Identification of Sliding Hiatus Hernia by High-Resolution Manometry and Upper Gastrointestinal Endoscopy in Patients with Gastro-Oesophageal Reflux Disease.

BACKGROUND/AIM: The aim of this study was to compare high-resolution manometry (HRM) and upper gastrointestinal (GI) endoscopy as diagnostic utilities in detecting a sliding hiatus hernia in patients with gastro-oesophageal reflux disease (GORD) symptoms. MATERIAL AND METHODS: For both diagnostic modalities, the data obtained from 31 patients (20 females; mean age 48.2) who qualified for Nissen fundoplication were analysed using oesophageal pressure topography in line with the Chicago Classification. Confirmation of hiatus hernia during the surgery was considered the gold standard. HRM protocol involved 10 consecutive boluses of 10 mL of water. RESULTS: Sliding hiatus hernia was confirmed intraoperatively in 29 out of 31 patients. In 14 patients, hiatus hernia was detected in HRM, while 19 patients were found to have hiatus hernia by upper GI endoscopy before surgery. No false positive results were obtained in HRM, while 15 false negative results were shown. In upper GI endoscopy, false positive data were observed in 1 patient, while false negative results were found in 10 patients. Thus, the sensitivity of HRM in detecting hiatus hernia was 48% (95%CIs: 29-67%), and sensitivity of upper GI endoscopy was 66% (95%CIs: 46-82%). It was not possible to assess the specificity of HRM or upper GI endoscopy because only 2 of 31 patients had no hiatus hernia during fundoplication (gold standard). False negative results (sensitivity) were not significantly different between compared diagnostic modalities HRM and upper GI endoscopy (52% vs. 34%, respectively, p = 0.29). CONCLUSIONS: Due to poor sensitivity, both modalities, i.e., HRM and upper GI endoscopy, are not reliable tools to diagnose sliding hiatus hernia in patients with GORD symptoms.

Preconditioning by isoflurane elicits mitochondrial protective mechanisms independent of sarcolemmal KATP channel in mouse cardiomyocytes.

Cardiac mitochondria and the sarcolemmal (sarc)KATP channels contribute to cardioprotective signaling of anesthetic-induced preconditioning. Changes in mitochondrial bioenergetics influence the sarcolemmal ATP-sensitive K (sarcKATP) channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATP channel (Kir6.2 KO) to investigate whether the functional sarcKATP channels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild-type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial reactive oxygen species production, and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180% ± 14% and 190% ± 15% and reactive oxygen species production to 118% ± 2% and 124% ± 6% of baseline in WT and Kir6.2 KO myocytes, respectively. Tetramethylrhodamine ethyl ester fluorescence decreased to 84% ± 6% in WT and to 86% ± 4% in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31% ± 1% to 21% ± 1% in WT and from 44% ± 2% to 35% ± 2% in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases the sensitivity of intact cardiomyocytes to oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATP channels in anesthetic-induced preconditioning by isoflurane.

Pharmacogenomic strain differences in cardiovascular sensitivity to propofol.

INTRODUCTION: A pharmacogenomic approach was used to further localize the genetic region responsible for previously observed enhanced cardiovascular sensitivity to propofol in Dahl Salt Sensitive (SS) versus control Brown Norway (BN) rats. METHODS: Propofol infusion levels that decreased blood pressure by 50% were measured in BN.13(SS) rats (substitution of SS chromosome 13 into BN) and in five congenic (partial substitution) strains of SS.13(BN). The effect of superfused 2,6 diisopropylphenol on small mesenteric arterial vascular smooth muscle transmembrane potential was measured in congenic strains before and during superfusion with Rp-adenosine-3',5'-cyclic monophosphorothioate and 2.5 µM (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, inhibitors of protein kinase A and G, respectively. The genetic locus and potential role of the renin gene in mediating vascular smooth muscle sensitivity to propofol were determined in three selected subcongenic SS.BN¹³ strains. RESULTS: A 30-32% smaller propofol infusion rate reduced blood pressure by 50% in BN.13(SS) compared with BN and the SS.13(BN) congenic containing an 80 BN gene substitution. Compared with the 80 BN gene-containing SS.13(BN) congenic, SS exhibited greater protein kinase A dependent vascular smooth muscle hyperpolarization in response to propofol. Using subcongenics, the increased propofol-induced cardiovascular sensitivity and hyperpolarization was further localized to an eight-gene region (containing the BN renin gene). Blockade of angiotensin receptors with losartan in this subcongenic increased propofol-induced hyperpolarization by threefold to that observed in SS. CONCLUSIONS: Enhanced cardiovascular sensitivity to propofol in SS (compared with BN) is caused by an altered renin gene. Through modified second messenger function, this differentially regulates vascular smooth muscle contractile state and reduces vascular tone, thereby exacerbating cardiovascular depression by propofol.

Differences in production of reactive oxygen species and mitochondrial uncoupling as events in the preconditioning signaling cascade between desflurane and sevoflurane.

BACKGROUND: Signal transduction cascade of anesthetic-induced preconditioning has been extensively studied, yet many aspects of it remain unsolved. Here, we investigated the roles of reactive oxygen species (ROS) and mitochondrial uncoupling in cardiomyocyte preconditioning by two modern volatile anesthetics: desflurane and sevoflurane. METHODS: Adult rat ventricular cardiomyocytes were isolated enzymatically. The preconditioning potency of desflurane and sevoflurane was assessed in cell survival experiments by evaluating myocyte protection from the oxidative stress-induced cell death. ROS production and flavoprotein fluorescence, an indicator of flavoprotein oxidation and mitochondrial uncoupling, were monitored in real time by confocal microscopy. The functional aspect of enhanced ROS generation by the anesthetics was assessed in cell survival and confocal experiments using the ROS scavenger Trolox. RESULTS: Preconditioning of cardiomyocytes with desflurane or sevoflurane significantly decreased oxidative stress-induced cell death. That effect coincided with increased ROS production and increased flavoprotein oxidation detected during acute myocyte exposure to the anesthetics. Desflurane induced significantly greater ROS production and flavoprotein oxidation than sevoflurane. ROS scavenging with Trolox abrogated preconditioning potency of anesthetics and attenuated flavoprotein oxidation. CONCLUSION: Preconditioning with desflurane or sevoflurane protects isolated rat cardiomyocytes from oxidative stress-induced cell death. Scavenging of ROS abolishes the preconditioning effect of both anesthetics and attenuates anesthetic-induced mitochondrial uncoupling, suggesting a crucial role for ROS in anesthetic-induced preconditioning and implying that ROS act upstream of mitochondrial uncoupling. Desflurane exhibits greater effect on stimulation of ROS production and mitochondrial uncoupling than sevoflurane.

Mechanism of differential cardiovascular response to propofol in Dahl salt-sensitive, Brown Norway, and chromosome 13-substituted consomic rat strains: role of large conductance Ca2+ and voltage-activated potassium channels.

Cardiovascular sensitivity to general anesthetics is highly variable among individuals in both human and animal models, but little is known about the genetic determinants of drug response to anesthetics. Recently, we reported that propofol (2,6-diisopropylphenol) causes circulatory instability in Dahl salt-sensitive SS/JRHsdMcwi (SS) rats but not in Brown Norway BN/NHsdMcwi (BN) rats and that these effects are related to genes on chromosome 13. Based on the hypothesis that propofol does target mesenteric circulation, we investigated propofol modulation of mesenteric arterial smooth muscle cells (MASMC) in SS and BN rats. The role of chromosome 13 was tested using SS-13(BN)/Mcwi and BN-13(SS)/Mcwi consomic strains with chromosome 13 substitution. Propofol (5 microM) produced a greater in situ hyperpolarization of MASMC membrane potential in SS than BN rats, and this effect was abrogated by iberiotoxin, a voltage-activated potassium (BK) channel blocker. In inside-out patches, the BK channel number, P(o), and apparent Ca(2+) sensitivity, and propofol sensitivity all were significantly greater in MASMC of SS rats. The density of whole-cell BK current was increased by propofol more in SS than BN myocytes. Immunolabeling confirmed higher expression of BK alpha subunit in MASMC of SS rats. Furthermore, the hyperpolarization produced by propofol, the BK channel properties, and propofol sensitivity were modified in MASMC of SS-13(BN)/Mcwi and BN-13(SS)/Mcwi strains toward the values observed in the background SS and BN strains. We conclude that differential function and expression of BK channels, resulting from genetic variation within chromosome 13, contribute to the enhanced propofol sensitivity in SS and BN-13(SS)/Mcwi versus BN and SS-13(BN)/Mcwi strains.

Age-related attenuation of isoflurane preconditioning in human atrial cardiomyocytes: roles for mitochondrial respiration and sarcolemmal adenosine triphosphate-sensitive potassium channel activity.

BACKGROUND: Clinical trials suggest that anesthetic-induced preconditioning (APC) produces cardioprotection in humans, but the mechanisms of APC and significance of aging for APC in humans are not well understood. Here, the impact of age on the role of two major effectors of APC, mitochondria and sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels, in preconditioning of the human atrial myocardium were investigated. METHODS: Right atrial appendages were obtained from adult patients undergoing cardiac surgery and assigned to mid-aged (MA) and old-aged (OA) groups. APC was induced by isoflurane in isolated myocardium and isolated cardiomyocytes. Mitochondrial oxygen consumption measurements, myocyte survival testing, and patch clamp techniques were used to investigate mitochondrial respiratory function and sarcKATP channel activity. RESULTS: After in vitro APC with isoflurane, the respiratory function of isolated mitochondria was better preserved after hypoxia-reoxygenation stress in MA than in OA. In isolated intact myocytes, APC significantly decreased oxidative stress-induced cell death in MA but not in OA, and isoflurane protection from cell death was attenuated by the sarcKATP channel inhibitor HMR-1098. Further, the properties of single sarcKATP channels were similar in MA and OA, and isoflurane sensitivity of pinacidil-activated whole cell KATP current was no different between MA and OA myocytes. CONCLUSION: Anesthetic-induced preconditioning with isoflurane decreases stress-induced cell death and preserves mitochondrial respiratory function to a greater degree in MA than in OA myocytes; however, sarcKATP channel activity is not differentially affected by isoflurane. Therefore, effectiveness of APC in humans may decrease with advancing age partly because of altered mitochondrial function of myocardial cells.

Role of sarcolemmal ATP-sensitive potassium channel in oxidative stress-induced apoptosis: mitochondrial connection.

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcK ATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcK ATP channel closing can lead to cardiac protection. Also, the role of the sarcK ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK ATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. sarcK ATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK ATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.

Volatile anesthetic-induced cardiac preconditioning.

Pharmacological preconditioning with volatile anesthetics, or anesthetic-induced preconditioning (APC), is a phenomenon whereby a brief exposure to volatile anesthetic agents protects the heart from the potentially fatal consequences of a subsequent prolonged period of myocardial ischemia and reperfusion. Although not completely elucidated, the cellular and molecular mechanisms of APC appear to mimic those of ischemic preconditioning, the most powerful endogenous cardioprotective mechanism. This article reviews recently accumulated evidence underscoring the importance of mitochondria, reactive oxygen species, and K(ATP) channels in cardioprotective signaling by volatile anesthetics. Moreover, the article addresses current concepts and controversies regarding the specific roles of the mitochondrial and the sarcolemmal K(ATP) channels in APC.

Isoflurane preconditioning uncouples mitochondria and protects against hypoxia-reoxygenation.

Ischemic cardiac injury can be substantially alleviated by exposing the heart to pharmacological agents such as volatile anesthetics before occurrence of ischemia-reperfusion. A hallmark of this preconditioning phenomenon is its memory, when cardioprotective effects persist even after removal of preconditioning stimulus. Since numerous studies pinpoint mitochondria as crucial players in protective pathways of preconditioning, the aim of this study was to investigate the effects of preconditioning agent isoflurane on the mitochondrial bioenergetic phenotype. Endogenous flavoprotein fluorescence, an indicator of mitochondrial redox state, was elevated to 195 +/- 16% of baseline upon isoflurane application in intact cardiomyocytes, indicating more oxidized state of mitochondria. Isoflurane treatment also elicited partial dissipation of mitochondrial transmembrane potential, which remained depolarized even after anesthetic withdrawal (tetramethylrhodamine fluorescence intensity declined to 83 +/- 3 and 81 +/- 7% of baseline during isoflurane exposure and washout, respectively). Mild uncoupling, with preserved ATP synthesis, was also detected in mitochondria that were isolated from animals that had been previously preconditioned by isoflurane in vivo, revealing its memory nature. These mitochondria, after exposure to hypoxia and reoxygenation, exhibited better preserved respiration and ATP synthesis compared with mitochondria from nonpreconditioned animals. Partial mitochondrial depolarization was paralleled by a diminished Ca(2+) uptake into isoflurane-treated mitochondria, as indicated by the reduced increment in rhod-2 fluorescence when mitochondria were challenged with increased Ca(2+) (180 +/- 24 vs. 258 +/- 14% for the control). In conclusion, isoflurane preconditioning elicits partial mitochondrial uncoupling and reduces mitochondrial Ca(2+) uptake. These effects are likely to reduce the extent of the mitochondrial damage after the hypoxic stress.

Mechanism of cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel activation by isoflurane in a heterologous expression system.

BACKGROUND: Activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channel during metabolic stress initiates cellular events that preserve cardiac performance. Previous studies showed that halogenated anesthetics prime KATP channels under whole cell voltage clamp and act in intracellular pH (pHi)-dependent manner on KATP channels in excised membrane patches. However, it is not known how halogenated anesthetics interact with these channels. METHODS: The authors evaluated the effect of pHi and isoflurane on the KATP channel subunits, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A, using HEK293 cells as a heterologous expression system. Single channel activity was recorded in the inside-out patch configuration. RESULTS: At pHi 7.4, isoflurane had negligible effect on activity of wild-type Kir6.2/SUR2A, but at pHi 6.8, the channel open probability was increased by isoflurane (0.177 +/- 0.077 to 0.364 +/- 0.164). By contrast, the open probability of truncated Kir6.2DeltaC26, which forms a functional channel without SUR2A, was attenuated by isoflurane at both pHi 7.4 and pHi 6.8. Coexpression of Kir6.2DeltaC26 with SUR2A restored pHi sensitivity of channel activation by isoflurane. Site-directed mutagenesis within the Walker motifs of SUR2A abolished isoflurane activation of KATP channel at pHi 6.8. In addition, the pancreatic-type channels expressing sulfonylurea receptor SUR1 could not be activated by isoflurane. CONCLUSIONS: The nucleotide binding domains of SUR2A play a crucial role in isoflurane facilitation of the KATP channel activity at moderately acidic pHi as would occur during early ischemia. These findings support direct and differential interaction of isoflurane with the subunits of the cardiac sarcolemmal KATP channel.

Distinct roles for sarcolemmal and mitochondrial adenosine triphosphate-sensitive potassium channels in isoflurane-induced protection against oxidative stress.

BACKGROUND: Cardiac preconditioning, including that induced by halogenated anesthetics, is an innate protective mechanism against ischemia-reperfusion injury. The adenosine triphosphate-sensitive potassium (K(ATP)) channels are considered essential in preconditioning mechanism. However, it is unclear whether K(ATP) channels are triggers initiating the preconditioning signaling, and/or effectors responsible for the cardioprotective memory and activated during ischemia-reperfusion. METHODS: Adult rat cardiomyocytes were exposed to oxidative stress with 200 microM H(2)O(2) and 100 microM FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial K(ATP) channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean +/- SD. RESULTS: Oxidative stress induced cell death in 47 +/- 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 +/- 8%. Blockade of the sarcolemmal K(ATP) channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 +/- 21%) or only during oxidative stress (50 +/- 12%), but not when applied during isoflurane pretreatment (29 +/- 13%). Inhibition of the mitochondrial K(ATP) channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 +/- 23, 45 +/- 23, and 46 +/- 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively. CONCLUSION: The authors conclude that both sarcolemmal and mitochondrial K(ATP) channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal K(ATP) channel seems to act as an effector of preconditioning, whereas mitochondrial K(ATP) channel plays a dual role as a trigger and an effector.

Impact of in vivo preconditioning by isoflurane on adenosine triphosphate-sensitive potassium channels in the rat heart: lasting modulation of nucleotide sensitivity during early memory period.

BACKGROUND: The early memory of anesthetic-induced preconditioning (APC) is a period when myocardial protection continues even after removal of the anesthetic. Because adenosine triphosphate-sensitive potassium (KATP) channels are important mediators of APC, the authors investigated the hypothesis that the memory involves channel priming by isoflurane via a long-term modulation of the sensitivity to intracellular adenosine nucleotides. METHODS: Ventricular cardiomyocytes were obtained from the rat hearts after 30 min in vivo APC with 1.4% isoflurane and from control non-APC rat hearts. Whole cell and excised inside-out patch clamp techniques were used to study the sarcolemmal KATP channel. Membrane expression of KATP channel proteins, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A were assessed in APC and non-APC hearts by Western blotting. RESULTS: Activation of whole cell KATP current by isoflurane was enhanced after in vivo APC. At the single-channel level, this was paralleled by a 12-fold decrease in adenosine 5'-triphosphate sensitivity and a 3-fold decrease in adenosine 5'-diphosphate sensitivity, without changing the probability of channel opening or single-channel conductance. The membrane expression of Kir6.2 and SUR2A subunits was not altered by in vivo APC. A direct in vitro application of isoflurane to excised membrane patches increased the channel open probability and produced a 4-fold decrease in adenosine 5'-triphosphate sensitivity only of channels in non-APC myocytes. CONCLUSIONS: In vivo APC by isoflurane decreases sensitivity of the sarcolemmal KATP channel to inhibition by adenosine 5'-triphosphate and decreases adenosine 5'-diphosphate sensitivity. These effects persist even after discontinuation of the anesthetic, suggesting a possible novel factor that may contribute to the mechanism of early memory of APC.

Preconditioning by isoflurane induces lasting sensitization of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel by a protein kinase C-delta-mediated mechanism.

BACKGROUND: Cardioprotective effects of volatile anesthetics in anesthetic-induced preconditioning involve activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels. This study addressed the memory phase of anesthetic preconditioning by investigating whether brief exposure to isoflurane produces lasting sensitization of the sarcKATP channel and whether protein kinase C mediates this effect. METHODS: Whole cell sarcKATP channel current (IKATP) was monitored from single isolated rat ventricular cardiomyocytes. Pinacidil was used to open the channel, and the magnitude of activated IKATP was an indicator of channel's ability to open. Involvement of protein kinase C was investigated using chelerythrine and isoform-specific peptide inhibitors and activators of protein kinase C-delta and protein kinase C-epsilon. RESULTS: The mean density of IKATP elicited by pinacidil (5 microm) in anesthetic-free conditions was 3.8 +/- 3.7 pA/pF (n = 11). After 10 min of exposure to isoflurane (0.56 mm) and 10 or 30 min of anesthetic washout, pinacidil-elicited IKATP was increased to 15.6 +/- 11.3 pA/pF (n = 12; P < 0.05) and 11.8 +/- 3.9 pA/pF (n = 6; P < 0.05), respectively. In the presence of chelerythrine (5 microm), isoflurane did not potentiate channel opening, and IKATP was 6.6 +/- 4.6 pA/pF (n = 11). Application of protein kinase C-delta peptide inhibitor also abolished isoflurane-induced sensitization of sarcKATP channel, and IKATP was 7.7 +/- 5.4 pA/pF (n = 12). In contrast, protein kinase C-epsilon peptide inhibitor did not affect channel sensitization, and pinacidil-elicited current was 14.8 +/- 9.6 pA/pF (n = 12). Interestingly, when both protein kinase C-delta and protein kinase C-epsilon activators were applied instead of isoflurane, they sensitized the channel to the same extent as isoflurane (18.9 +/- 7.2 pA/pF, n = 11, and 18.6 +/- 11.1 pA/pF, n = 10, respectively). CONCLUSION: Isoflurane induces prolonged sensitization of the sarcKATP channel to opening that persists even after anesthetic withdrawal. Our results indicate that protein kinase C-delta, rather than protein kinase C-epsilon, is a likely mediator of isoflurane effects, although both protein kinase C-delta and protein kinase C-epsilon can modulate the channel function.

The interaction of isoflurane and protein kinase C-activators on sarcolemmal KATP channels.

Protein kinase C (PKC)-dependent signaling pathways may be involved in the "memory" effect of anesthetic and ischemic preconditioning, which facilitates activation of cardioprotective adenosine triphosphate (ATP)-sensitive potassium channels during later ischemic challenge and ATP depletion. Using patch-clamp techniques, we found that exposure of isolated guinea pig cardiomyocytes to 1 mM of isoflurane after phorbol ester stimulation of PKC facilitates the induction of larger (P < or = 0.05) sarcolemmal K(ATP) channel currents (IKATP) during cell dialysis with 0.5, compared to 1.0, mM of ATP in the pipette (10 +/- 5 versus 2 +/- 1 pA/pF in five and six cells, respectively). A PKC inhibitor, bisindolylmaleimide, abolished the induction of IKATP by a second brief isoflurane exposure under these conditions. A diacylglycerol PKC activator applied via the pipette elicited concentration-related activation of IKATP. The diacylglycerol alone (0.5 microM) elicited I(KATP), averaging 5 +/- 3 pA/pF in nine cells. Briefly treating myocytes on the microscope stage with isoflurane, followed by washout and patching with the same diacylglycerol solution, elicited larger (P < or = 0.01) IKATP, averaging 40 +/- 9 pA/pF (10 cells), with an onset 48 +/- 2 min after anesthetic pretreatment. Facilitation of IKATP by isoflurane during the reduction of intracellular ATP is dependent on PKC, whereas "preconditioning" myocytes with isoflurane causes persistent changes in sarcolemmal KATP channel function, which enhance the induction of IKATP by a diacylglycerol.

Contribution of reactive oxygen species to isoflurane-induced sensitization of cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to pinacidil.

BACKGROUND: Myocardial protection by volatile anesthetics involves activation of cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channels. The authors have previously shown that isoflurane enhances sensitivity of the sarcolemmal K(ATP) channel to the opener, pinacidil. Because reactive oxygen species seem to be mediators in anesthetic preconditioning, the authors investigated whether they contribute to the mechanism of the sensitization effect by isoflurane. METHODS: Ventricular myocytes were isolated from guinea pig hearts for the whole cell patch clamp recordings of the sarcolemmal K(ATP) channel current (I(KAPT)). Free radical scavengers N-acetyl-L-cysteine, carnosine, superoxide dismutase, and catalase were used to investigate whether reactive oxygen species mediate isoflurane facilitation of the channel opening by pinacidil. A possible role of the mitochondrial K(ATP) channels was tested using a blocker of these channels, 5-hydroxydecanoate. RESULTS: The mean density (+/- SEM) of I(KAPT) elicited by pinacidil (20 microM) was 18.9 +/- 1.8 pA/pF (n = 11). In the presence of isoflurane (0.55 mM), the density of pinacidil-activated I(KAPT) increased to 38.5 +/- 2.4 pA/pF (n = 9). Concurrent application of isoflurane and N-acetyl-L-cysteine decreased the sensitization effect by isoflurane in a concentration-dependent manner, whereby the densities of I(KAPT) were 32.6 +/- 1.4 (n = 6), 26.2 +/- 2.3 (n = 6), and 19.4 +/- 2.1 pA/pF (n = 8) at 100, 250, and 500 microM N-acetyl-L-cysteine, respectively. Concurrent application of isoflurane and carnosine (100 microM), superoxide dismutase (100 U/ml), or catalase (100 U/ml) attenuated the densities of I(KAPT) to 27.9 +/- 2.6, 27.2 +/- 2.9, and 25.9 +/- 2.2 pA/pF, respectively. None of the scavengers affected activation of I(KAPT) by pinacidil alone. 5-Hydroxydecanoate (100 microM) did not alter the sensitization effect by isoflurane, and the density of I(KAPT) in this group was 37.1 +/- 3.8 pA/pF (n= 6). CONCLUSION: These results suggest that reactive oxygen species contribute to the mechanism by which isoflurane sensitizes the cardiac sarcolemmal K(ATP) channel to the opener, pinacidil.

Isoflurane decreases ATP sensitivity of guinea pig cardiac sarcolemmal KATP channel at reduced intracellular pH.

BACKGROUND: Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel. METHODS: The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts. RESULTS: At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm. CONCLUSIONS: Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.

Isoflurane sensitizes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to pinacidil.

BACKGROUND: Cardioprotective effects of isoflurane are partially mediated by the sarcolemmal adenosine triphosphate-sensitive potassium (sarcK ATP ) channel. The authors tested the hypothesis that isoflurane sensitizes sarcK ATP channels to a potassium channel opener, pinacidil, adenosine- and phospholipid-mediated pathways. METHODS: Activation by pinacidil of the K ATP current (I KATP ) was monitored in guinea pig ventricular myocytes at 0.5 and 5 mm intracellular ATP in the whole cell configuration of the patch clamp technique. The sensitization effect was evaluated by pretreating each myocyte with isoflurane (0.57 +/- 0.04 mm) before application of pinacidil (5 micro m) in the continued presence of the anesthetic. To investigate whether intracellular signaling pathways may be involved in isoflurane sensitization, the authors used the adenosine receptor antagonist theophylline (100 micro m) and the phosphatidylinositol kinase inhibitor wortmannin (100 micro m). RESULTS: The density of pinacidil-activated I KATP was higher at 0.5 mm ATP (20.7 +/- 3.2 pA/pF) than at 5 mm ATP (2.0 +/- 0.3 pA/pF). At 0.5 mm ATP, pretreatment with isoflurane caused an increase in density of pinacidil-activated I KATP (42.4 +/- 6.2 pA/pF) and accelerated the rate of current activation (from 5.4 +/- 1.2 to 39.0 +/- 7.9 pA. pF(-1). min(-1) ). Theophylline attenuated current activation by pinacidil (9.4 +/- 3.9 pA/pF) and abolished the sensitization effect of isoflurane on I KATP (10.0 +/- 2.5 pA/pF). Wortmannin did not alter pinacidil activation of I KATP (13.2 +/- 1.7 pA/pF) but prevented sensitization by isoflurane (15.8 +/- 4.5 pA/pF). CONCLUSIONS: These results suggest that isoflurane increases sensitivity of cardiac sarcK ATP channels to the potassium channel opener pinacidil. Blockade of adenosine receptors or phosphatidylinositol kinases abolishes the sensitization effect, suggesting that the adenosine and phospholipid signaling pathways may be involved in the actions by isoflurane.

Protein tyrosine kinase-dependent modulation of isoflurane effects on cardiac sarcolemmal K(ATP) channel.

BACKGROUND: Cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channels and protein tyrosine kinases (PTKs) are mediators of ischemic preconditioning, but the interaction of both and a role in myocardial protection afforded by volatile anesthetics have not been defined. METHODS: Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal K(ATP) channel in acutely dissociated guinea pig ventricular myocytes. RESULTS: At 0.5 mm internal ATP, genistein (50 microm) elicited whole cell K(ATP) current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 microm. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 microm), did not elicit K(ATP) current. Isoflurane (0.5 mm) increased genistein (35 microm)-activated whole cell K(ATP) current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on K(ATP) current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 microm) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 microg/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114. CONCLUSIONS: These results suggest that the PTK-protein tyrosine phosphatase signaling pathway may be one of the regulators of cardiac sarcolemmal K(ATP) channel and may play a role in modulating its responsiveness to isoflurane. Relative importance of this modulation for cardioprotection by volatile anesthetics remains to be established.

Differential modulation of the cardiac adenosine triphosphate-sensitive potassium channel by isoflurane and halothane.

BACKGROUND: The cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channel is activated during pathophysiological episodes such as ischemia and hypoxia and may lead to beneficial effects on cardiac function. Studies of volatile anesthetic interactions with the cardiac K(ATP) channel have been limited. The goal of this study was to investigate the ability of volatile anesthetics halothane and isoflurane to modulate the cardiac sarcolemmal K(ATP) channel. METHODS: The K(ATP) channel current (I(KATP)) was monitored using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated from guinea pig hearts. I(KATP) was elicited by extracellular application of the potassium channel openers 2,4-dinitrophenol or pinacidil. RESULTS: Volatile anesthetics modulated I(KATP) in an anesthetic-dependent manner. Isoflurane facilitated the opening of the K(ATP) channel. Following initial activation of I(KATP) by 2,4-dinitrophenol, isoflurane at 0.5 and 1.3 mm further increased current amplitude by 40.4 +/- 11.1% and 58.4 +/- 20.6%, respectively. Similar results of isoflurane were obtained when pinacidil was used to activate I(KATP). However, isoflurane alone was unable to elicit K(ATP) channel opening. In contrast, halothane inhibited I(KATP) elicited by 2,4-dinitrophenol by 50.6 +/- 5.8% and 72.1 +/- 11.6% at 0.4 and 1.0 mm, respectively. When I(KATP) was activated by pinacidil, halothane had no significant effect on the current. CONCLUSIONS: The cardiac sarcolemmal K(ATP) channel is differentially modulated by volatile anesthetics. Isoflurane can facilitate the further opening of the K(ATP) channel following initial channel activation by 2,4-dinitrophenol or pinacidil. The effect of halothane was dependent on the method of channel activation, inhibiting I(KATP) activated by 2,4-dinitrophenol but not by pinacidil.