Isoflurane neuroprotection in hypoxic hippocampal slice cultures involves increases in intracellular Ca2+ and mitogen-activated protein kinases

BACKGROUND: The volatile anesthetic isoflurane reduces acute and delayed neuron death in vitro models of brain ischemia, an action that the authors hypothesize is related to moderate increases in intracellular calcium concentration ([Ca2+]i). Specifically, the authors propose that during hypoxia, moderate increases in [Ca2+]i in the presence of isoflurane stimulates the Ca2+-dependent phosphorylation of members of the mitogen-activated protein kinase (MAP) kinase Ras-Raf-MEK-ERK pathway that are critical for neuroprotective signaling and suppression of apoptosis. METHODS: Death of CA1, CA3, and dentate neurons in rat hippocampal slice cultures was assessed by propidium iodide fluorescence 48-72 h after 60-75 min of hypoxia. [Ca2+]i in CA1 neurons was measured with fura-2 and fura-2 FF. Concentrations of the survival-signaling proteins Ras, MEK, MAP kinase p42/44, and protein kinase B (Akt) were assessed by immunostaining, and specific inhibitors were used to ascertain the role of Ca2+ and MAP kinases in mediating survival. RESULTS: Isoflurane, 1%, decreased neuron death in CA1, CA3, and dentate gyrus neurons after 60 but not 75 min of hypoxia. Survival of CA1 neurons required an inositol triphosphate receptor-dependent increase in [Ca2+]i of 30-100 nm that activated the Ras-Raf-MEK-ERK (p44/42) signaling pathway. Isoflurane also increased the phosphorylation of Akt during hypoxia. CONCLUSIONS: Isoflurane stimulates the phosphorylation of survival signaling proteins in hypoxic neurons. The mechanism involves a moderate increase in [Ca2+]i from release of Ca from inositol triphosphate receptor-dependent intracellular stores. The increase in [Ca2+]i sets in motion signaling via Ras and the MAP kinase p42/44 pathway and the antiapoptotic factor Akt. Isoflurane neuroprotection thus involves intracellular signaling well known to suppress both excitotoxic and apoptotic/delayed cell death.     

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 Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca 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 Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]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 [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. CONCLUSIONS: The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor.     

Isoflurane preconditions hippocampal neurons against oxygen-glucose deprivation: role of intracellular Ca2+ and mitogen-activated protein kinase signaling

BACKGROUND: Isoflurane preconditions neurons to improve tolerance of subsequent ischemia in both intact animal models and in in vitro preparations. The mechanisms for this protection remain largely undefined. Because isoflurane increases intracellular Ca2+ concentrations and Ca2+ is involved in many processes related to preconditioning, the authors hypothesized that isoflurane preconditions neurons via Ca2+-dependent processes involving the Ca2+- binding protein calmodulin and the mitogen-activated protein kinase-ERK pathway. METHODS: The authors used a preconditioning model in which organotypic cultures of rat hippocampus were exposed to 0.5-1.5% isoflurane for a 2-h period 24 h before an ischemia-like injury of oxygen-glucose deprivation. Survival of CA1, CA3, and dentate neurons was assessed 48 later, along with interval measurements of intracellular Ca2+ concentration (fura-2 fluorescence microscopy in CA1 neurons), mitogen-activated protein kinase p42/44, and the survival associated proteins Akt and GSK-3beta (in situ immunostaining and Western blots). RESULTS: Preconditioning with 0.5-1.5% isoflurane decreased neuron death in CA1 and CA3 regions of hippocampal slice cultures after oxygen-glucose deprivation. The preconditioning period was associated with an increase in basal intracellular Ca2+ concentration of 7-15%, which involved Ca2+ release from inositol triphosphate-sensitive stores in the endoplasmic reticulum, and transient phosphorylation of mitogen-activated protein kinase p42/44 and the survival-associated proteins Akt and GSK-3beta. Preconditioning protection was eliminated by the mitogen-activated extracellular kinase inhibitor U0126, which prevented phosphorylation of p44 during preconditioning, and by calmidazolium, which antagonizes the effects of Ca2+-bound calmodulin. CONCLUSIONS: Isoflurane, at clinical concentrations, preconditions neurons in hippocampal slice cultures by mechanisms that apparently involve release of Ca2+ from the endoplasmic reticulum, transient increases in intracellular Ca2+ concentration, the Ca2+ binding protein calmodulin, and phosphorylation of the mitogen-activated protein kinase p42/44.     

Sevoflurane does not alter norepinephrine-induced intracellular Ca2+ changes in the diabetic rat aorta

Purpose

The effect of volatile anesthetics on the mechanism(s) of vascular contraction in diabetes mellitus (DM) has not been fully understood. The current study was designed to determine the effects of sevoflurane on the norepinephrine (NE)-induced changes in contractile state and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in the spontaneously developing type 2 DM rat.

Methods

The effects of sevoflurane on NE (10^?6M)-induced vasoconstriction and increase in [Ca²⁺]_i in the aortas from Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 DM model, and from age-matched control Long-Evans Tokushima Otsuka (LETO) rats were investigated using an isometric force transducer and fluorometer with fura-2 as an indicator of [Ca²⁺]_i.

Results

Norepinephrine-induced increases in tension and [Ca²⁺]_i in OLETF rats were 54.8%, 95% confidence interval (CI) 36.9-72.6% and 58.8%, 95% CI 51.5-66.1%, respectively, and in LETO rats they were 46.4%, 95% CI 39.0-53.7% and 53.8%, 95% CI 46.9-60.7%, respectively, when expressed as the percentage relative to that induced by KCl 30 mM. In LETO rats, sevoflurane at a concentration of 3.4% inhibited the vascular contraction (9.4%, 95% CI 6.3-12.6%; P < 0.001) and the increase in [Ca²⁺]_i (33.3%, 95% CI 27.4-39.2%; P = 0.002). In OLETF rats, however, sevoflurane failed to affect either the NE-induced contraction (43.6%, 95% CI 28.3-58.9%; P = 0.68) or the elevation in [Ca²⁺]_i (60.5%, 95% CI 56.3-64.8%; P = 0.93).

Conclusion

Sevoflurane at clinically relevant concentrations inhibited the NE-induced increase in [Ca²⁺]_i in the aortic smooth muscle from normal rats but not in that from type 2 DM rats. Thus, a Ca²⁺- signalling pathway resistant to sevoflurane appears to exist in the type 2 DM rat aorta.     

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.     

Adrenergic regulation of the intracellular [Ca2+] and voltage-operated Ca2+ channel currents in the rat prostate neuroendocrine cells

BACKGROUND: The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neurons. PNECs are believed to influence the growth and physiological function of the prostate gland via paracrine release of hormones. MATERIALS AND METHODS: Using fura-2 fluorescence measurement and patch-clamp techniques, we investigated the effects of adrenergic stimulation on cytosolic concentration of Ca2+ ([Ca2+]c) and high voltage-activated Ca2+ channel currents (HVA-I(Ca)) of the putative rat prostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion. RESULTS: Noradrenaline (NA, 1 microM) induced a sharp, transient increase of [Ca2+]c measured by the fura-2 fluorescence. Pharmacological studies showed that alpha1-adrenoceptors (alpha1-ARs) coupled with PLC/IP3 signaling pathway induce the release of stored Ca2+, which subsequently recruits store-operated Ca2+ entry pathways. In the whole-cell voltage clamp experiment, NA decreased the amplitude of HVA-I(Ca) by 40%, which was mimicked by an alpha2-AR agonist (UK14304) but not by an alpha1-AR agonist (phenyleprine). After selective blockade of N-type Ca2+ channels by omega-conotoxin GVIA, the addition of NA showed no further inhibition on the remaining L-type Ca2+ channel currents. The adrenergic inhibition of HVA-I(Ca) was partially prevented by the pretreatment with pertussis toxin (PTX) (5 microg/ml, 4 hr, 37 degrees C). CONCLUSIONS: RPNECs express both alpha1- and alpha2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively.     

Corticotropin-releasing factor modulation of Ca2+ influx in rat pancreatic beta-cells.

The effects of corticotropin-releasing factor (CRF) on the intracellular concentration of Ca2+ were studied in isolated single beta-cells of the rat islet. Immunohistochemical staining using CRF-receptor antibodies revealed the presence of both type 1 (CRF-R1) and type 2 (CRF-R2) receptors for CRF in the majority of islet cells. CRF (2 nmol/l) increased cytosolic Ca2+ concentration under 2.8 mmol/l glucose, dependent upon extracellular Ca2+. CRF caused depolarization of the cell membrane, which was followed by action potentials under 2.8 mmol/l glucose. The dose-response relationships of CRF-induced depolarization in the presence of 1 micromol/l nifedipine produced a bell-shaped curve, showing the peak response at 2 nmol/l. In the whole-cell patch-clamp recording, CRF enhanced Ca2+ currents through L-type Ca2+ channels in a dose-dependent manner similar to that for depolarization. In cells pretreated with Rp-deastereomer of adenosine cyclic 3',5'-phosphorothiolate (100 micromol/l), neither depolarization nor an increase in the Ca2+ current was caused by CRF at concentrations <2 nmol/l. In these cells, CRF at 20 nmol/l reduced the Ca2+ current. These results suggest that in single beta-cells of rat islets, CRF, through its own receptor, potentiates Ca2+ influx through the L-type Ca2+ channel by activation of the cAMP/protein kinase A signaling pathway. CRF at a high concentration also shows an inhibitory effect on the Ca2+ current through an unknown signaling pathway.     

降钙素对体外培养大鼠成骨细胞内钙及钙通道电流影响的研究

目的观察降钙素(CT)对培养的大鼠成骨细胞内钙离子和钙通道电流的影响,揭示其对骨代谢的作用并初步探讨其机制.方法采用去势法制备大鼠骨质疏松模型,二次酶消化法分离培养新生及去势大鼠的成骨细胞,应用激光扫描共聚焦显微镜测定不同组不同浓度降钙素(1×10-8～1×10-12mol/L)即时给药及长期给药后细胞内钙的变化规律([Ca2+]i)(以平均荧光强度表示),同时应用全细胞膜片钳技术记录成骨细胞膜钙电流(ICa)的变化.结果不同浓度的降钙素即时给药后新生组及去势组成骨细胞内钙离子浓度较空白组均有显著增高,且呈剂量依赖关系,给药10min后开始增高,15min后达峰值,之后至30min未见明显衰减,从CT≥1×10-9mol/L时有显著性差异(P＜0.05);不同浓度CT加入成骨细胞培养两周后,成骨细胞内钙浓度较空白组增高并有显著性差异(P＜0.05),较即时给药方法增高幅度较低但无显著性差异(P＞0.05);新生组同去势组各浓度均无显著性差异(P＞0.05);应用全细胞膜片钳技术发现CT1×10-12mol/L即可引起成骨细胞膜钙通道的开放,随着CT浓度的增高Ca2+通道电流的幅值增大(P＜0.05),且CT对Ca2+通道电流的兴奋作用呈剂量依赖性.结论降钙素可使成骨细胞内钙离子浓度增高从而提高成骨细胞生物学活性,并与降钙素浓度相关;降钙素对新生组和去势组大鼠成骨细胞内钙离子浓度的影响无差别;降钙素长期给药同即时给药对大鼠成骨细胞内钙离子浓度的影响无差别;降钙素增高成骨细胞内钙离子浓度的机制可能与引起成骨细胞膜上Ca2+通道的开放有关.     

Droperidol inhibits intracellular Ca2+, myofilament Ca2+ sensitivity, and contraction in rat ventricular myocytes

BACKGROUND: Droperidol has recently been associated with cardiac arrhythmias and sudden cardiac death. Changes in action potential duration seem to be the cause of the arrhythmic behavior, which can lead to alterations in intracellular free Ca concentration ([Ca]i). Because [Ca]i and myofilament Ca sensitivity are key regulators of myocardial contractility, the authors' objective was to identify whether droperidol alters [Ca]i or myofilament Ca sensitivity in rat ventricular myocytes and to identify the cellular mechanisms responsible for these effects. METHODS: Freshly isolated rat ventricular myocytes were obtained from adult rat hearts. Myocyte shortening, [Ca]i, nitric oxide production, intracellular pH, and action potentials were monitored in cardiomyocytes exposed to droperidol. Langendorff perfused hearts were used to assess overall cardiac function. RESULTS: Droperidol (0.03-1 mum) caused concentration-dependent decreases in peak [Ca]i and shortening. Droperidol inhibited 35 mm KCl-induced increase in [Ca]i, with little direct effect on sarcoplasmic reticulum Ca stores. Droperidol had no effect on action potential duration but caused a rightward shift in the concentration-response curve to extracellular Ca for shortening, with no concomitant effect on peak [Ca]i. Droperidol decreased pHi and increased nitric oxide production. Droperidol exerted a negative inotropic effect in Langendorff perfused hearts. CONCLUSION: These data demonstrate that droperidol decreases cardiomyocyte function, which is mediated by a decrease in [Ca]i and a decrease in myofilament Ca sensitivity. The decrease in [Ca]i is mediated by decreased sarcolemmal Ca influx. The decrease in myofilament Ca sensitivity is likely mediated by a decrease in pHi and an increase in nitric oxide production.     

Differential effects of bupivacaine on intracellular Ca2+ regulation: potential mechanisms of its myotoxicity

BACKGROUND: Bupivacaine produces skeletal muscle damage in clinical concentrations. It has been suggested that this may be caused by an increased intracellular level of [Ca2+]. Therefore, the aim of this study was to investigate direct intracellular effects of bupivacaine on Ca2+ release from the sarcoplasmic reticulum (SR), on Ca2+ uptake into the SR, and on Ca2+ sensitivity of the contractile proteins. METHODS: Saponin skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure described previously. For the assessment of effects on Ca2+ uptake and release from the SR, bupivacaine was added to the loading solution and the release solution, respectively. Force transients and force decays were monitored, and the position of the curve relating relative isometric force free [Ca2+] was evaluated in the presence or absence of bupivacaine. RESULTS: Bupivacaine induces Ca2+ release from the SR. In addition, the Ca2+ loading procedure is suppressed, resulting in smaller caffeine-induced force transients after loading in the presence of bupivacaine. The decay of caffeine-induced force transients is reduced by bupivacaine, and it also shifts [Ca2+]-force relation toward lower [Ca2+]. CONCLUSIONS: These data reveal that bupivacaine does not only induce Ca2+ release from the SR, but also inhibits Ca2+ uptake by the SR, which is mainly regulated by SR Ca2+ adenosine triphosphatase activity. It also has a Ca2+ -sensitizing effect on the contractile proteins. These mechanisms result in increased intracellular [Ca2+] concentrations and may thus contribute to its pronounced skeletal muscle toxicity.     

Isoflurane enhances spontaneous Ca2+ oscillations in developing rat hippocampal neurons in vitro

Background: During the nervous system development, spontaneous synchronized Ca²⁺ oscillations are thought to possess integrative properties because their amplitude and frequency can influence the patterning of neuronal connection, neuronal differentiation, axon outgrowth, and long-distance wiring. Accumulating studies have confirmed that some drugs such as volatile anesthetic isoflurane produced histopathologic changes in the central nervous system in juvenile animal models. Because the hippocampus plays an important role in learning and memory, the present work was designed to characterize the Ca²⁺ oscillations regulated by volatile anesthetic isoflurane in primary cultures of developing hippocampal neurons (5-day-cultured).
Methods: Primary cultures of rat hippocampal neurons (5-day-cultured) were loaded with the Ca²⁺ indicator Fluo-4AM (4 μM) and were studied with a confocal laser microscope.
Results: Approximately 22% of 5-day-cultured hippocampal neurons exhibited typical Ca²⁺ oscillations. These oscillations were dose-dependently enhanced by isoflurane (EC50 0.5 MAC, minimum alveolar concentration) and this effect could be reverted by bicuculline (50 μM), a specific γ-aminobutyric acid (GABA_A) receptor antagonist.
Conclusion: Unlike its depressant effect on the Ca²⁺ oscillations in adult neurons in previous researches, isoflurane dose-dependently enhanced calcium oscillations in developing hippocampal neurons by activating GABA_A receptors, a major excitatory receptor in synergy with N -methyl- d -aspartate receptors at the early stages of development. It may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.     

Dual functional role of membrane depolarization/Ca2+ influx in rat pancreatic B-cell.

Transient exposure of rat pancreatic B-cell to 50 mM K+ ([K+50]) makes exocytosis unresponsive to further depolarization, i.e., stimulation with 100 mM K+ or 1 uM glyburide, which closes the ATP-sensitive K+ (K+ATP) channel, simultaneously with [K+50] does not produce any greater insulin secretion compared with [K+50] alone. In sharp contrast, 16.7 mM glucose ([G16.7]) applied simultaneously with [K+50] elicits an insulin response markedly greater than that produced by [K+50] alone, which is not attenuated by 100 uM diazoxide, an inhibitor of K+ATP channel closure. [G16.7]-induced insulin secretion at the basal K+ concn of 4.7 mM was greatly (93%) suppressed by 100 uM diazoxide. Insulin secretion induced by [K+50] plus [G16.7] ([K+50 + G16.7]) was markedly suppressed (70%) by 1 uM nifedipine, a Ca(2+)-channel blocker and was completely abolished by 2 mM 2-cyclohexen-1-one, which reportedly decreases reduced glutathione level and blocks glucokinase. This finding indicates that insulin release induced by [K+50 + G16.7] is not due to leakage produced by toxic stimuli but to activation of exocytosis. When graded concentrations (25 and 50 mM) of K+ were applied simultaneously with [G16.7] in the presence of 100 uM diazoxide, insulin response was clearly dependent on K+ concentration, indicating that the physiological range of membrane depolarization also activates the glucose-responsive effector. Membrane depolarization/Ca2+ influx directly stimulates hormone exocytosis on one hand and activates the K+ATP channel-independent glucose-responsive effector or effectors on the other in the B-cell. The nature of the glucose-responsive effector or effectors remains to be established.     

Glucose-induced changes in histochemically determined Ca2+ in B-cell granules, 45Ca uptake, and total Ca2+ of rat pancreatic islets

Rat pancreatic islets contain an ionized or readily ionizable calcium fraction that can be determined by the metallochromic indicator glyoxal-bis-(2- hydroxyanil ) ( GBHA ). This calcium fraction is mainly localized in the secretory granules. The relationship between the effects of glucose on 45Ca uptake and on this ionized calcium fraction was investigated. In addition, the effects of glucose on total islet calcium content were also studied. Stimulation of isolated islets for 30 min with 15 mM glucose in the presence of 2.5 mM CaCl2 increased the 45Ca uptake but decreased the GBHA -Ca content, while the total calcium content was not affected. Deletion of CaCl2 caused, at 2.5 mM glucose, an abrupt decrease of GBHA -Ca, which did not occur at 15 mM glucose. Total islet calcium content decreased slowly at 2.5 mM glucose, but this was not significantly affected by glucose stimulation. Islet GBHA -Ca can be reduced by 70% and total islet calcium by 30% by means of washing with calcium-free buffer. Reintroduction of calcium at 2.5 mM glucose partly restored, but glucose 15 mM completely restored, the GBHA -Ca level within 5 min. The total calcium content was restored within 15 min independent of the glucose concentration. The increase of the islet calcium content equalled the 45Ca uptake at 2.5 mM glucose. The 45Ca uptake at 15 mM glucose was higher than the increase of the islet calcium content. The results indicate that the intragranular Ca2+ pool, as measured by GBHA , is rapidly and dramatically altered by glucose stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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。     

Digital imaging microscopy for intracellular Ca2+ in cultured single rat vascular smooth muscle cells using fluorescent Ca2+ indicator "fura-2"]

We presented the principle and methodology of digital imaging microscopy for intracellular Ca2+ in cultured single vascular smooth muscle cells of the rat using fluorescent Ca2+ indicator "fura-2". The methods seemed useful for studying the physiological and pathological phenomena in a single smooth muscle cell. Analysis of the spatial and temporal dynamics of intracellular Ca2+ might be crucial for studying the active sites of vasoactive or anesthetic drugs in a vascular smooth muscle cell as well as for understanding pathophysiology of the arterial spasm.     

Mechanism of the ropivacaine-induced increase in intracellular Ca2+ concentration in rat aortic smooth muscle

BACKGROUND: Ropivacaine is a long-acting local anesthetic with low cardiac toxicity that induces vasoconstriction in vitro and in vivo. Vascular smooth muscle tone is regulated by changes in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity. Therefore, the aim of this study was to examine the mechanism underlying the increase in [Ca(2+)](i) in ropivacaine-induced vascular contraction. METHODS: Ropivacaine-induced contractile responses and changes in [Ca(2+)](i) were examined using an isometric force transducer and a fluorometer, respectively. RESULTS: Ropivacaine induced a biphasic, concentration-dependent change in [Ca(2+)](i) and contractile response in rat aortic smooth muscles: an increase in [Ca(2+)](i) occurred at lower ropivacaine concentrations (3 x 10(-5) to 3 x 10(-4) M) and a decrease was observed at higher concentrations (10(-3) to 3 x 10(-3) M). Contraction and the [Ca(2+)](i) increase induced by ropivacaine were attenuated significantly by a voltage-dependent Ca(2+) channel antagonist, an inositol 1,4,5-triphosphate receptor antagonist and Ca(2+)-free solution (P < 0.01, n = 6). CONCLUSION: Ropivacaine-induced contraction of rat aortic smooth muscle is, in part, regulated by Ca(2+) influx from the extracellular space and Ca(2+) release from the sarcoplasmic reticulum.     

Differential effects of bupivacaine and ropivacaine enantiomers on intracellular Ca2+ regulation in murine skeletal muscle fibers

BACKGROUND: Increased intracellular Ca concentrations are considered to be a major pathomechanism in local anesthetic myotoxicity. Racemic bupivacaine and S-ropivacaine cause Ca release from the sarcoplasmic reticulum of skeletal muscle fibers and simultaneously inhibit Ca reuptake. Examining the optical isomers of both agents, the authors investigated stereoselective effects on muscular Ca regulation to get a closer insight in subcellular mechanisms of local anesthetic myotoxicity. METHODS: R- and S-enantiomers as well as racemic mixtures of both agents were tested in concentrations of 1, 5, 10, and 15 mm. Saponin-skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure. For the assessment of effects on Ca uptake and release from the sarcoplasmic reticulum, agents were added to the loading solution and the release solution, respectively, and force and Ca transients were monitored. RESULTS: The effects of S-enantiomers on both Ca release and reuptake were significantly more pronounced than those of racemic mixtures and R-enantiomers, respectively. In addition, the effects of racemates were markedly stronger than those of R-enantiomers. With regard to Ca release, the effects of bupivacaine isomers were more pronounced than the isomers of ropivacaine. CONCLUSIONS: These data show that stereoselectivity is involved in alterations of intracellular Ca regulation by bupivacaine and ropivacaine. S-enantiomers seem to be more potent than R-enantiomers, with intermediate effects of racemic mixtures. In addition, lipophilicity also seems to determine the extent of Ca release by local anesthetics.     

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.     

Ca2+ signaling mediated by IP3-dependent Ca2+ releasing and store-operated Ca2+ channels in rat odontoblasts.

In the phospholipase-C (PLC) signaling system, Ca2+ is mobilized from intracellular Ca2+ stores by an action of inositol 1,4,5-trisphosphate (IP3). The depletion of IP3-sensitive Ca2+ stores activates a store-operated Ca2+ entry (SOCE). However, no direct evidence has been obtained about these signaling pathways in odontoblasts. In this study, we investigate the characteristics of the SOCE and IP3-mediated Ca2+ mobilizations in rat odontoblasts using fura-2 microfluorometry and a nystatin-perforated patch-clamp technique. In the absence of extracellular Ca2+ ([Ca2+]o), thapsigargin (TG) evoked a transient rise in intracellular Ca2+ concentration ([Ca2+]i). After TG treatment to deplete the store, the subsequent application of Ca2+ resulted in a rapid rise in [Ca2+]i caused by SOCE. In the absence of TG treatment, no SOCE was evoked. The Ca2+ influx was dependent on [Ca2+]o (KD = 1.29 mM) and was blocked by an IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (2-APB), as well as La3+ in a concentration-dependent manner (IC50 = 26 microM). In TG-treated cells, an elevation of [Ca2+]o from 0 to 2.5 mM elicited an inwardly rectifying current at hyperpolarizing potentials with a positive reversal potential. The currents were selective for Ca2+ over the other divalent cations (Ca2+ > Ba2+ > Sr2+ > Mn2+). In the absence of [Ca2+]o, carbachol, bradykinin, and 2-methylthioadenosine 5'triphosphate activated Ca2+ release from the store; these were inhibited by 2-APB. These results indicate that odontoblasts possessed Ca2+ signaling pathways through the activation of store-operated Ca2+ channels by the depletion of intracellular Ca2+ stores and through the IP3-induced Ca2+ release activated by PLC-coupled receptors.