Protein Tyrosine Kinase-Dependent Modulation of Isoflurane Effects on Cardiac Sarcolemmal K^ATPChannel

After we obtained approval from the Institutional Animal Use and Care Committee of the Medical College of Wisconsin, we isolated single ventricular myocytes from guinea pig hearts by an enzymatic dissociation procedure as described previously. 33Briefly, either male or female guinea pigs weighing 150–300 g were injected intraperitoneally with heparin (1,000 U/ml) and anesthetized with sodium pentobarbital (275 mg/kg). Each heart was rapidly excised, mounted on a cannula of the Langendorff apparatus (Radnoti, Monrovia, CA), and perfused via  the aorta with oxygenated (95% O²–5% CO²) Joklik medium (Gibco BRL, Life Technologies, Grand Island, NY) containing heparin (2.5 U/ml), at a constant flow of 7 ml/min at 37°C. After washout of blood, hearts were perfused for 14 min with Joklik medium containing 0.4 mg/ml collagenase (Type II; Gibco BRL), 0.13 mg/ml protease (Type XIV; Sigma, St. Louis, MO), and 1 mg/ml bovine serum albumin (Serologicals, Kankakee, IL) at pH 7.23. The ventricles were then minced and incubated for additional 3–10 min in the enzyme solution. Isolated myocytes were washed in a Tyrode solution and used for patch clamp experiments within 10 h after isolation.

The modified Tyrode solution contained 132 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1 mm CaCl², 10 mm HEPES, and 5 mm glucose, at pH 7.4 adjusted with NaOH.

For the whole cell recordings, the external-bath solution contained 132 mm N -methyl-d-glucamine, 5 mm KCl, 2 mm MgCl², 1 mm CaCl², 10 mm HEPES (N -2-hydroxyethylpiperazine-N ′-2 ethanesulphonic acid), and 0.0002 mm nisoldipine, at pH 7.4 adjusted with HCl. N -methyl-d-glucamine replaced sodium, and nisoldipine blocked the L-type Ca²⁺channel currents. The intracellular-pipette solution contained 60 mm K glutamate, 50 mm KCl, 1 mm MgCl², 1 mm CaCl², 11 mm EGTA, 10 mm HEPES, and 0.5 or 5 mm K²ATP, at pH 7.4 adjusted with KOH.

For the single channel recordings in the excised inside-out patch configuration, the bath solution facing the cytosolic side of the membrane patch contained 140 mm KCl, 0.5 mm MgCl², 2 mm EGTA, 10 mm HEPES, 0.2 mm K²ATP, at pH 7.4 adjusted with KOH. The pipette solution facing the outer side of the membrane patch contained 140 mm KCl, 0.5 mm MgCl², 0.5 mm CaCl², 10 mm HEPES, at pH 7.4 adjusted with KOH.

All standard chemicals, ATP, sodium orthovanadate (Na³VO⁴), and nerve growth factor (NGF; from Vipera lebetina venom) were obtained from Sigma. Nisoldipine was supplied by Miles-Pentex (West Haven, CT). Genistein (4′,5,7-trihydroxyisoflavone), daidzein (4′,7-dihydroxyisoflavone), tyrphostin B42 [AG490, α-cyano-(3,4-dihydroxy)-N -benzylcinnamide], and murine epidermal growth factor (EGF) were obtained from Calbiochem (Calbiochem-Novabiochem, San Diego, CA). Recombinant PTP1B was purchased from Upstate Biotechnology (Lake Placid, NY). Inhibitors of PTK and daidzein were dissolved in dimethyl sulfoxide. Nisoldipine was prepared as 1 mm stock in polyethylene glycol. Control experiments with the vehicles have shown that neither dimethyl sulfoxide (0.02–0.06%) nor polyethylene glycol (0.02%) at their final dilution in the recording buffers had any effect on whole cell I^KATPand single K^ATPchannel activity (data not shown).

Measured volumes of isoflurane (Baxter Healthcare, Deerfield, IL) were added to appropriate bath solutions, and the anesthetic was dispersed by sonication. The solutions were then transferred to gas-tight glass syringe reservoirs and delivered to the recording chamber by a gravity-fed perfusion system. Isoflurane was used at a clinically relevant concentration of 0.5 ± 0.03 mm, equivalent to 1.05 vol% as determined at 20–23°C. Samples (1 ml) of anesthetic-containing perfusate were withdrawn from the recording chamber to determine effective millimolar concentrations of isoflurane by the headspace analysis method using a Shimadzu GC 8A flame ionization detection gas chromatograph (Shimadzu, Kyoto, Japan).

Conventional configurations of the patch clamp technique were used to measure whole cell K^ATPcurrent and single K^ATPchannel activity. Currents were recorded using a LIST EPC-7 patch clamp amplifier (ALA Scientific Instruments, Westbury, NY) and a Digiata 1200B (Axon Instruments, Foster City, CA) interfaced to a PC computer. Data were acquired using pClamp8 software (Axon Instruments). The recording chamber (RC-16; Warner, Hamden, CT) was mounted on the stage of an inverted IMT-2 microscope (Olympus, Tokyo, Japan). Patch pipettes were pulled from borosilicate glass tubing (Garner Glass, Claremont, CA) with a PC-84 micropipette puller (Sutter Instruments, Novato, CA), and the pipette tips were heat-polished with a MF-83 microforge (Narishige, Tokyo, Japan). The resistance of pipettes filled with the appropriate pipette solution was 2–3 MΩ for the whole cell recordings and 7–12 MΩ for single channel recordings. All experiments were conducted at room temperature (20–23°C).

For the whole cell I^KATPmeasurements, the series resistance was compensated electronically to obtain the fastest cell capacity transient. Whole cell currents were monitored over time by applying a 100-ms voltage step to 0 mV from a holding potential of −40 mV every 15 s. Current amplitude, measured at the end of the voltage step, was normalized to cell capacitance for I^KATPdensity (pA/pF) determination.

Single K^ATPchannel currents were recorded from the excised inside-out patches at the transmembrane patch potential of +40 mV. Currents were sampled at 1 kHz and low-pass filtered at 500 Hz through an eight-pole Bessel filter. The 120-s-long recordings were made at each experimental step. The K^ATPchannels were identified by single channel conductance, sensitivity to inhibition by intracellular ATP, and blockade by 1 μm glibenclamide. The threshold for detecting the open state was set at half of the single channel amplitude. Amplitude of single channel current was determined from the all-points amplitude histograms. The number of channels (N) in the patch was estimated from the mean single channel amplitude and the maximal current. The channel open probability (Po) was calculated as a fraction of the total time the active channels in the patch were in the open state during the recording. Po was determined from the ratios of the area under the peaks in the all-points amplitude histograms fitted with a Gaussian function. Because of a variable number (N) of channels in the patches reported here, Po is expressed as cumulative Po. The experimental protocols were completed within 10–15 min after patch excision to minimize the influence of rundown. Recordings from patches exhibiting large differences in channel activity between the control and the final washout that suggested a significant rundown were excluded from further analyses.

Whole cell and single channel data were analyzed using pClamp8 software (Axon Instruments) and Origin6 software (ORIGINLAB, Northampton, MA). Data were presented as means ± SEM. Comparisons between two groups of means were made using a paired or unpaired Student t  test. Multiple group means were compared using one-way analysis of variance with a Student-Newman-Keuls test. Differences with a P < 0.05 were considered statistically significant.

To assess a possible role of the PTK-PTP signaling pathway for isoflurane interaction with the cardiac sarcolemmal K^ATPchannel, we first evaluated the effects of genistein, an inhibitor of PTKs, on the whole cell K⁺current in ventricular myocytes. Applied in the external solution, genistein consistently activated whole cell outward K⁺current in myocytes dialyzed for 20 min with the pipette solution containing low (0.5 mm) but no high (5 mm) ATP. Genistein-activated current was identified as I^KATPbecause of sensitivity to blockade by 1 μm glibenclamide. Activation of I^KATPby 50 μm genistein was biphasic, with a rapidly rising peak followed by a more gradually established plateau. Figure 1shows the effects of genistein on whole cell I^KATP: sample traces of glibenclamide-sensitive current recorded during a 100-ms depolarizing voltage step from −40 mV to 0 mV in the absence and presence of 50 μm genistein (fig. 1A), a time course of genistein activation of I^KATP(fig. 1B), and mean current density data from 10 myocytes (fig. 1C). The density of genistein-activated I^KATPwas 37.4 ± 11.3 pA/pF at peak and 22.5 ± 7.9 pA/pF at plateau. Because of high variability in the magnitude of genistein-activated current and a lack of significant differences between mean peak and plateau values, only plateau values were subsequently used in the evaluation of genistein effects on I^KATP. In another group of experiments, daidzein, an inactive structural analog of genistein, was used to determine whether genistein effects were mediated by PTK. Time course and mean current density data in figures 1D and Eshow that externally applied daidzein (50 μm, n = 5) did not activate whole cell I^KATP.

Genistein activation of I^KATPwas concentration-dependent (1–200 μm) with maximal current activation at 50 μm. Less activation at 100 and 200 μm genistein resulted in a bell-shaped concentration-response relation. Fitting the ascending part of a normalized concentration-response relation to a Hill equation gave a concentration of genistein required for half-maximal activation (EC⁵⁰) of 32.3 ± 1.4 μm and a Hill coefficient of 11.7 ± 5.1 (fig. 2). Tyrphostin B42, a specific inhibitor of EGF receptor-associated tyrosine kinases but structurally unrelated to genistein compound, had a similar effect and activated I^KATPwith an EC⁵⁰of 30.2 ± 0.2 μm and a Hill coefficient of 4.9 ± 0.3 (n = 4).

Whether the effects of genistein on I^KATPcould, in part, be explained by PTP activity to reverse kinase effects was tested by using sodium orthovanadate (1 mm), the inhibitor of PTPs that prevents tyrosine dephosphorylation and thus stabilizes the phosphorylated state. Orthovanadate alone had no effect on I^KATP. However, applied prior to genistein, orthovanadate antagonized I^KATPactivation, and in its continued presence genistein failed to activate I^KATPin all tested cells (n = 5). An inhibitor of serine-threonine phosphatases, 0.1 μm okadaic acid was used in the pipette solution to assess the specificity of orthovanadate effects (n = 3). In contrast to orthovanadate, okadaic acid did not prevent activation of whole cell I^KATPby genistein (data not shown), suggesting lack of involvement of serine-threonine phosphatases in genistein effects.

A possible role of the PTK-PTP pathway for isoflurane actions on the sarcolemmal K^ATPchannel was assessed by first determining whether isoflurane modulates genistein-activated I^KATP. Isoflurane (0.5 mm) alone did not activate whole cell I^KATPeither at 5 mm or 0.5 mm internal ATP (n = 5 in each group). A sample time course of current in figure 3Ashows that isoflurane alone did not activate whole cell I^KATPat 0.5 mm in-ternal ATP. However, activated by 35 μm genistein, I^KATP(14.5 ± 3.1 pA/pF, n = 10) was further increased in the presence of 0.5 mm isoflurane to 32.5 ± 6.6 pA/pF (n = 10) at 0.5 mm internal ATP (figs. 3B and C). Isoflurane also enhanced I^KATPactivated by tyrphostin B42 (n = 4, data not shown). Furthermore, at high (5 mm) internal ATP and 35 μm genistein in the bath, isoflurane activated I^KATPin three of the five cells examined. Isoflurane, however, did not activate I^KATPwhen daidzein was substituted for genistein (n = 5, data not shown). The effects of genistein alone and genstein plus isoflurane were prevented by orthovanadate (1 mm, n = 5) applied prior to genistein and present in the bath solution throughout the experiment (fig. 3D), while okadaic acid (0.1 μm) was without effect (data not shown).

If genistein effects are PTK-mediated and PTKs negatively modulate K^ATPchannel, then stimulation of receptor-associated PTKs should alter or prevent the effects of genistein and isoflurane on I^KATP. To test this hypothesis, the growth factor receptor PTKs and insulin receptor PTKs were stimulated with EGF (10 ng/ml), NGF (0.5 μg/ml), and insulin (5 μm), respectively. A sample time course in figure 4Ashows that, when present in the bath solution throughout the experiment, EGF (10 ng/ml) suppressed the effects of genistein and isoflurane on whole cell I^KATP. Not only EGF, but also NGF and insulin attenuated activation of I^KATPby genistein (fig. 4B). However, potentiating effect of isoflurane was attenuated by EGF and NGF but was not altered by insulin (n = 3 in each group). A gradual decrease in current was also observed when EGF (20 ng/ml) was applied in the external solution at the plateau of genistein-activated and isoflurane-enhanced whole cell I^KATP(fig. 4C).

To determine whether genistein and isoflurane may directly modulate activity of K^ATPchannels, we tested their effects under cell-free conditions in excised inside-out membrane patches. Single channel activity was monitored at patch potential of +40 mV in the presence of 0.2 mm internal ATP. As shown in figure 5, applied to the inside-out patches (n = 4), isoflurane alone did not change unitary current amplitude but tended to reversibly decrease single channel activity (fig. 5A). With isoflurane, Po decreased from control 0.032 ± 0.01 to 0.017 ± 0.006 (n = 4;fig. 5B). This effect, however, was not statistically significant.

Genistein applied alone at 30–50 μm facilitated K^ATPchannel opening. Figure 6shows recordings of single K^ATPchannel activity and the corresponding all-points histograms from a representative inside-out patch. While unitary current amplitude was not altered (control, 2.06 ± 0.04 pA; genistein, 2.1 ± 0.06 pA; n = 10), Po was increased from 0.053 ± 0.016 to 0.183 ± 0.039 in the presence of genistein (fig. 6A; n = 10). When coapplied with genistein, isoflurane did not alter unitary current amplitude (2.11 ± 0.04 pA), and unlike during whole cell conditions, failed to increase channel activity. With genistein plus isoflurane, Po was 0.162 ± 0.051 (n = 10;figs. 6A and B). Daidzein at 50 μm (n = 3) did not alter Po in the absence or presence of isoflurane, as shown in figure 6C. Since receptor-associated PTKs and PTPs may exist in excised membrane patches as channel regulatory proteins, we also tested whether orthovanadate affects single K^ATPchannel activity. At 0.2 mm internal ATP, orthovanadate (1 mm) prevented Po increase by genistein, and the effect was reversible (n = 3, data not shown).

Inability of isoflurane to potentiate single, genistein-activated K^ATPchannel in inside-out patches suggested that another factor, not available during cell-free conditions, might be required for this effect. The excised membrane patches may contain receptor PTKs and membrane-associated PTPs, but will lack soluble PTPs. Since PTP1B has recently been shown to modulate activity of ventricular sarcolemmal K^ATPchannel, 21we tested whether this phosphatase might be needed for isoflurane potentiation of genistein effects. Figure 7summarizes the results obtained from four single channel experiments in which genistein (50 μm) increased Po from control 0.018 ± 0.005 to 0.114 ± 0.040, and at coapplication of PTP1B (1 μg/ml), Po remained at 0.143 ± 0.046. Notably, isoflurane applied to genistein-activated channels in the presence of PTP1B caused a marked increase in Po (0.473 ± 0.184, n = 4). These results suggest a possibility of direct modulation of cardiac K^ATPchannel by genistein and PTP1B, which may, in part, play a role in isoflurane potentiation of channel activity.

Possible contribution of the PTK-PTP signaling pathway to modulation of isoflurane effects on the cardiac sarcolemmal K^ATPchannel was investigated in guinea pig ventricular myocytes using the PTK inhibitor genistein. The results show that extracellularly applied genistein activates the whole cell I^KATPwith an EC⁵⁰of 32 μm, a concentration at which the activity of serine-threonine protein kinases A and C are not affected. 34Furthermore, applied to the cytosolic side of excised membrane patches, genistein increases single K^ATPchannel activity by increasing channel Po. Several lines of evidence suggest a possible involvement of PTK signaling pathway in these actions: (1) genistein activation of the whole cell I^KATPis ATP-dependent; (2) a structurally unrelated inhibitor of PTKs, tyrphostin B42, may also activate whole cell I^KATP; (3) the inactive structural analog of genistein, daidzein, does not activate I^KATPat the whole cell or single channel level; (4) a specific inhibitor of PTPs, orthovanadate, prevents genistein activation of whole cell or single K^ATPchannel currents, whereas the serine-threonine phosphatase inhibitor, okadaic acid, does not alter genistein effects; and (5) activation of the whole cell I^KATPby genistein is markedly attenuated during stimulation of receptor-associated PTKs with EGF, NGF, or insulin. These results taken together suggest a possibility of inhibitory control of cardiac sarcolemmal K^ATPchannels by basal PTK activity.

The finding that genistein increases single K^ATPchannel activity could be explained either by the presence of closely associated tyrosine kinases and phosphatases in the membrane patches 35or by a direct action of genistein on the channel protein. The former mechanism appears to be supported by the finding that inhibition of PTP prevents the effects of genistein not only during whole cell conditions but also in excised patches. If membrane-bound PTK and PTP activities are balanced in a patch, genistein inhibition of basal PTK would shift balance toward PTP-dependent dephosphorylation, promoting channel activation. However, orthovanadate inhibition of PTP would produce an opposite effect. As predicted, during cell-free conditions, orthovanadate prevented activation of the K^ATPchannel by genistein. High variability in the magnitude of genistein-activated whole cell current in this study might reflect different levels of cellular expression of the proteins involved. The patch-to-patch variability in activation of the single channel current could be explained by differences in distribution of kinases and phosphatases in randomly excised membrane patches. Thus, similar to the results obtained in the whole cell configuration, activation of single K^ATPchannels by genistein could be explained by inhibition of basal PTK activity, and prevention of tyrosine phosphorylation on the channel protein.

The cardiac K^ATPchannel is an octameric complex of four Kir6.2 subunits of the inward rectifier K channel forming the channel pore, and four SUR2A receptor subunits. A member of the ATP binding cassette superfamily, SUR2A confers channel sensitivity to nucleotides, K channel openers, and sulfonylureas. The amino acid sequence of cardiac K^ATPchannel shows 7 tyrosine residues on each Kir6.2 subunit and approximately 34 tyrosine residues located in various regions of the SUR2A receptor protein. 36,37It is possible that PTKs may target some of these residues, and tyrosine phosphorylation attenuates channel activity. By inhibiting PTK and preventing tyrosine phosphorylation in a particular region of the channel, genistein might promote its opening. However, it is difficult to speculate about sites of genistein action, because potential PTK phosphorylation sites on the cardiac sarcolemmal K^ATPchannel have not been identified.

The concentration-response curves for genistein and tyrphostin B42 were steep, with high Hill coefficients that were greater for genistein (11.7 ± 5.1) than tyrphostin B42 (4.9 ± 0.3). This may partly reflect characteristics of a ligand-gated ion channel where allosteric interaction of several molecules of these compounds with the channel may be involved.

Genistein inhibits PTK via  competition with ATP at its binding site, the Walker A motif in the nucleotide binding domain (NBD) of the kinase catalytic subunit. 8Genistein may interact with NBDs of other proteins, inhibiting, for example, DNA topoisomerase II, 38but increasing activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. 37In the CFTR channel, where ATP hydrolysis at NBD1 is thought essential for channel opening and ATP hydrolysis at NBD2 causes channel closure, genistein increases the single channel activity by prolonging bursting activity. This effect is also a recognized consequence of the CFTR mutation that specifically prevents ATP hydrolysis at NBD2 but not at NBD1. Thus, genistein activation of the CFTR channels is thought to result from inhibition of ATP hydrolysis at NBD2 that delays channel closure. 39 

In contrast to the CFTR channel, genistein inhibition of the ATPase activity in cardiac K^ATPchannels would be expected to cause channel closure. Our study, however, shows activation of the K^ATPchannel in the presence of genistein. This may be explained by differences in the function and modulation of NBDs between these channels. 40In the cardiac K^ATPchannel, binding of ATP at NBD1, and Mg²ADP at NBD2, and cooperativity between NBDs are essential for channel activation. 40,41Local activities of ATPase and creatine kinase modulate cardiac K^ATPchannel function. A marked inhibition of creatine kinase activity that occurs during metabolic or ischemic stress may increase local adenosine diphosphate concentration and facilitate channel activation. 41Thus, the mechanism of genistein action could involve modulation of SUR2A activity via  altering some of these enzymes. At the present, however, it is not known whether genistein modulates creatine kinase or ATPase activity in ventricular myocytes.

During our experimental conditions, isoflurane alone did not induce I^KATPin guinea pig ventricular myocytes, but the genistein-activated whole cell I^KATPwas further increased in the presence of isoflurane at low internal ATP. Furthermore, even at high internal ATP, where genistein alone does not activate I^KATP, coapplication of isoflurane may induce whole cell current, suggesting that both genistein modulation of PTK activity and interaction with the channel may be important for isoflurane potentiation. This is further supported by the finding that stimulation of receptor-associated PTKs with EGF and NGF markedly attenuates both genistein activation and isoflurane potentiation of the whole cell I^KATP. These results also suggest that EGF or NGF receptor PTKs or their downstream signaling pathways might be involved in isoflurane potentiation, because stimulation of insulin receptor-associated PTKs attenuated only the effects of genistein, but not isoflurane potentiation. Such differential effects of EGF and NGF stimulation versus  insulin receptor stimulation may imply yet-unknown but distinct mechanisms of isoflurane potentiation of the cardiac K^ATPchannel.

In contrast to the results obtained in the whole cell configuration, isoflurane had no potentiating effect on K^ATPchannel in excised membrane patches. One possible explanation is that this effect may require not only inhibition of PTKs, but also input from another signaling pathway, or a cytosolic factor that is missing during cell-free conditions. Since dynamic and opposing activities of both PTKs and PTPs are important for catalyzing phosphorylation-dephosphorylation processes, we focused on cytosolic PTPs that are accessible in intact cells 42and during whole cell recording conditions but absent during cell-free conditions. The phosphatase PTP1B was previously reported to prevent single K^ATPchannel rundown in membrane patches from rat ventricular myocytes. 21Our study shows that, in the presence of PTP1B, isoflurane may potentiate genistein-activated channels. These results may suggest possible modulatory role for both PTKs and PTPs for cardiac K^ATPchannel and enhancement of its activity by isoflurane. However, we cannot exclude a possibility of direct interaction of PTP1B or other tyrosine phosphatases with the channel.

Protein tyrosine kinases are an important component of intracellular signaling in ischemic preconditioning and may also play an important role in cardioprotection mediated by volatile anesthetics. Genistein has been shown to prevent the beneficial action of ischemic preconditioning via  inhibition of PTKs. However, the role of PTKs for sarcolemmal K^ATPchannel activation in ischemic preconditioning or anesthetic preconditioning has not been defined. Recent whole-animal studies have demonstrated that PTKs are activated in ischemic preconditioning. 43Activation of both PTKs and protein kinase C during multiple ischemic preconditioning stimuli has been shown to reduce infarct size by opening the K^ATPchannel. This is in apparent contradiction to the current findings, which suggest inhibitory control of PTKs over sarcolemmal K^ATPchannel. However, the in vivo  studies have demonstrated that PTKs enhance the activity of mitochondrial K^ATPchannels and not necessarily sarcolemmal K^ATPchannels. 44Cardiac K^ATPchannels are important contributors to ischemic preconditioning, and recent evidence points to a predominant role of mitochondrial K^ATPchannels in the triggering of preconditioning, while sarcolemmal K^ATPchannels appear to mediate protection during the reperfusion phase. Although both types of K^ATPchannel are thought to be involved in cardioprotection by volatile anesthetics, 28their exact roles are not clarified, and the mechanism by which anesthetic enhancement of K^ATPchannel activity protects the heart has been a focus of intense investigation.

In conclusion, the results from this study suggest a modulatory role of the PTK-PTP signaling pathway in regulation of the cardiac sarcolemmal K^ATPchannel, possibly via  inhibitory control by basal PTK activity. At reduced intracellular ATP, inhibition of PTKs could promote channel opening. This further suggests that physiologic-metabolic conditions that modify endogenous PTK and PTP activities could shift the channel into an activatable state. Although our results suggest that inhibition of PTKs may underlie genistein actions on the cardiac sarcolemmal K^ATPchannel, a direct interaction of this isoflavone and also tyrosine phosphatases with the channel cannot be ruled out. This study also shows that isoflurane may potentiate genistein-activated I^KATPduring whole cell conditions but not in cell-free membrane patches, implying a possibility of another cytosolic factor requirement for isoflurane effects. The study suggests that a tyrosine phosphatase, such as PTP1B, may be a candidate factor to play a role in isoflurane potentiation of the cardiac sarcolemmal K^ATPchannel.