Anna Stadnicka

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.

Region of Epidural Blockade Determines Sympathetic and Mesenteric Capacitance Effects in Rabbits

Background The mechanisms producing hemodynamic changes during epidural anesthesia are incompletely understood. The role of capacitance changes in the splanchnic venous bed can be clarified by comparing blocks of differing segmental distributions. Specifically, we speculated that blocks that include the innervation to the mesenteric circulation alter hemodynamics, sympathetic activity, and venous capacitance to a greater extent than blocks without blockade of sympathetic nerves to this critical vascular bed.

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 KATP 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 KATP channels in APC.

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.

Differential Modulation of the Cardiac Adenosine Triphosphate-sensitive Potassium Channel by Isoflurane and Halothane

Background The cardiac adenosine triphosphate–sensitive potassium (KATP) 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 KATP 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 KATP channel. Methods The KATP channel current (IKATP) was monitored using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated from guinea pig hearts. IKATP was elicited by extracellular application of the potassium channel openers 2,4-dinitrophenol or pinacidil. Results Volatile anesthetics modulated IKATP in an anesthetic-dependent manner. Isoflurane facilitated the opening of the KATP channel. Following initial activation of IKATP 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 IKATP. However, isoflurane alone was unable to elicit KATP channel opening. In contrast, halothane inhibited IKATP elicited by 2,4-dinitrophenol by 50.6 ± 5.8% and 72.1 ± 11.6% at 0.4 and 1.0 mm, respectively. When IKATP was activated by pinacidil, halothane had no significant effect on the current. Conclusions The cardiac sarcolemmal KATP channel is differentially modulated by volatile anesthetics. Isoflurane can facilitate the further opening of the KATP channel following initial channel activation by 2,4-dinitrophenol or pinacidil. The effect of halothane was dependent on the method of channel activation, inhibiting IKATP activated by 2,4-dinitrophenol but not by pinacidil.

Effects of Epidural and Systemic Lidocaine on Sympathetic Activity and Mesenteric Circulation in Rabbits

BackgroundThe mechanisms producing hemodynamic changes during epidural anesthesia are incompletely understood. This study examines the sympathetic block and splanchnic venodilatation that result from extensive thoracolumbar epidural anesthesia in rabbits using direct measurements of sympathetic efferent nerve activity (SENA) and mesenteric vein diameter (VD). MethodsEpidural catheters were inserted in rabbits anesthetized with α-chloralose, paralyzed with vecuronium, and receiving mechanical ventilation. Arterial pressure was monitored with a femoral cannula, heart rate was determined from the pressure signal, SENA was measured from a postganglionic splanchnic nerve, and VD was measured from segments of ileum externalized in situ. Epidural anesthesia was induced with 0.4 ml/kg lidocaine, using concentrations of either 0.5, 1, or 1.5%. Control animals received intramuscular lidocaine in a dose of either 6 or 15 mg/kg. After recovery from epidural anesthesia, complete sympathetic blockade was induced by systemic administration of the ganglionic blocker hexamethonium (HX). Individual groups included from five to eight animals. ResultsA mild decrease in arterial pressure and SENA followed the larger dose of intramuscular lidocaine, but no changes occurred in VD in the control animals exposed to systemic lidocaine at levels comparable to that in the epidural groups (0.96–3.58 μg/ml). Epidural injectate extended from T2 to L5. All concentrations of epidural lidocaine produced comparable degrees of hypotension (-53.5 to-61.4%), decreased SENA (-82.6 to-95.5%), and increased VD (7.5 to 10.2%). The duration of the changes was greater with more concentrated lidocaine. Hexamethonium produced changes in arterial pressure and VD comparable to those evoked by epidural anesthesia. ConclusionsEpidural anesthesia increases splanchnic venous capacitance by markedly decreasing splanchnic sympathetic nerve activity.

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 (KATP) channels are considered essential in preconditioning mechanism. However, it is unclear whether KATP 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 &mgr;m H2O2 and 100 &mgr;m 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 KATP 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 KATP 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 KATP 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 KATP channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal KATP channel seems to act as an effector of preconditioning, whereas mitochondrial KATP channel plays a dual role as a trigger and an effector.

Preconditioning by Isoflurane Induces Lasting Sensitization of the Cardiac Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channel by a Protein Kinase C-δ–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-&dgr; and protein kinase C-ϵ. Results:The mean density of IKATP elicited by pinacidil (5 &mgr;m) 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 &mgr;m), isoflurane did not potentiate channel opening, and IKATP was 6.6 ± 4.6 pA/pF (n = 11). Application of protein kinase C-&dgr; 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-ϵ 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-&dgr; and protein kinase C-ϵ 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-&dgr;, rather than protein kinase C-ϵ, is a likely mediator of isoflurane effects, although both protein kinase C-&dgr; and protein kinase C-ϵ can modulate the channel function.

Modulation of cardiac inward rectifier K(+)current by halothane and isoflurane.

UNLABELLED The cellular mechanisms that underlie general anesthetic actions on the inward rectifier K(+) current (IKir), a determinant of the resting potential in myocardium, are not fully understood. Using the whole-cell patch clamp technique, therefore, we investigated the effects of halothane and isoflurane on IKir in guinea pig ventricular myocytes. At membrane potentials negative to the equilibrium potential for potassium both anesthetics decreased amplitude of the steady-state inward IKir in a concentration- and voltage-dependent manner. The slope conductance was reduced, but the activation kinetics of the inward current were not altered. At potentials positive to the equilibrium potential for potassium, the outward current was increased by both anesthetics, which also caused small depolarizing shifts in the activation curve. With high internal magnesium concentration, the outward current increase by isoflurane was abolished, and the inward current block by halothane was attenuated. Spermine prevented the effects of both anesthetics on IKir at all membrane potentials tested. The results show voltage-dependent modulation of cardiac IKir channel by volatile anesthetics. Distinct modification of anesthetic effects by inward rectification gating agents, magnesium and spermine, suggests anesthetic interactions with the IKir channel protein. IMPLICATIONS Differential modulation of myocardial inward rectifier potassium current by volatile anesthetics under normal and altered rectification may contribute to the mechanism of dysrhythmic actions by these anesthetics.

Enflurane, halothane, and isoflurane attenuate contractile responses to exogenous and endogenous norepinephrine in isolated small mesenteric veins of the rabbit

Background:Volatile anesthetics exert both direct and indirect (neurally mediated) effects to produce splanchnic venodilation. These effects may result in clinically relevant hemodynamic changes. The present study examined the direct effects of isoflurane, halothane, and enflurane on rabbit mesenteric venous smooth muscle. Methods:Changes in isometric tension, in response to exogenous and endogenous norepinephrine, were measured in isolated mesenteric vein rings before and during the administration of volatile anesthetics. Results:Exogenous and electrically evoked endogenous norepinephrine produced an increase in tension with superimposed rhythmic oscillations in tension. The exogenous norepincphrine-induced increase in tension was augmented in the presence of NG-nitro-L-arginine methyl ester (L-NAME, 5 X 10-5 M). The oscillatory activity was not altered by L-NAME. The increase in isometric tension in response to electrical stimulation was inhibited by phentolamine (5 X 10-6 M) and tetrodotoxin (3 X 10-6 M). Equianesthetic (1 MAC) concentrations of isoflurane, halothane, and enflurane significantly attenuated contractile responses to exogenous and endogenous norepinephrine, with isoflurane demonstrating a more depressant effect than halothane or enflurane. Volatile anesthetics also suppressed the amplitude and frequency of oscillations in the control as well as L-NAME-treated veins. The inhibitory effects of volatile anesthetics on the oscillations were comparable to the effects of ryanodine, a specific Mocker of calcium channels in sarcoplasmic reticulum. Conclusions:These results suggest that: 1) vascular endothelium, via endothelium-derived relaxing factor, modulates exogenous norepinephrine responses of the venous smooth muscle; 2) the oscillatory behavior of mesenteric veins may be attributed to calcium fluxes in the venous smooth muscle cells; and 3) the norepinephrine-dependent increases in contractile and oscillatory activity are attenuated more by isoflurane than halothane or enflurane. This indicates that volatile anesthetic-mediated splanchnic venodilation is, at least in part, due to a direct action on vascular smooth muscle as well as withdrawal of sympathetic tone.