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 microM) 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. CONCLUSIONS: These results suggest that propofol increases the sensitivity of myofibrillar actomyosin ATPase to Ca2+ (ie., increases myofilament Ca2+ sensitivity), at least in part by increasing pHi via protein kinase C-dependent activation 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. CONCLUSIONS: Depressed myofilament Ca2+ sensitivity contributes to the negative inotropic effects of isoflurane and halothane but not sevoflurane. The decrease in the Ca2+ transient is either responsible for or contributory to the negative inotropic effects of all three anesthetics and is either primarily the result of a decrease in fractional release (isoflurane and sevoflurane) or primarily the result of a decrease in SR Ca2+ content (halothane).     

The differential effects of midazolam and diazepam on intracellular Ca2+ transients and contraction in adult rat ventricular myocytes

We investigated the direct effects of midazolam and diazepam on cardiac excitation-contraction coupling in adult rat ventricular myocytes. Freshly isolated rat ventricular myocytes were loaded with fura-2/AM and field-stimulated at 28 degrees C. Intracellular Ca(2+) transients (340:380 ratio) and myocyte shortening (video edge detection) were simultaneously monitored in individual cells. Midazolam (3-100 micro M) caused a dose-dependent decrease in both peak intracellular Ca(2+) and cell shortening. Diazepam (30 and 100 micro M) increased myocyte shortening and peak Ca(2+) concomitant with a decrease in time to peak Ca(2+). A larger concentration of diazepam (>300 micro M) nearly abolished intracellular Ca(2+) and cell shortening. Midazolam (100 micro M) and diazepam (300 micro M) decreased the amount of Ca(2+) released from intracellular stores in response to caffeine. Diazepam (30 micro M), but not midazolam (10 micro M), caused a downward shift in the dose-response curve to extracellular Ca(2+) for shortening, with no concomitant effect on peak intracellular Ca(2+) transient. These results indicate that midazolam and diazepam have different inotropic effects on cardiac excitation-contraction coupling at the cellular level, which is mediated by altering the availability of intracellular-free Ca(2+). However, the benzodiazepines have no direct influence on excitation-contraction coupling in rat ventricular myocytes, except at very large doses. Inhibition of Ca(2+) release from caffeine-sensitive intracellular Ca(2+) stores may play some part in myocardial depression at the larger concentrations of benzodiazepines. Diazepam, but not midazolam, decreased myofilament responsiveness to Ca(2+). IMPLICATIONS: Midazolam and diazepam differentially alter the cardiac excitation-contraction coupling at the cellular level, which is mediated by altering the availability of intracellular free Ca(2+) in adult rat ventricular myocytes. In addition, diazepam, but not midazolam, decreases myofilament Ca(2+) sensitivity. However, the benzodiazepines have no direct influence on excitation-contraction coupling, except at very large doses.     

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 approximately 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 Ca 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). CONCLUSIONS: Ketamine attenuates acetylcholine contraction by inhibiting the acetylcholine-induced increase in myofilament Ca2+ sensitivity, which is mediated by the protein kinase C signaling pathway.     

Halothane alters control of intracellular Ca2+ mobilization in single rat ventricular myocytes.

In an attempt to understand the cellular mechanisms underlying volatile anesthetic-induced myocardial depression, halothane-induced negative inotropy was investigated in an animal model through continuous monitoring of intracellular Ca2+ concentration [( Ca2+]i) in rat ventricular myocytes loaded with fura-2. Single cells were stimulated with 15 mM caffeine or 15 mM extracellular K+ (K+O) or were paced by extracellular glass suction pipette electrode. With each stimulus modality, halothane (0.6-1.5%) caused a significant (P less than 0.05) and dose-dependent depression of the Ca2+ transient. Caffeine and electrically stimulated Ca2+ transients were reduced, in 1.5% halothane, to 35 +/- 14 and 42 +/- 8% of control, respectively. Resting or basal [Ca2+]i was unaffected by halothane. Halothane did not elicit spontaneous Ca2+ transients in these cells. Single cells stimulated by trains of electrical stimuli at 1.0, 1.5, and 2.0 Hz showed a change in [Ca2+]i from prestimulus levels to a stimulated baseline steady state that appeared to increase with stimulus frequency. Halothane at 0.7% increased the change in resting to stimulated baseline [Ca2+]i and depressed net transients (P less than 0.05) at 1.0 and 1.5 Hz. In contrast, 0.1 microM ryanodine depressed the Ca2+ transients in myocytes stimulated by trains of stimuli, but did not potentiate the change in stimulated baseline [Ca2+]i at any pacing rate. The results are consistent with the hypothesis that halothane reduces Ca2+i availability by causing a net loss of Ca2+ from the sarcoplasmic reticulum. The results from experiments using onset of pacing to induce a sudden increase in Ca2+i load in previously quiescent myocytes suggest that halothane may act to limit sarcoplasmic reticulum and/or sarcolemmal uptake/extrusion mechanisms, as compared to ryanodine, which depletes sarcoplasmic reticulum Ca2+ stores without affecting reuptake and extrusion.     

The effects of alfentanil on cytosolic Ca(2+) and contraction in rat ventricular myocytes

Previous investigations of the effects of potent opioid analgesics on the heart have concentrated on effects on contraction magnitude and time course, but little is known about their effects on cytosolic Ca(2+) regulation in cardiac tissue. In this study, we sought to assess the effects of alfentanil on contractility and the cytosolic Ca(2+) transient in ventricular myocytes isolated from the rat ventricle by enzymatic dispersion. Cells were loaded with fura-2 and electrically stimulated at 1 Hz, and Ca(2+) transients and contractions were recorded optically at 30 degrees C. Alfentanil 10(-8) and 10(-7) M had no effect on the magnitude or time course of contraction or the cytosolic Ca(2+) transient. In contrast, 10(-6) M alfentanil induced a significant (P < 0.001) positive inotropic effect, increasing the mean (+/-SEM) unloaded shortening from 7.3 +/- 1.3 microm to 8.7 +/- 1.4 microm (an increase of 20%), with no change in the cytosolic Ca(2+) transient. Myofilament Ca(2+) sensitivity was significantly (P = 0.027) increased by 10(-6) M alfentanil but unaffected at 10(-7) M alfentanil. These data show that 10(-6) M alfentanil, a concentration close to the maximum clinical free plasma concentration, induced a positive inotropic effect due to sensitization of the myofilaments to Ca(2+) rather than to modified cytosolic Ca(2+) regulation. IMPLICATIONS: Alfentanil, at concentrations achieved in clinical practice, increased contraction in ventricular cells by a mechanism involving an increase in the sensitivity of the contractile apparatus to Ca(2+).     

Metformin but not glyburide prevents high glucose-induced abnormalities in relaxation and intracellular Ca2+ transients in adult rat ventricular myocytes.

We have recently demonstrated that adult rat ventricular myocytes maintained in a high glucose (HG) culture medium exhibit abnormalities in excitation-contraction coupling similar to myocytes from diabetic rats. Metformin, an insulin-sensitizing biguanide, enhances peripheral insulin action and lowers blood pressure in hyperinsulinemic animals, but its direct impact on cardiac function is not fully understood. To examine the role of metformin on HG-induced cardiac dysfunction at the cellular level, normal adult ventricular myocytes were cultured for 1 day in a serum-free insulin-containing medium with either normal glucose (5.5 mmol/l glucose) or HG (25.5 mmol/l glucose) in the presence or absence of metformin or the sulfonylurea glyburide. Mechanical properties were evaluated using a high-speed video-edge detection system, and intracellular Ca2+ transients were recorded in fura-2-loaded myocytes. As previously reported, culturing myocytes in HG depresses peak shortening, prolongs time to 90% relengthening, and slows Ca2+ transient decay. Culturing cells with metformin (50 micromol/l) prevented the HG-induced abnormalities in relaxation without ameliorating depressed peak-shortening amplitudes. Incubation of the cells with metformin also prevented slower intracellular Ca2+ clearing induced by HG. However, the HG-induced relaxation defects were not improved by glyburide (50-300 micromol/l). Interestingly, metformin also improved HG-induced relaxation abnormalities in the absence of insulin, whereas it failed to protect against HG in the presence of the tyrosine kinase inhibitor genistein (50 micromol/l). These data demonstrate that, unlike glyburide, metformin provides cardioprotection against HG-induced abnormalities in myocyte relaxation, perhaps through tyrosine kinase-dependent changes in intracellular Ca2+ handling, independent of its insulin sensitizing action.     

Antagonistic actions of halothane and sevoflurane on spontaneous Ca2+ release in rat ventricular myocytes

BACKGROUND: Halothane has been reported to sensitize Ca(2+) 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 Ca(2+) release process, which may contribute to the inotropic profile of these anesthetics. METHODS: Mild Ca(2+) overload was induced in isolated rat ventricular myocytes by increase of extracellular Ca(2+) to 2 mM. The resultant Ca(2+) transients due to spontaneous Ca(2+) 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 Ca(2+) transients were measured. RESULTS: Halothane caused a temporary threefold increase in frequency and decreased the amplitude (to 54% of control) of spontaneous Ca(2+) transients. Removal of halothane inhibited spontaneous Ca release before it returned to control. In contrast, sevoflurane initially inhibited frequency of Ca(2+) release (to 10% of control), whereas its removal induced a burst of spontaneous Ca(2+) release. Isoflurane had no significant effect on either frequency or amplitude of spontaneous Ca(2+) release on application or removal. Sevoflurane was able to ameliorate the effects of halothane on the frequency and amplitude of spontaneous Ca(2+) release both on application and wash-off. CONCLUSIONS: Application of halothane and removal of sevoflurane sensitize the SR Ca(2+) release process (and vice versa on removal). Sevoflurane reversed the effects of halothane, suggesting they may act at the same subcellular target on the SR.     

Effects of halothane on membrane potential, Ca2+ current and intracellular cAMP content in single guinea pig ventricular myocytes

In order to assess directly the actions of halothane on myocardium, especially on the Ca2+ channel we studied effects of halothane on electrophysiological and biochemical properties in single ventricular myocytes isolated enzymatically from guinea pig hearts. Membrane potentials and the slow inward Ca2+ current (ICa) were recorded with a suction microelectrode technique and a whole cell voltage clamp technique. The plateau duration of the action potential, maintained by ICa, and ICa was depressed by 2% halothane (to 58% and 29% of control respectively). To define the site on which halothane acts in the cell membrane, we measured cyclic adenosine monophosphate (cAMP) content of single ventricular myocytes using radioimmunoassay. One percent (1%) and 2% halothane directly produced a dose-dependent decrease in myocardial cAMP content (79% and 65% of control respectively). In conclusion, the present results suggest that the decrease of ICa by halothane participates in the observed depression of the action potential plateau phase and demonstrate that halothane depression of ICa is in part due to an inactivation of phosphorylation dependent gate in the Ca2+ channel resulting from the decrease in cAMP content by halothane.     

Transient and sustained changes in myofilament sensitivity to Ca2+ contribute to the inotropic effects of sevoflurane in rat ventricle

Background. The volatile anaesthetics isoflurane and sevofluraneinduce both negative and positive inotropic effects in ventricularmyocytes, the mechanisms of which are not fully understood.Previous data suggest that changes in myofilament Ca²⁺ sensitivitycontribute to their sustained negative inotropic effects. Inthis study, the role of changes in myofilament Ca²⁺ sensitivityin both positive and negative inotropic effects of these agentswas examined in intact ventricular myocytes. Methods. Contractility and cytosolic Ca²⁺ (fura-2) were recordedoptically in ventricular myocytes stimulated electrically (1Hz) at 30°C. Myofilament Ca²⁺ sensitivity was assessed fromplots of cell length against fura-2 fluorescence ratio (Fr)from individual twitches at various points before, during andafter a 1 or 4 min exposure to 0.6 mM anaesthetic. Results. Isoflurane reduced mean (SD) myofilament Ca²⁺ sensitivityfrom 10.3 (1.9) to 5.9 (1.6) µm Fr^–1 (P<0.001)throughout a 1 min exposure, which returned to control on removal.In contrast, on initial exposure to sevoflurane, Ca²⁺ sensitivitywas reduced from 10.8 (1.3) to 4.3 (0.9) µm Fr^–1(P<0.001) but this recovered partially towards control over3 min. On removal, sensitivity was increased above control (to17.7 (2.2) µm Fr^–1; P<0.001) before preanaestheticlevels were restored. Conclusions. These data show that both isoflurane and sevofluranereduce apparent myofilament Ca²⁺ sensitivity at steady state.However, sevoflurane (but not isoflurane) induced transientchanges in apparent myofilament Ca²⁺ sensitivity, which wouldcontribute to its inotropic profile.     

瑞芬太尼对电刺激诱导大鼠心室肌细胞钙瞬变的影响

目的观察不同浓度瑞芬太尼对电刺激诱导的大鼠心室肌细胞钙瞬变的影响。方法雄性SD大鼠，体重190—210g，分离单个心室肌细胞，在细胞逐步复钙后行细胞指标剂的负载，采用钙敏感荧光探针Fluo-2／AM结合激光扫描共聚焦显微镜测定胞浆游离钙浓度（[Ca^2＋]i），分别用含0．1、0．3、1、3、10、30、100、300、1000ng／ml瑞芬太尼的Kreb’8液灌流细胞，每个浓度8个细胞，以波长340／380nm时荧光比值反映[Ca^2＋]i），用0．2Hz的电流刺激细胞，产生的[Ca^2＋]i峰值即为电刺激激发的瞬时[Ca^2＋]i，给予瑞芬太尼后瞬时[Ca^2＋]i与基础值的比值表示钙瞬变振幅，绘出量．效关系曲线和100ng／ml瑞芬太尼的时．效关系曲线。结果瑞芬太尼剂量依赖性抑制钙瞬变，Sigmoidal方程为Y=78．49＋25．31／[1＋10^（-6.41-x）]（P〈0．01），半数有效浓度为0．4ng／ml，R^2为0．9833；100ng／ml瑞芬太尼时间依赖性抑制钙瞬变，Sigmoidal方程为Y=68．5＋45．7／[1＋10^（-0.358-x）]（P〈0．01），达半数最大效应的时间为2．3min，R^2为0．937。结论瑞芬太尼对电刺激诱发的大鼠心室细胞钙瞬变产生剂量和时间依赖性抑制。     

Ca2+ channel modulation alters halothane-induced depression of ventricular myocytes

Purpose

This study examined the direct myocardial depressant effect of halothane and determined whether an L-type Ca²⁺ channel agonist and antagonists altered the myocardial depression induced by halothane in cultured rat ventricular myocytes.

Methods

Ventricular myocytes were obtained from neonatal rats by enzymatic digestion with collagenase and then cultured for 6 to 7 days. The myocytes were stabilized in a serum-free medium, and the spontaneous beating rate and amplitude were measured. To assess the halothane-induced conformational changes in L-type Ca²⁺ channel, receptor binding study was performed using a dihydropyridine derivative, [³H] PN 200-110, in cardiac membrane preparation.

Results

Halothane (1%, 2%, 3%, 4%) decreased the beating rate and amplitude in a concentration-dependent manner (P < 0.05). The myocardial depressant effects of halothane were potentiated by nifedipine or verapamil (P < 0.05). Bay K 8644, an L-type Ca²⁺ channel agonist, completely prevented the halothane-induced depression in amplitude (P < 0.05), but affected the beating rate less. Adding halothane (2%) decreased (P < 0.05) the maximum binding site density for [³H] PN 200-110 (from 198.6 ± 23.7 fmol·mg^?1 protein to 115.3 ± 21.6 fmol·mg^?1 protein) but did not affect binding affinity (from 0.461 ± 0.077 nM to 0.307 ± 0.055 nM).

Conclusion

The reduction of Ca²⁺ current via sarcolemmal L-type Ca²⁺ channel, probably due to conformational changes in dihydropyridine binding sites, plays an important role in halothane-induced myocardial depression in living heart cells.     

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 microm 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 microm 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 microm) significantly (P < 0.05) shifted the midpoint of the pMgATP-tension relation to the higher pMgATP side. CONCLUSIONS: Bupivacaine decreases myofibrillar Ca2+ sensitivity in ventricular muscle, and this is coupled with the compound's inhibitory effect on the pathway beyond Ca2+ binding to troponin C, possibly on the actomyosin interaction. The current results may partly explain the overall cardiodepressant effect of bupivacaine in vivo.     

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 microM) 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-epsilon, and constitutive NOS were up-regulated and inducible NOS was expressed in diabetic cardiomyocytes. NOS inhibition attenuated the propofol-induced decrease in shortening. CONCLUSION: Myofilament Ca2+ sensitivity and, to a lesser extent, peak [Ca2+]i are decreased in diabetic cardiomyocytes. Increases in PKC and NOS expression in combination with TnI phosphorylation seem to contribute to the decrease in [Ca2+]i and myofilament Ca2+ sensitivity. Propofol decreases [Ca2+]i and shortening via a PKC-, NOS-dependent pathway.     

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 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 G? 6976 (10 M) but was not inhibited by the nonselective PKC inhibitor Ro 31-8425 (10 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. CONCLUSION: The current study indicates that Ca2+ and cPKC-alpha are involved in angiotensin II-induced vascular contraction. Sevoflurane dose-dependently inhibited the angiotensin II-stimulated, cPKC-mediated but not Ca2+-elicited contraction of rat aortic smooth muscle.     

The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic-mediated contraction of mammalian urinary bladder smooth muscle

OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions. MATERIALS AND METHODS: Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release). RESULTS: Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions. CONCLUSION: These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.     

Effects of halothane on contraction and intracellular calcium in ventricular myocytes from streptozotocin-induced diabetic rats

Background. Some of the cellular targets affected by volatileanaesthetics (e.g. halothane) which contribute to the negativeinotropic effects of these agents are also affected during theprogression of diabetic cardiomyopathy. A previous report suggestedthat halothane inhibited contraction to a lesser extent in papillarymuscle from diabetic animals and so the aim of this study wasto investigate possible mechanisms underlying this effect. Methods. Contractility and cytosolic calcium ion (Ca²⁺) transientswere measured (fura-2) in ventricular myocytes isolated fromcontrol and streptozotocin (STZ)-induced diabetic rats in theabsence and presence of halothane 0.6 mmol litre^–1 at1 Hz stimulation. Sarcoplasmic reticulum (SR) Ca²⁺ content wasassessed by rapid application of caffeine. All experiments werecarried out at 36–37°C. Results. The amplitude of shortening, the electrically evokedCa²⁺ transient, SR Ca²⁺ content and myofilament Ca²⁺ sensitivity,though not altered by STZ treatment, were significantly reducedby halothane to a similar extent in control and STZ myocytes.The time course of contraction and Ca²⁺ transient were prolongedin myocytes from STZ-treated rats compared with controls butthis was not altered further by halothane. STZ treatment appearedto reduce Ca²⁺ efflux from the cell, an effect reversed by halothane. Conclusions. In contrast to a previous report, we could findno evidence of amelioration of the negative inotropic effectof halothane in myocytes from the STZ-induced diabetic rat.Contractility, the cytosolic Ca²⁺ transient, SR Ca²⁺ contentand myofilament Ca²⁺ sensitivity were qualitatively similarin control and STZ myocytes and were all depressed to the sameextent by halothane. Br J Anaesth 2004; 92: 246–53     

Droperidol inhibits tracheal contraction induced by serotonin,histamine or carbachol in guinea pigs

Purpose

Droperidol (D) is effective in the treatment of patients with status asthmaticus. It has been reported that D inhibits the bronchoconstriction induced by serotonin (5-HT) but not that by histamine (H) or acetylcholine. However, haloperidol, another butyrophenone, is known to interact with and inhibit calmodulin, an intracellular Ca⁺⁺-binding protein which is important in the contraction of smooth muscles. The present study was designed to investigate the effects of D on tracheal contractions induced by 5-HT, H or carbachol (C) and to determine the contribution of α-adrenoceptors to the relaxant effect of D in vitro.

Methods

Tracheas of female guinea pigs were cut spirally into strips and mounted in water-jacketed organ baths in Tyrode’s solution, aerated with a mixture of 95% O₂ and 5% CO₂ at 37°C. The changes in isometric tension induced by each spasmogen in the strips were measured with a transducer and a polygraph.

Results

We found that D inhibited the tracheal contractions induced by 5-HT, H or C in a concentration-dependent manner. At 1.25 × 10^?6 M D blocked the effect of 10^?4 M 5-HT by 44.1 ± 4.3% and at 2.5 × 10^?6 M by 63.8 ± 3.8%. Similarly, at 5.0 × 10^?6 M concentration, D blocked the effect of 10^?5 M H by 27.7 ± 5.3% and at 10^?5 M by 56.2 ± 2.6%. Furthermore, 5 ×10^?6 M of D reduced the contractions produced by 10^?7 M C by 37.1 ± 3.0% and 70^?5 M of D by 76.1 ± 3.2%. The inhibiting effect of D was strongest on contractions induced by 5-HT. Prazosin (70^?6 M) affected neither 5-HT-induced contractions nor the inhibition by D.

Conclusion

Our data indicate that D partially blocks the contractile responses not only to 5-HT, an effect which would be mediated through a blockade of the 5-HT receptors, but also to H or C, probably through inhibition of calmodulin. Our data support previous reports indicating that droperidol may be an important therapeutic agent in the treatment of patients with hyperreactive airways.     

Effects of L-type Ca2+ channel modulation on direct myocardial effects of diazepam and midazolam in adult rat ventricular myocytes

Purpose Our objective was to determine whether an L-type Ca²⁺ channel modulation could alter myocardial depression induced by midazolam or diazepam in adult rat ventricular myocytes. Methods Freshly isolated rat ventricular myocytes were loaded with fura-2/AM and field-stimulated (0.3 Hz) at 28°C. Amplitude and timing of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and myocyte shortening were simultaneously monitored in individual cells. Results Midazolam (3–100 μM) caused a decrease in both peak [Ca²⁺]_i and shortening. Diazepam (30, 100 μM) increased myocyte shortening and peak [Ca²⁺]_i; however, higher concentration of diazepam (300 μM) decreased shortening and peak [Ca²⁺]_i. Bay K 8644 (0.01–10 μM), an L-type Ca²⁺ channel agonist, caused dose-dependent increases in peak [Ca²⁺]_i and shortening. In contrast, verapamil (0.1–50 μM), an L-type Ca²⁺ channel antagonist, caused dose-dependent decreases in peak [Ca²⁺]_i and shortening. Dose–response curves to benzodiazepines on peak [Ca²⁺]_i and shortening were not affected by pretreatment with Bay K 8644 (0.1 μM) or verapamil (1 μM). Diazepam (30, 100 μM), but not midazolam (3–30 μM), increased shortening and [Ca²⁺]_i in the presence or absence of L-type Ca²⁺ channel modulators. Diazepam (30 μM) and midazolam (10 μM) had no effect on peak [Ca²⁺]_i of a caffeine-induced [Ca²⁺]_i transient, which was used as a measure of SR Ca²⁺ content. Conclusion Midazolam and diazepam have differential effects on cardiac E-C coupling. Diazepam, but not midazolam, enhances cardiac E-C coupling independent of L-type Ca²⁺ channel modulation.