Lawrence A. Turner

Differential Effects of Halothane, Enflurane, and Isoflurane on Ca2+transients and Papillary Muscle Tension in Guinea Pigs

These studies were designed to examine the effects of inhalational anesthetics on rapid changes in myocardial intracellular Ca2+ and Ca2+ sensitivity of the contractile apparatus. The effects of halothane, enflurane, and isoflurane on rapid changes in intracellular Ca2+ (Ca2+ transients as measured with bioluminescent protein aequorin) and contractile characteristics were compared in guinea pig right ventricular papillary muscles. In addition to examination of their potencies at equianesthetic concentrations, the effects of these agents on alterations in Ca2+ sensitivity at myofilaments were also investigated. The negative inotropic effects of halothane (0.65 and 1.15%) and enflurane (1.0 and 2.2%) were dose-dependent and closely related to a decrease in Ca2+ transients. In the presence of isoflurane (0.77 and 1.6%), the contractile force decreased in a dose-dependent manner, but the decrease was significantly less as compared to that with equianesthetic concentrations of halothane and enflurane. An additional feature observed in the presence of isoflurane was a dissociation between intracellular Ca2+ availability and contractile force. Although the magnitude of the Ca2+ transients did not change when the percentage of isoflurane was increased from 0.77 to 1.6, the contractile force decreased. Because of these findings, the effects of halothane (1.2%), enflurane (2.2%), and isoflurane (1.6%) on the relationship between intracellular Ca2+ and tension developed in the papillary muscle were examined in order to assess myofibrillar responsiveness to Ca2+. The results indicate that only isoflurane slightly but significantly shifted the Ca2+/isometric tension curve toward higher intracellular Ca2+ concentrations; no differences were observed in the absence and presence of equianesthetic concentrations of halothane and enflurane.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-Dependent Effects of Volatile Anesthetics on Cardiac Sodium Current

Cardiac dysrhythmias during inhaled anesthesia are well documented and may, in part, involve depression of the fast inward Na+ current (INa) during the action potential upstroke.In this study, we examined the effects of halothane, isoflurane, and sevoflurane at clinically relevant concentrations on INa in single ventricular myocytes isolated enzymatically from adult guinea pig hearts. INa was recorded using standard whole-cell configuration of the patch clamp technique. Halothane at 0.6 mM and 1.2 mM produced significant (P < 0.05) depressions of peak INa of 12.3% +/- 1.8% and 24.4% +/- 4.1% (mean +/- SEM, n = 12), respectively. Isoflurane (0.5 mM, n = 12; 1.0 mM, n = 15) and sevoflurane (0.6 mM, n = 14; 1.2 mM, n = 12) were less potent than halothane, decreasing peak INa by 4.8% +/- 1.1% and 11.4% +/- 1.4% (isoflurane) and 3.0% +/- 0.7% and 10.7% +/- 3.9% (sevoflurane). The depressant effects on INa were reversible in all cases. For all anesthetics tested, the degree of block increased at more depolarizing potentials. Anesthetics induced significant shifts in the steady-state inactivation and activation of the channel toward more hyperpolarizing potentials. The present findings indicate that volatile anesthetics at clinical concentrations decrease the cardiac INa in a dose- and voltage-dependent manner. At approximately equianesthetic concentrations, the decrease of INa caused by halothane was twice that observed with isoflurane or sevoflurane. (Anesth Analg 1997;84:285-93)

Protein kinase C-epsilon primes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to modulation by isoflurane.

Background:Cardioprotection by volatile anesthetic–induced preconditioning is known to involve intracellular signaling pathways. Recent studies have shown that protein kinase C (PKC) plays an important role in anesthetic-induced preconditioning. In this study, the effects of the activation of specific isozymes of PKC, specifically PKC-&egr; and -&dgr;, on the modulation of the sarcolemmal adenosine triphosphate–sensitive potassium (sarcKATP) channel by isoflurane were investigated. Methods:The sarcKATP current was measured in ventricular myocytes isolated from guinea pig hearts using the whole cell configuration of the patch clamp technique. Peptides that induced the translocation of specific PKC isozymes were used to activate PKC-&egr; and PKC-&dgr;. Results:Under whole cell conditions, isoflurane alone was unable to elicit the opening of the sarcKATP channel. Pretreatment with the specific PKC-&egr; activator, PP106, primed the sarcKATP channel to open in the presence of isoflurane. The resulting sarcKATP current densities in the presence of 0.88 mm isoflurane were 6.5 ± 6.0 pA/pF (n = 7) and 40.4 ± 18.2 pA/pF (n = 7) after pretreatment with 100 and 200 nm PP106, respectively. The PKC-&egr; antagonist PP93 abolished this effect. A scrambled peptide of the PKC-&egr; activator PP105 did not prime the sarcKATP channel. The PKC-&dgr; activator PP114 was significantly less effective in priming the sarcKATP channel. 5-Hydroxydecanoate significantly attenuated the effect of the PKC-&egr; activator on the sarcKATP channel. In addition, immunohistochemical analysis showed that the PKC-&egr; isoform translocated to both the mitochondria and sarcolemma after anesthetic-induced preconditioning, whereas the PKC-&dgr; isoform translocated to the mitochondria. Conclusion:The PKC-&egr; isozyme primed the sarcKATP channel to open in the presence of isoflurane. The PKC-&dgr; isozyme was significantly less effective in modulating the isoflurane effect on this channel.

Effects of hypothermia, potassium , and verapamil on the action potential characteristics of canine cardiac Purkinje fibers.

Background Hypothermia may induce hypokalemia and increase Intracellular Calcium2+ by affecting serum Potassium sup + and Calcium2+ fluxes across the cell membrane. These ionic alterations may significantly change the electrophysiologic characteristics of the cardiac action potential and may induce cardiac arrhythmias. The current study was undertaken to determine whether electrophysiologic changes in Purkinje fibers induced by hypothermia could be reversed by manipulating the extracellular Potassium sup + and transmembrane Calcium2+ fluxes by Calcium2+ channel blockade with verapamil. Methods A conventional microelectrode method was used to determine the effects of hypothermia (32 + 0.5 degrees Celsius and 28 + 0.5 degrees Celsius) and various external Potassium sup + concentrations ([Potassium sup +]o) (2.3, 3.8, and 6.8 mM) on maximum diastolic potential, maximum rate of phase 0 depolarization (Vmax), and action potential duration (APD) at 50% (APD50) and at 95% (APD95) repolarization in isolated canine cardiac Purkinje fibers. To evaluate the contribution of the slow inward Calcium2+ current to action potential changes in hypothermia, the experiments were repeated in the presence of the Calcium2+ ‐channel antagonist verapamil (1 micro Meter). Results Variations of [Potassium sup +]o induced the expected shifts in maximum diastolic potential, and hypothermia (28 degrees Celsius) induced moderate depolarization, but only when [Potassium sup +]o was *symbol* 3.9 mM (P < 0.05). Hypothermia decreased Vmax at all [Potassium sup +]o studied (P < 0.05). Regardless of the temperature, Vmax was not affected by verapamil when [Potassium sup +]o *symbol* was 3.9 mM, but at 6.8 mM [Potassium sup +]o in hypothermia Vmax was significantly lower in the presence of verapamil. Hypothermia increased both the APD50 and the APD95. The effects of verapamil on APD were temperature and [Potassium sup +] sub o dependent; between 37 degrees Celsius and 28 degrees Celsius with 2.3 mM [Potassium sup +]o in the superfusate, verapamil did not affect APD. At 28 degrees Celsius in the presence of verapamil, the APD sub 50 and APD95 decreased only if the [Potassium sup +]o was *symbol* 3.9 mM. Conclusions Verapamil and Potassium sup + supplementation in hypothermia may exert an autiarrhythmic effect, primarily by reducing the dispersion of prolonged APD.

Actions of Halothane on the Electrical Activity of Purkinji Fibers Derived from Normal and Interfact Canine Hearts

The effects of 0.39 mM halothane (approx. 1.1 vol%) on the action potentials of proximal (false tendon) and distal (apical) left ventricular Purkinje fibers were compared in analogous in vitro preparations derived from normal dogs and animals surviving 1 day following acute myocardial infarction. In ten noninfarcted hearts, halothane reduced regional differences in repolarization by decreasing action potential duration (APD90, mean +/- SE) in proximal fibers from 300 +/- 7 to 277 +/- 6 msec (P less than or equal to 0.01) without decreasing APD90 in distal fibers (control 240 +/- 4 msec, halothane 249 +/- 5 msec). In ten infarcted hearts, halothane accentuated pathologic differences in repolarization by decreasing APD90 in the non-ischemic proximal fibers from 311 +/- 8 to 287 +/- 7 msec (P less than or equal to 0.01), while increasing APD90 in the ischemic distal fibers from 375 +/- 15 to 406 +/- 18 msec (P less than or equal to 0.01). Halothane also decreased the overshoot from 32.9 +/- 1.0 to 28.4 +/- 0.8 m V (P less than or equal to 0.01) and Vmax from 356 +/- 28 to 300 +/- 22 V/s (P less than or equal to 0.05) in ischemic fibers. In seven infarcts evaluated by extrastimulus techniques, halothane slowed the conduction of premature impulses and prolonged refractoriness, while, in five of the seven hearts, it reversibly increased the range of coupling intervals which induced probable reentrant responses. In a separate study of seven infarcts, halothane decreased the rate of spontaneous activity originating in the ischemic region. It is concluded that halothane facilitates the occurrence of re-entry while inhibiting the initiation of abnormal impulses in the in vitro canine infarction model.

Modulation of Cardiac Sodium Current by α1-stimulation and Volatile Anesthetics

Background: alpha1 ‐adrenoceptor stimulation is known to produce electrophysiologic changes in cardiac tissues, which may involve modulations of the fast inward Na sup + current (INa). A direct prodysrhythmic alpha1 ‐mediated interaction between catecholamines and halothane has been demonstrated, supporting the hypothesis that generation of halothane‐epinephrine dysrhythmias may involve slowed conduction, leading to reentry. In this study, we examined the effects of a selective alpha1 ‐adrenergic receptor agonist, methoxamine, on cardiac INa in the absence and presence of equianesthetic concentrations of halothane and isoflurane in single ventricular myocytes from adult guinea pig hearts. Methods: INa was recorded using the standard whole‐cell configuration of the patch‐clamp technique. Voltage clamp protocols initiated from two different holding potentials (VH) were applied to examine state‐dependent effects of methoxamine in the presence of anesthetics. Steady state activation and inactivation and recovery from inactivation were characterized using standard protocols. Results: Methoxamine decreased INa in a concentration‐ and voltage‐dependent manner, being more potent at the depolarized VH. Halothane and isoflurane interacted synergistically with methoxamine to suppress INa near the physiologic cardiac resting potential of ‐80 mV. The effect of methoxamine with anesthetics appeared to be additive when using a VH of ‐110 mV, a potential where no Na sup + channels are in the inactivated state. Methoxamine in the absence and presence of anesthetics significantly shifted the half maximal inactivation voltage in the hyperpolarizing direction but had no effect on steady‐state activation. Conclusion: The present results show that methoxamine (alpha1 ‐adrenergic stimulation) decreases cardiac Na sup + current in a concentration‐ and voltage‐dependent manner. Further, a form of synergistic interaction between methoxamine and inhalational anesthetics, halothane and isoflurane, was observed. This interaction appears to depend on the fraction of Na sup + channels in the inactivated state.

The effects of halothane and isoflurane on slowly inactivating sodium current in canine cardiac Purkinje cells

The effects of halothane (0.45 and 0.9 mM, equivalent to 0.7 and 1.5 vol%, respectively) and isoflurane (0.56 and 1.23 mM, equivalent to 0.9 and 2.0 vol%) on slowly inactivating Na+ current were examined by whole-cell voltage-clamp techniques. This approach allows evaluation of the role of anesthetic inhibition of inward Na+ current on the action potentials of canine Purkinje fibers isolated from the false tendons. Cells were super-fused with Tyrodes solution containing nifedipine and Ni2+ to block Ca2+ channel currents and internally dialyzed with Cs+ to block outward K+ currents. Veratridine (0.5 μM) was used throughout the experiments to enhance the slow Na+ current. Na+ current was elicited by depolarizing pulses from a holding potential of −100 mV to stepwise (10 mV increments) more positive membrane potentials and measured at 200 ms after initiation of the voltage pulse before, during, and after exposure to anesthetics. Slowly inactivating Na+ current showed threshold activation at −80 mV and peak activation around −55 to −40 mV. This current was abolished by 5 μM tetrodotoxin, showing the characteristic feature of Na+ channel current. Halothane and isoflurane depressed the amplitude of slowly inactivating Na+ current in a concentration-dependent manner and did not shift the current-voltage relationship for slow Na+ current activation. There was no significant difference between the sensitivities of slow Na+ current to halothane or isoflurane at equianesthetic concentrations. Inhibition of slow inward Na+ current may contribute to the marked decrease of action potential duration produced by volatile anesthetics in false tendon Purkinje fibers but does not account for the larger decreases of duration produced by isoflurane than halothane. This anesthetic action may facilitate reexcitation of Purkinje fibers by abbreviating repolarization and have an antiarrhythmic influence on after-depolarization-related arrhythmias during repolarization of the Purkinje fiber action potential.

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 ≤ 0.05) sarcolemmal KATP 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 &mgr;M) elicited IKATP, 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 ≤ 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.

Differential Effects of Halothane and Isoflurane on Contractile Force and Calcium Transients in Cardiac Purkinje Fibers

BackgroundHalothane and isoflurane have been shown to differentially effect mechanisms of contraction in working myocardial fibers. The aim of this study was to compare effects of halothane and isoflurane on contractile force (CF) and Ca2+ transients in canine cardiac Purkinje fiber preparations. MethodsFiber preparations (n = 29) were superfused with Krebs-Ringer solution and stimulated at 40–60 pulses/min at 30°C in the absence and presence of 0.55% and 1.01% isoflurane or 0.27% and 0.78% halothane. Isometric tension (CF), and intracellular Ca2+ transients (luminescence, L) were measured after microinjecting the Ca2+ sensitive photoprotein aequorin into Purkinje fibers. Peak CF and peak L, rate of rise (slope) of CF and L, time to attain peak CF and L, and duration of CF and L at half-peak CF and L, were measured at 5 mM extracellular CaCl2. Changes in peak CF and peak L also were measured during incremental increases in CaCl2 from 3.6 to 9–0 mM. ResultsBoth anesthetics depressed peak CF and peak L and the rate of increase in peak CF and L in a concentration-dependent fashion, and effects of halothane were greater than those of isoflurane. Time to attain peak L and duration of L at half-peak L was decreased or unchanged by isoflurane and was increased by halothane, whereas time to attain peak CF and duration of CF at half-peak CF was shortened by both. The change in peak CF response as a function of the change in peak L with increasing extracellular CaCl2 was attenuated similarly by both halothane and isoflurane. ConclusionsHalothane depresses peak CF and Ca2+ transients and prolongs Ca2+ transients more than does isoflurane at equivalent minimum alveolar concentration in Purkinje fibers. This suggests Ca2+ concentration is differentially altered by anesthetics in this tissue. Peak CF at equivalent peak Ca2+ transients, however, appears to be attenuated similarly by both anesthetics. These differences in anesthetic effect are qualitatively similar to those found in cardiac tissue of other species. These findings add to our understanding of effects of volatile anesthetics on contractile properties and myoplasmic Ca2+ in cardiac Purkinje fibers.

A Comparison of the Effects of Halothane and Tetrodotoxin on the Regional Repolarization Characteristics of Canine Purkinje Fibers

The effects of halothane, tetrodotoxin (TTX), veratridine (VTD), and alterations of extracellular calcium ion concentration [Ca++]0) on regional differences of canine Purkinje fiber action potential duration (APD50 and APD90) were investigated in vitro at a paced rate of 75 beats per min. Under control conditions (n = 15 hearts) APD90 of proximal (false tendon) fibers (289 +/- 6 ms) always exceeded (P less than or equal to 0.01) that of distal (apical) fibers (213 +/- 4 ms). Halothane (0.35-1.07 mM) reduced regional differences of APD90 by producing dose-dependent decreases of proximal APD90 without decreases of distal APD90. The regional actions of halothane were similar to those of low (0.33-1.0 microM) concentrations of the Na+ channel antagonist TTX, which also decreased proximal APD90 more than distal APD90. The actions of halothane in combination with TTX further decreased proximal APD90, whereas the Na+ channel agonist VTD, which increased proximal APD90 more than distal APD90, reversed the regional actions of halothane. Decreasing Ca++ influx by reducing [Ca++]0 from 1.8 to 0.6 mM increased proximal APD90 more than distal APD90 in a manner opposite to the regional actions of halothane. Although there was no difference between the values of APD90 obtained for each region in the presence of halothane at 0.6, 1.8, and 3.6 mM [Ca++]0, the action of halothane decreasing APD90 of proximal fibers was more prominent at 0.6 mM [Ca++]0 because of the increased APD90 of fibers under this condition. The findings are consistent with, but do not definitively prove, the hypothesis that halothane may decrease APD90 of proximal Purkinje fibers by a mechanism similar to that of TTX involving inhibition of plateau-phase inward Na+ current.