to opening by isoflurane in quite the same way as did PMA for reasons that are not entirely clear. The pattern of PKC isoform translocation varies between different preconditioning models (21). The different responses of IKATP to DAG versus phorbol stimulation might be explained by variations in drug efficacy and affinity at the DAG binding sites of different PKC isoforms, processes of auto- and cross-phosphorylation of the isoforms (25), or potential antagonistic effects of specific PKC isoforms (9).
Finally, we found a preconditioning-like effect of isoflurane facilitating IKATP after anesthetic washout during later cell dialysis with a DAG to stimulate PKC and reduced intracellular ATP. This pretreatment effect occurred independent of any potential neurohumoral activation of receptors, as might occur in whole tissues, and may be best explained by an intracellular regulatory change because there seemed to be no direct membrane delimited actions of isoflurane altering sarcKATP channel activity in excised membrane patches under our recording conditions at a pH value of 7.4 (18). Although further studies are required to define the role of specific PKC isoform activation in this pretreatment effect, it is clear that isoflurane can produce persistent changes in sarcKATP channel function, lasting 30–45 minutes, which sensitize or produce larger magnitudes of whole-cell IKATP in response to DAG stimulation of PKC than dialysis with the DAG alone at the same concentration.
The addition of a PKC agonist during reduction of ATP in this cardiomyocyte model only, in part, simulates kinase activation during the test or effector phase of ischemia but may permit examination of the coupling between mito- and sarcKATP channel activation because we found that the IKATP response to isoflurane, after selective PKC-ε stimulation, is reduced by the mitoKATP channel antagonist 5-hydroxy decanoate (23). Inhibition of isoflurane’s pretreatment effects, followed by testing by cell dialysis with a PKC agonist at a reduced ATP, may permit evaluation of mechanisms of anesthetic-induced changes in KATP channel function occurring at sites upstream of PKC (5,6,19). The model is subject to possible artifacts because of enzymatic cell isolation and is limited in that single-cell studies in the guinea pig may not reflect the different pattern of receptor, phospholipase, and kinase activation induced by myocardial ischemia in humans. In addition, prolonged dialysis to reduce cellular ATP levels may not mimic important effects of substrate deprivation leading to oxidative uncoupling, reactive oxygen species generation, mitoKATP channel opening, and focal decreases of intracellular ATP concentrations. The model may also be subject to large differences in the intracellular concentrations of ATP or other regulatory substances between individual cells and possible differences in expression and posttranslational modification of regulatory components between cells from different regions of the heart.
In conclusion, under patch-clamp conditions, isoflurane facilitates the opening of sarcKATP channels in a manner dependent on PKC and the degree of reduction of intracellular ATP but only after effective priming of the channels by phorbol ester activation of PKC. Isoflurane pretreatment also preconditions or sensitizes sarcKATP channels to subsequent opening by a synthetic DAG activator of PKC applied via the patch pipette.
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