Thiopental Alters Contraction, Intracellular Ca2+, and pH in Rat Ventricular Myocytes

Background: Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2+]i) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes.

Methods: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+]i were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied.

Results: Field stimulation increased [Ca2+]i from 85 +/- 10 nM to 355 +/- 22 nM (mean +/- SEM). Myocytes shortened by 10% of resting cell length (127 +/- 5 [micro sign]m). Times to peak [Ca2+]i and shortening were 139 +/- 6 and 173 +/- 7 msec, respectively. Times to 50% recovery for [Ca2+]i and shortening were 296 +/- 6 and 290 +/- 6 ms, respectively. Addition of thiopental (30-1,000 [micro sign]M) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+]i and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 [micro sign]M) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+ -H+ exchange.     

Effects of Isoflurane, Sevoflurane, and Halothane on Myofilament Ca2+ Sensitivity and Sarcoplasmic Reticulum Ca2+ Release in Rat Ventricular Myocytes

Background: The aim of this study was to describe and compare the effects of isoflurane, sevoflurane, and halothane at selected concentrations (i.e., concentrations that led to equivalent depression of the electrically evoked Ca2+ transient) on myofilament Ca2+ sensitivity, sarcoplasmic reticulum (SR) Ca2+ content, and the fraction of SR Ca2+ released during electrical stimulation (fractional release) in rat ventricular myocytes.

Methods: Single rat ventricular myocytes loaded with fura-2 were electrically stimulated at 1 Hz, and the Ca2+ transients and contractions were recorded optically. Cells were exposed to each anesthetic for 1 min. Changes in myofilament Ca2+ sensitivity were assessed by comparing the changes in the Ca2+ transient and contraction during exposure to anesthetic and low Ca2+. SR Ca2+ content was assessed by exposure to 20 mm caffeine.

Results: Isoflurane and halothane caused a depression of myofilament Ca2+ sensitivity, unlike sevoflurane, which had no effect on myofilament Ca2+ sensitivity. All three anesthetics decreased the electrically stimulated Ca2+ transient. SR Ca2+ content was reduced by both isoflurane and halothane but was unchanged by sevoflurane. Fractional release was reduced by both isoflurane and sevoflurane, but was unchanged by halothane.     

Propofol Increases Myofilament Ca2+ Sensitivity and Intracellular pH via Activation of Na+-H+ Exchange in Rat Ventricular Myocytes

Background: The objectives were to determine the extent and mechanism of action by which propofol increases myofilament Ca2+ sensitivity and intracellular pH (pHi) in ventricular myocytes.

Methods: Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of myofibrillar actomyosin adenosine triphosphatase (ATPase) activity. Myocyte shortening (video edge detection) and pHi (2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein, 500/440 ratio) were monitored simultaneously in individual cells field-stimulated (0.3 Hz) and superfused with HEPES-buffered solution (pH 7.4, 30[degrees]C).

Results: Propofol (100 [mu]m) reduced the Ca2+ concentration required for activation of myofibrillar actomyosin ATPase from pCa 5.7 +/- 0.01 to 6.6 +/- 0.01. Increasing pHi (7.05 +/- 0.03 to 7.39 +/- 0.04) with NH4Cl increased myocyte shortening by 35 +/- 12%. Washout of NH4Cl decreased pHi to 6.82 +/- 0.03 and decreased myocyte shortening to 52 +/- 10% of control. Propofol caused a dose-dependent increase in pHi but reduced myocyte shortening. The propofol-induced increase in pHi was attenuated, whereas the decrease in myocyte shortening was enhanced after pretreatment with ethylisopropyl amiloride, a Na+-H+ exchange inhibitor, or bisindolylmaleimide I, a protein kinase C inhibitor. Propofol also attenuated the NH4Cl-induced intracellular acidosis, increased the rate of recovery from acidosis, and attenuated the associated decrease in myocyte shortening. Propofol caused a leftward shift in the extracellular Ca2+-shortening relation, and this effect was attenuated by ethylisopropyl amiloride.     

Differential Effects of Fentanyl and Morphine on Intracellular Ca2+ Transients and Contraction in Rat Ventricular Myocytes

Background: Our objective was to elucidate the direct effects of fentanyl and morphine on cardiac excitation-contraction coupling using individual, field-stimulated rat ventricular myocytes.

Methods: Freshly isolated myocytes were loaded with fura-2 and field stimulated (0.3 Hz) at 28 [degree sign]C. Amplitude and timing of intracellular Ca2+ concentration (at a 340:380 ratio) and myocyte shortening (video edge detection) were monitored simultaneously in individuals cells. Real time Ca2+ uptake into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment.

Results: The authors studied 120 cells from 30 rat hearts. Fentanyl (30-1,000 nm) caused dose-dependent decreases in peak intracellular Ca2+ concentration and shortening, whereas morphine (3-100 [micro sign]M) decreased shortening without a concomitant decrease in the Ca2+ transient. Fentanyl prolonged the time to peak and to 50% recovery for shortening and the Ca2+ transient, whereas morphine only prolonged the timing parameters for shortening. Morphine (100 [micro sign]M), but not fentanyl (1 [micro sign]M), decreased the amount of Ca2+ released from intracellular stores in response to caffeine in intact cells, and it inhibited the rate of Ca2+ uptake in isolated sarcoplasmic reticulum vesicles. Fentanyl and morphine both caused a downward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on the Ca2+ transient.     

Ketamine Attenuates Acetylcholine-induced Contraction by Decreasing Myofilament Ca2+ Sensitivity in Pulmonary Veins

Background: The authors investigated the extent and cellular mechanisms by which the intravenous anesthetic ketamine alters acetylcholine-induced contraction in pulmonary veins (PVs). They tested the hypothesis that ketamine inhibits acetylcholine contraction in PVs.

Methods: Canine PV rings with endothelium (E+) and without endothelium (E-) were isolated for measurement of isometric tension. The effects of ketamine (10-5 m~10-3 m) on acetylcholine contraction were assessed in E+ and E- rings. The effects of inhibiting nitric oxide synthase on ketamine-induced changes in acetylcholine contraction were investigated in E+ rings, whereas the effects of Ca2+ influx and Ca2+ release were investigated in E- rings. In fura-2 loaded E- PV strips, the effects of ketamine (10-4 m) on the intracellular Ca2+ concentration-tension relation (i.e., myofilament Ca2+ sensitivity) were assessed in the presence or absence of acetylcholine. The roles of the protein kinase C and rho-kinase signaling pathways in ketamine-induced changes in myofilament Ca2+ sensitivity were also investigated.

Results: Ketamine caused dose-dependent (P < 0.001) inhibition of acetylcholine contraction in E+ and E- PV rings. The ketamine-induced attenuation of acetylcholine contraction was still observed after inhibition of nitric oxide synthase (P = 0.002), Ca2+ influx (P < 0.001), and Ca2+ release (P = 0.021). Ketamine alone had no effect on myofilament Ca2+ sensitivity (P = 0.892) but attenuated (P = 0.038) the acetylcholine-induced increase in myofilament Ca2+ sensitivity. This attenuation was still observed after rho-kinase inhibition (P = 0.039), whereas it was abolished by protein kinase C inhibition (P = 0.798).     

Antagonistic Actions of Halothane and Sevoflurane on Spontaneous Ca2+ Release in Rat Ventricular Myocytes

Background: Halothane has been reported to sensitize Ca2+ release from the sarcoplasmic reticulum (SR), which is thought to contribute to its initial positive inotropic effect. However, little is known about whether isoflurane or sevoflurane affect the SR Ca2+ release process, which may contribute to the inotropic profile of these anesthetics.

Methods: Mild Ca2+ overload was induced in isolated rat ventricular myocytes by increase of extracellular Ca2+ to 2 mm. The resultant Ca2+ transients due to spontaneous Ca2+ release from the SR were detected optically (fura-2). Cells were exposed to 0.6 mm anesthetic for a period of 4 min, and the frequency and amplitude of spontaneous Ca2+ transients were measured.

Results: Halothane caused a temporary threefold increase in frequency and decreased the amplitude (to 54% of control) of spontaneous Ca2+ transients. Removal of halothane inhibited spontaneous Ca2+ release before it returned to control. In contrast, sevoflurane initially inhibited frequency of Ca2+ release (to 10% of control), whereas its removal induced a burst of spontaneous Ca2+ release. Isoflurane had no significant effect on either frequency or amplitude of spontaneous Ca2+ release on application or removal. Sevoflurane was able to ameliorate the effects of halothane on the frequency and amplitude of spontaneous Ca2+ release both on application and wash-off.     

Isoflurane Neuroprotection in Rat Hippocampal Slices Decreases with Aging: Changes in Intracellular Ca2+ Regulation and N-methyl-d-aspartate Receptor-mediated Ca2+ Influx

Background: Most in vitro neuroprotection studies with isoflurane have involved cells obtained during the embryonic or early postnatal period. However, in mature rodents, isoflurane neuroprotection does not persist. The authors determined whether neuroprotection of hippocampal slices with isoflurane decreases with aging and is due to decreased intracellular Ca2+ regulation and survival protein phosphorylation.

Methods: Hippocampal slices from 5-day-old, 1-month-old, and 19- to 23-month-old rats were deprived of oxygen and glucose for 5-30 min in media bubbled with 1% isoflurane. Cell death was assessed in the CA1, CA3, and dentate regions, and intracellular Ca2+ concentration was measured in CA1 neurons. N-methyl-d-aspartate receptor (NMDAR)-dependent Ca2+ influx was measured and the phosphorylation of NMDARs, and the survival proteins Akt and mitogen-activated protein kinase p42/44 were quantified.

Results: Twenty minutes of oxygen and glucose deprivation killed approximately 40-60% of neurons in CA3 and dentate in all age groups. Isoflurane, 1%, reduced death of CA1, CA3, and dentate neurons in slices from 5-day-old rats but not those from 23-month-old rats. In 5-day slices, isoflurane attenuated NMDAR-mediated Ca2+ influx, whereas in aging slices, Ca2+ influx was increased protein kinase C. In aging slices, isoflurane did not increase the phosphorylation of Akt and p42/44.     

Involvement of Ca2+ Sensitization in Ropivacaine-induced Contraction of Rat Aortic Smooth Muscle

Background: The mechanisms of amino-amide local anesthetic agent-induced vasoconstriction remain unclear. The current study was designed to examine the roles of the protein kinase C (PKC), Rho kinase, and p44/42 mitogen-activated protein kinase (p44/42 MAPK) signaling pathways in calcium (Ca2+)-sensitization mechanisms in ropivacaine-induced vascular contraction.

Methods: Endothelium-denuded rat aortic rings, segments, and strips were prepared. The cumulative dose-response relations of contraction and intracellular Ca2+ concentration to ropivacaine were tested, using isometric force transducers and a fluorometer, respectively. The dose-dependent ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and the membrane translocation of Rho kinase were also detected using Western blotting.

Results: Ropivacaine induced a dose-dependent biphasic contractile response and an increase in intracellular Ca2+ concentration of rat aortic rings, increasing at concentrations of 3 x 10-5 m to 3 x 10-4 m and decreasing from 10-3 m to 3 x 10-3 m, with a greater tension/intracellular Ca2+ concentration ratio than that induced with potassium chloride. The contraction was attenuated in a dose-dependent manner, by the PKC inhibitors bisindolylmaleimide I and calphostin C, the Rho-kinase inhibitor Y 27632, and the p44/42 MAPK inhibitor PD 098059. Ropivacaine also induced an increase in phosphorylation of PKC and p44/42 MAPK, and membrane translocation of Rho kinase in accordance with the contractile responses, which were also significantly inhibited by bisindolylmaleimide I and calphostin C, Y 27632, and PD 098059, correspondingly.     

Bupivacaine Attenuates Contractility by Decreasing Sensitivity of Myofilaments to Ca2+, in Rat Ventricular Muscle

Background: Bupivacaine exhibits a cardiodepressant effect, the molecular mechanism(s) of which have yet to be fully understood. Bupivacaine may directly act on contractile proteins and thereby decrease myofibrillar Ca2+ sensitivity.

Methods: Rat ventricular muscle was used. First, the effect of bupivacaine was examined on tetanic contractions in isolated intact myocytes. Next, Triton X-100-treated ventricular trabeculae were used to investigate the effect of bupivacaine on the pCa (= -log [Ca2+])-tension relation as well as on maximal Ca2+-activated tension. Furthermore, to test whether bupivacaine inhibits the pathway downstream from Ca2+ binding to troponin C, tension was elicited in the skinned preparations by lowering the Mg-adenosine triphosphate (MgATP) concentration in the absence of Ca2+. The effect of bupivacaine on the pMgATP (= -log [MgATP])-tension relation was examined.

Results: In myocytes, 3 [mu]m bupivacaine significantly (P < 0.01) increased intracellular Ca2+ concentration required for 5% cell shortening from the resting cell length. In skinned preparations, bupivacaine shifted the pCa-tension relation to the lower pCa side; the midpoint of the pCa curve (pCa50) was significantly (P < 0.05) changed by 10 and 100 [mu]m bupivacaine. A highly correlated linear relation (R = 0.81;P < 0.0005) was present between pCa50 and maximal Ca2+-activated tension. Bupivacaine (10 and 100 [mu]m) significantly (P < 0.05) shifted the midpoint of the pMgATP-tension relation to the higher pMgATP side.     

Anesthetic Preconditioning Enhances Ca2+ Handling and Mechanical and Metabolic Function Elicited by Na+-Ca2+ Exchange Inhibition in Isolated Hearts

Background: Anesthetic preconditioning (APC) is well known to protect against myocardial ischemia-reperfusion injury. Studies also show the benefit of Na+-Ca2+ exchange inhibition on ischemia-reperfusion injury. The authors tested whether APC plus Na+-Ca2+ exchange inhibitors given just on reperfusion affords additive protection in intact hearts.

Methods: Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 [mu]m Na+-Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 [mu]m SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 +/- 0.03 mm) given for 15 min and washed out for 15 min before ischemia-reperfusion; (6, 7) APC plus KBR or SEA.

Results: The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion-induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA.     

Ca2+- and Myosin Phosphorylation-independent Relaxation by Halothane in K+-depolarized Rat Mesenteric Arteries

Background: Volatile anesthetics inhibit vascular smooth muscle contraction, but the mechanisms responsible are uncertain. In this study, the effects of halothane on Ca2+ signaling and Ca2+ activation of contractile proteins were examined in high K+-depolarized smooth muscle from rat mesenteric resistance arteries.

Methods: Vessels were cannulated and held at a constant transmural pressure (40 mmHg). Image analysis and microfluorimetry were used to simultaneously measure vessel diameter and smooth muscle intracellular [Ca2+] concentration ([Ca2+]i). Myosin light chain (MLC) phosphorylation was measured using the Western blotting technique.

Results: Step increases in extracellular [Ca2+] concentration (0-10 mm) in high K+ (40 mm)-depolarized smooth muscle produced incremental increases in [Ca2+]i, MLC phosphorylation, and contraction. Halothane (0.5-4.5%) inhibited contraction in a concentration-dependent manner, but the decrease in [Ca2+]i was small, and there was a marked shift in the [Ca2+]i-contraction relationship to the right, indicating an important Ca2+ desensitizing effect. Halothane (0.5-4.5%) did not affect MLC phosphorylation or the [Ca2+]-MLC phosphorylation relationship, but the MLC phosphorylation-contraction relationship was also shifted rightward, indicating an "MLC phosphorylation" desensitizing effect. In contrast, control relaxations produced by the Ca2+ channel blocker nifedipine were accompanied by decreases in both [Ca2+]i and MLC phosphorylation, and nifedipine had no affect on the [Ca2+]i-contraction, [Ca2+]i-MLC phosphorylation, and MLC phosphorylation-contraction relationships.     

Role of Intracellular Ca2+ Stores in the Inhibitory Effect of Halothane on Airway Smooth Muscle Contraction

Background: Halothane directly inhibits contraction of airway smooth muscle, mainly by decreasing the intracellular concentration of free Ca2+ ([Ca2+]i). The role of intracellular Ca2+ stores, sarcoplasmic reticulum, is still unclear. We investigated the role of sarcoplasmic reticulum in the inhibitory effect of halothane on contraction of airway smooth muscle by measuring [Ca2+]i and intracellular concentration of inositol 1,4,5-triphosphate ([IP3]i), a second messenger for release of Ca2+ from sarcoplasmic reticulum.

Methods: [Ca2+]i was monitored by measuring the 500-nm light emission ratio (F340/F380) of a Ca2+ indicator fura-2 with isometric tension of canine tracheal smooth muscle strip. During Ca2+-free conditions, carbachol (10-5 M) was introduced with pretreatment of halothane (0-3%). During Ca2+-free conditions, 20 mM caffeine, a Ca (2+-induced) Ca2+ release channel opener, was introduced with or without halothane. We measured [IP3]i during exposure to carbachol and halothane by radioimmunoassay technique.

Results: Pretreatment with halothane significantly diminished carbachol-induced increases in [Ca2+]i by 77% and muscle tension by 83% in a dose-dependent manner. Simultaneous administration of halothane significantly enhanced caffeine-induced transient increases in [Ca2+] (i) and muscle tension in a dose-dependent manner, by 97% and 69%, respectively. Pretreatment with halothane abolished these responses. Rapid increase in [IP3]i produced by carbachol was significantly inhibited by 32% by halothane in a dose-dependent manner.     

Sevoflurane Inhibits Angiotensin II-induced, Protein Kinase C-mediated but Not Ca2+-elicited Contraction of Rat Aortic Smooth Muscle

Background: Whether volatile anesthetics attenuate angiotensin II-mediated vascular tone has not been determined. The current study was designed to investigate the effects of sevoflurane on the angiotensin II-stimulated, Ca2+- and protein kinase C (PKC)-mediated contraction of rat aortic smooth muscle.

Methods: The dose-dependent effects of sevoflurane on angiotensin II (10-7 m)-induced contraction, the increase in intracellular Ca2+ concentration, and PKC phosphorylation of rat aortic smooth muscle were measured using an isometric force transducer, a fluorometer, and Western blotting, respectively.

Results: Angiotensin II induced a transient increase in intracellular Ca2+ concentration, phosphorylation of Ca2+-dependent PKC (cPKC)-[alpha], and consequently, a transient contraction of rat aortic smooth muscle. Phosphorylation of the Ca2+-independent PKC-[epsilon] was not detected. The angiotensin II-induced contraction was almost completely abolished by removing extracellular Ca2+ and was significantly inhibited by the selective cPKC inhibitor Go 6976 (10-5 m) but was not inhibited by the nonselective PKC inhibitor Ro 31-8425 (10-5 m). Sevoflurane dose-dependently inhibited the angiotensin II-induced contraction, with reductions of 14.2 +/- 5.2% (P > 0.05), 26.7 +/- 8.9% (P < 0.05), and 38.5 +/- 12.8% (P < 0.01) (n = 10) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively. The angiotensin II-elicited increase in intracellular Ca2+ concentration was not significantly influenced by 3.4, 5.1, or 8.5% sevoflurane. However, cPKC-[alpha] phosphorylation induced by angiotensin II was inhibited dose dependently by 1.7, 3.4, and 5.1% sevoflurane, with depressions of 20.5 +/- 14.2% (P > 0.05), 37.0 +/- 17.8% (P < 0.05), and 62.5 +/- 12.2% (P < 0.01) (n = 4), respectively.     

Effects of Sevoflurane on the Intracellular Ca2+ Transient in Ferret Cardiac Muscle

Background: Sevoflurane depresses myocardial contractility by decreasing transsarcolemmal Ca2+ influx. In skinned muscle fibers, sevoflurane affects actin-myosin cross-bridge cycling, which might contribute to the negative inotropic effect. It is uncertain to what extent decreases in Ca2+ sensitivity of the contractile proteins play a role in the negative inotropic effect of sevoflurane in intact cardiac muscle tissue. The aim of this study was to assess whether sevoflurane decreases myofibrillar Ca2+ sensitivity in intact living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability by sevoflurane.

Methods: The effects of sevoflurane 0-4.05% vol/vol (0-1.5 minimum alveolar concentration [MAC]) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscles microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium.

Results: Sevoflurane decreased contractility, time to peak force, time to half isometric relaxation, and the [Ca2+]i transient in a reversible, concentration-dependent manner. Increasing [Ca2+]o in the presence of sevoflurane to produce peak force equal to control increased intracellular Ca2+ transient higher than control.     

Inhibitory Effects of Diazepam and Midazolam on Ca2+ and K+ Channels in Canine Tracheal Smooth Muscle Cells

Background: Benzodiazepines have a direct bronchodilator action in airway smooth muscle, but the mechanisms by which these agents produce muscle relaxation are not fully understood. The current study was performed to identify the effects of the benzodiazepines diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells.

Methods: Whole-cell patch-clamp recording techniques were used to evaluate the effects of the benzodiazepines diazepam (10-8 to 10-3 M) and midazolam (10-8 to 10-3 M) on inward Ca2+ and outward K (+) channel currents in dispersed canine tracheal smooth muscle cells. The effects of the antagonists flumazenil (10-5 M) and PK11195 (10-5 M) on these channels were also studied.

Results: Each benzodiazepine tested significantly inhibited Ca2+ currents in a dose-dependent manner, with 10-6 M diazepam and 10-5 M midazolam each causing approximately 50% depression of peak voltage-dependent Ca2+ currents. Both benzodiazepines promoted the inactivated state of the channel at more-negative potentials. The Ca2+ -activated and voltage-dependent K+ currents were inhibited by diazepam and midazolam (> 10-5 M and > 10-4 M, respectively). Flumazenil and PK11195 had no effect on these channel currents or on the inhibitory effects of the benzodiazepines.     

Halothane Inhibits an Intermediate Conductance Ca2+-activated K+ Channel by Acting at the Extracellular Side of the Ionic Pore

Background: Actions of volatile anesthetics on ligand-gated ion channels, such as [gamma]-aminobutyric acid type A receptors, have been studied extensively. However, actions on other types of channels, such as K+ channels, are poorly understood. The authors previously showed that a Ca2+-activated K+ channel, IK, is sensitive to halothane, whereas SK1, another Ca2+-activated K+ channel, is insensitive. To explore how halothane acts on Ca2+-activated K+ channels, chimeras between IK and SK1 were constructed, and halothane sensitivity was analyzed.

Methods: IK, SK1, and chimera channels were expressed in Xenopus laevis oocytes. Currents of expressed channels were measured in the presence of 10 [mu]m Ca2+ by excised patch clamp analysis. Time constants of inhibition by halothane were compared between inside-out and outside-out patch configurations.

Results: Currents from chimera channels possessing the pore domain derived from IK were inhibited by halothane, whereas those possessing the SK1 pore domain were insensitive. Time constants of inhibition by halothane were significantly smaller in the outside-out patches than in the inside-out patches of both wild-type IK and a chimera with pore domain of IK.     

Propofol Decreases Myofilament Ca2+ Sensitivity via a Protein Kinase C-, Nitric Oxide Synthase-dependent Pathway in Diabetic Cardiomyocytes

Background: The authors' objective was to assess the role of protein kinase C (PKC) and nitric oxide synthase (NOS) in mediating the effects of propofol on diabetic cardiomyocyte contractility, intracellular free Ca2+ concentration ([Ca2+]i), and myofilament Ca2+ sensitivity.

Methods: Freshly isolated ventricular myocytes were obtained from normal and diabetic rat hearts. [Ca2+]i and cell shortening were simultaneously measured in electrically stimulated, ventricular myocytes using fura-2 and video-edge detection, respectively. Actomyosin adenosine triphosphatase activity and troponin I (TnI) phosphorylation were assessed in [32P]orthophosphate-labeled myofibrils. Western blot analysis was used to assess expression of PKC and NOS.

Results: Propofol (10 [mu]m) decreased peak shortening by 47 +/- 6% with little effect on peak [Ca2+]i (92 +/- 5% of control) in diabetic myocytes. Maximal actomyosin adenosine triphosphatase activity was reduced by 43 +/- 7% and TnI phosphorylation was greater (32 +/- 6%) in diabetic myofibrils compared with normal. Propofol reduced actomyosin adenosine triphosphatase activity by 17 +/- 7% and increased TnI phosphorylation in diabetic myofibrils. PKC inhibition prevented the propofol-induced increase in TnI phosphorylation and decrease in shortening. Expression of PKC-[alpha], PKC-[delta], PKC-[varepsilon], and constitutive NOS were up-regulated and inducible NOS was expressed in diabetic cardiomyocytes. NOS inhibition attenuated the propofol-induced decrease in shortening.     

Isoflurane Reduces the Carbachol-evoked Ca2+ Influx in Neuronal Cells

Background: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca2+ transient increase ([Ca2+]cyt) was eliminated by K+ or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca2+]cyt transient involved Ca2+ influx through the plasma membrane.

Methods: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca2+ (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La3+ (100 [mu]m), of the L-type Ca2+ channel blocker nitrendipine (10 [mu]m), and of the N-type Ca2+ channel blocker [omega]-conotoxin GVIA (0.1 [mu]m) on isoflurane modulation of the carbachol-evoked [Ca2+]cyt transient. [Ca2+]cyt was detected with fura-2 and experiments were carried out at 37[degrees]C.

Results: Isoflurane reduced the peak and area of the carbachol-evoked [Ca2+]cyt transient in the presence but not in the absence of extracellular Ca2+. La3+ had a similar effect as the removal of extracellular Ca2+. [omega]-Conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response.     

Effects of Halothane and Isoflurane on the Intracellular Ca2+ Transient in Ferret Cardiac Muscle

Background: Halothane and isoflurane depress myocardial contractility by decreasing transsarcolemmal Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum. Decreases in Ca2+ sensitivity of the contractile proteins have been shown in skinned cardiac fibers, but the relative importance of this effect in intact living myocardium is unknown. The aims of this study were to assess whether halothane and isoflurane decrease myofibrillar Ca2+ sensitivity in intact, living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability caused by these anesthetics.

Methods: The effects of halothane and isoflurane (0-1.5 times the minimum alveolar concentration (MAC) in three equal increments) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscle microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium.

Results: Halothane and isoflurane decreased contractility, time-to-peak force, time to half-isometric relaxation, and intracellular Ca2+ transient in a reversible, concentration-dependent manner. Halothane, but not isoflurane, slowed the increase and the decrease of the intracellular Ca2+ transient. Increasing extracellular Ca2+ in the presence of anesthetic to produce peak force equal to control values increased intracellular Ca2+ to values higher than control values.     

Painful Nerve Injury Shortens the Intracellular Ca2+ Signal in Axotomized Sensory Neurons of Rats

Background: Neuropathic pain is inadequately treated and poorly understood at the cellular level. Because intracellular Ca2+ signaling critically regulates diverse neuronal functions, the authors examined effects of peripheral nerve injury on the Ca2+ transient that follows neuronal activation.

Methods: Cytoplasmic Ca2+ levels were recorded by digital microfluorometry from dissociated dorsal root ganglion neurons of hyperalgesic animals after ligation of the fifth lumbar spinal nerve and control animals. Neurons were activated by field stimulation or by K+ depolarization.

Results: Transients in presumptively nociceptive, small, capsaicin-sensitive neurons were diminished after axotomy, whereas transient amplitude increased in axotomized nonnociceptive neurons. Axotomy diminished the upward shift in resting calcium after transient recovery. In contrast, nociceptive neurons adjacent to axotomy acquired increased duration of the transient and greater baseline shift after K+ activation. Transients of nonnociceptive neurons adjacent to axotomy showed no changes after injury. In nociceptive neurons from injured rats that did not develop hyperalgesia, transient amplitude and baseline offset were large after axotomy, whereas transient duration in the adjacent neurons was shorter compared with neurons excised from hyperalgesic animals, which show normalization of these features.