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-3[beta] (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-3[beta]. 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.     

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 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.     

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.     

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 Ca 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 Ca concentration was measured in CA1 neurons. N-methyl-d-aspartate receptor (NMDAR)-dependent Ca 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 Ca influx, whereas in aging slices, Ca influx was increased protein kinase C. In aging slices, isoflurane did not increase the phosphorylation of Akt and p42/44. CONCLUSIONS: Isoflurane neuroprotection of hippocampal slices during oxygen and glucose deprivation decreases with age. Isoflurane does not prevent large increases in intracellular Ca concentration during oxygen and glucose deprivation and does not induce the phosphorylation of the prosurvival proteins in aging slices. A protein kinase C-mediated increase in NMDAR activity may result in increased excitotoxicity and decreased neuroprotection by volatile anesthetics in the aging brain.     

Isoflurane Preconditioning Improves Long-term Neurologic Outcome after Hypoxic-Ischemic Brain Injury in Neonatal Rats

Background: Preconditioning the brain with relatively safe drugs seems to be a viable option to reduce ischemic brain injury. The authors and others have shown that the volatile anesthetic isoflurane can precondition the brain against ischemia. Here, the authors determine whether isoflurane preconditioning improves long-term neurologic outcome after brain ischemia.

Methods: Six-day-old rats were exposed to 1.5% isoflurane for 30 min at 24 h before the brain hypoxia-ischemia that was induced by left common carotid arterial ligation and then exposure to 8% oxygen for 2 h. The neuropathology, motor coordination, and learning and memory functions were assayed 1 month after the brain ischemia. Western analysis was performed to quantify the expression of the heat shock protein 70, Bcl-2, and survivin 24 h after isoflurane exposure.

Results: The mortality was 45% after brain hypoxia-ischemia. Isoflurane preconditioning did not affect this mortality. However, isoflurane preconditioning attenuated ischemia-induced loss of neurons and brain tissues, such as cerebral cortex and hippocampus in the survivors. Isoflurane also improved the motor coordination of rats at 1 month after ischemia. The learning and memory functions as measured by performance of Y-maze and social recognition tasks in the survivors were not affected by the brain hypoxia-ischemia or isoflurane preconditioning. The expression of Bcl-2, a well-known antiapoptotic protein, in the hippocampus is increased after isoflurane exposure. This increase was reduced by the inhibitors of inducible nitric oxide synthase. Inducible nitric oxide synthase inhibition also abolished isoflurane preconditioning-induced neuroprotection.     

gamma-Aminobutyric acid-A receptors contribute to isoflurane neuroprotection in organotypic hippocampal cultures

The mechanisms by which anesthetics such as isoflurane reduce cell death in rodent models of cerebral ischemia remain incompletely defined. Reduction in glutamate excitotoxicity explains some but not all of isoflurane's neuroprotection. Because isoflurane potentiates gamma-aminobutyric acid (GABA) receptor-mediated ion fluxes and GABA(A) receptor agonists have neuroprotective effects, we hypothesized that GABA(A) receptors contribute to isoflurane neuroprotection. As a model of cerebral ischemia and recovery, we used rat hippocampal slice cultures. Survival of CA1, CA3, and dentate neurons was examined 2 and 3 days after 1-h combined oxygen-glucose deprivation (OGD) at 37 degrees C. To define the role of GABA(A) receptors in mediating protection, the effect of 1% isoflurane on cell survival was examined in the presence of the GABA(A) antagonist bicuculline during OGD. Cell death was measured with propidium iodide fluorescence. Isoflurane and the selective GABA(A) agonist muscimol (25 micro M) reduced cell death after OGD to values similar to slices not exposed to OGD, with the exception that muscimol did not reduce cell death in CA3 neurons 2 days after OGD. The GABA(A) antagonist bicuculline reduced the neuroprotective effects of isoflurane on hippocampal neurons 2 and 3 days after OGD. We conclude that GABA(A) receptors contribute to neuroprotection against OGD produced by isoflurane in the hippocampal slice model. Based on this and other studies, it is likely that neuroprotection produced by isoflurane is multifactorial and includes actions at both GABA(A) and glutamate receptors and possibly other mechanisms. IMPLICATIONS: Isoflurane is neuroprotective in rodent brain ischemia models, but the mechanisms for this effect remain incompletely defined. In organotypic cultures of rat hippocampus, we show that protection of CA1, CA3, and dentate neurons by 1% isoflurane from death caused by oxygen and glucose deprivation involves GABA(A) receptors.     

The inhibitory effects of isoflurane on protein tyrosine phosphorylation-modulated contraction of rat aortic smooth muscle

BACKGROUND: Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in initiating and modulating vascular smooth muscle contraction. The aim of the current study was to examine the effects of isoflurane on sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor-induced, tyrosine phosphorylation-mediated contraction of rat aortic smooth muscle. METHODS: The Na3VO4-induced contraction of rat aortic smooth muscle and tyrosine phosphorylation of proteins including phospholipase Cgamma-1 (PLCgamma-1) and p44/p42 mitogen-activated protein kinase (MAPK) were assessed in the presence of different concentrations of isoflurane, using isometric force measurement and Western blotting methods, respectively. RESULTS: Na3VO4 (10(-4) m) induced a gradually sustained contraction and significant increase in protein tyrosine phosphorylation of a set of substrates including PLCgamma-1 and p42MAPK, all of which were markedly inhibited by genistein (5 x 10(-5) m), a tyrosine kinase inhibitor. Isoflurane (1.2-3.5%) dose-dependently depressed the Na3VO4-induced contraction (P < 0.05-0.005; n = 8). Isoflurane also attenuated the total density of the Na3VO4-induced, tyrosine-phosphorylated substrate bands and the density of tyrosine-phosphorylated PLCgamma-1 band and p42MAPK band (P < 0.05-0.005; n = 4) in a concentration-dependent manner. CONCLUSION: The findings of the current study, that isoflurane dose-dependently inhibits both the Na3VO4-stimulated contraction and tyrosine phosphorylation of a set of proteins including PLCgamma-1 and p42MAPK in rat aortic smooth muscle, suggest that isoflurane depresses protein tyrosine phosphorylation-modulated contraction of vascular smooth muscle, especially that mediated by the tyrosine-phosphorylated PLCgamma-1 and MAPK signaling pathways.     

Volatile anesthetics increase intracellular calcium in cerebrocortical and hippocampal neurons

BACKGROUND: An increase in intracellular calcium concentration ([Ca2+]i) in neurons has been proposed as an important effect of volatile anesthetics, because they alter signaling pathways that influence neurotransmission. However, the existing data for anesthetic-induced increases in [Ca2+]i conflict. METHODS: Changes in [Ca2+]i were measured using fura-2 fluorescence spectroscopy in rat cortical brain slices at 90, 185, 370, and 705 microM isoflurane. To define the causes of an increase in [Ca2+]i, slices were studied in Ca2+-free medium, in the presence of Ca2+-channel blockers, and in the presence of the Ca2+-release inhibitor azumolene. The authors compared the effect of the volatile anesthetic with that of the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane. Single-dose experiments in CA1 neurons in hippocampal slices with halothane (360 microM) and in acutely dissociated CA1 neurons with halothane (360 microM) and isoflurane (445 microM) also were performed. RESULTS: Isoflurane at 0.5, 1, and 2 minimum alveolar concentrations increased basal [Ca2+]i in cortical slices in a dose-dependent manner (P < 0.05). This increase was not altered by Ca2+-channel blockers or Ca2+-free medium but was reduced 85% by azumolene. The nonanesthetic 1,2-dichlorohexafluorocyclobutane did not increase [Ca2+]i. In dissociated CA1 neurons, isoflurane reversibly increased basal [Ca2+]i by 15 nM (P < 0.05). Halothane increased [Ca2+]i in dissociated CA1 neurons and CA1 neurons in hippocampal slices by approximately 30 nM (P < 0.05). CONCLUSIONS: (1) Isoflurane and halothane reversibly increase [Ca2+]i in isolated neurons and in neurons within brain slices. (2) The increase in [Ca2+]i is caused primarily by release from intracellular stores. (3) Increases in [Ca2+]i occur with anesthetics but not with the nonanesthetic 1,2-dichlorohexafluorocyclobutane.     

Intracellular mechanism of mitochondrial adenosine triphosphate-sensitive potassium channel activation with isoflurane

The precise mechanism of isoflurane and mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK(ATP)) interaction is still unclear, although the mitoK(ATP) is involved in isoflurane-induced preconditioning. We examined the role of various intracellular signaling systems in mitoK(ATP) activation with isoflurane. Mitochondrial flavoprotein fluorescence (MFF) was measured to quantify mitoK(ATP) activity in guinea pig cardiomyocytes. To confirm isoflurane-induced MFF, cells were exposed to Tyrode's solution containing either isoflurane (1.0 +/- 0.1 mM) or diazoxide and then both drugs together (n = 10 each). In other studies, the following drugs were each added during isoflurane administration: adenosine or the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (SPT); the protein kinase C (PKC) activators phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu); the PKC inhibitors polymyxin B and staurosporine; the tyrosine kinase inhibitor lavendustin A; or the mitogen-activated protein kinase inhibitor SB203580 (n = 10 each). Isoflurane potentiated MFF induced by diazoxide (100 micro M), and diazoxide also increased isoflurane-induced MFF. PMA (0.2 micro M), PDBu (1 micro M), and adenosine (100 micro M) induced MFF. However, SPT (100 micro M), polymyxin B (50 micro M), staurosporine (200 nM), lavendustin A (0.5 micro M), and SB203580 (10 micro M) all failed to inhibit the effect of isoflurane. Our results show that isoflurane, adenosine, and PKC activate mitoK(ATP). However, our data do not support an action of isoflurane through pathways involving adenosine, PKC, tyrosine kinase, or mitogen-activated protein kinase. These results suggest that isoflurane may directly activate mitoK(ATP). IMPLICATIONS: Our results show that isoflurane activates mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, but not through pathways involving adenosine, protein kinase C, tyrosine kinase, or p38 mitogen-activated protein kinase. Isoflurane may directly activate mitoK(ATP) channels.     

JNK信号通路在异氟醚预处理和七氟醚预处理减轻大鼠海马脑片缺氧无糖损伤中的作用

目的 评价c-Jun氨基末端激酶(JNK)信号通路在异氟醚预处理和七氟醚预处理减轻大鼠海马脑片缺氧无糖(OGD)损伤中的作用.方法雄性成年SD大鼠,体重270～290 g,断头处死,剥离海马,制备海马脑片.取大鼠海马脑片96张,采用随机数字表法,将其随机分为8组(n=12):对照组(C组)、OGD组、异氟醚预处理组(Iso组)、七氟醚预处理组(Sevo组)、SP600125+异氟醚预处理组(SP+Iso组)、SP600125+七氟醚预处理组(SP+Sevo组)、二甲基亚砜(DMSO)+异氟醚预处理组(DMSO+Iso组)和DMSO+七氟醚预处理组(DMSO+Sevo组).采用电生理技术,细胞外记录CA1区群锋电位(PS)波幅,计算PS恢复程度.采用碘化丙啶染色法,测定细胞活力.结果 与C组比较,其余各组PS恢复程度和细胞活力降低(P＜0.01);与OGD组比较,Iso组、Sevo组、SP+Iso.组、SP+Sevo组、DMSO+Iso组和DMSO+Sevo组PS恢复程度和细胞活力升高(P＜0.01);与180组比较,SP+Iso组PS恢复程度和细胞活力升高(P＜0.01),DMSO+Iso组PS恢复程度和细胞活力差异无统计学意义(P＞0.05);与Sevo组比较,SP+Sevo组PS恢复程度和细胞活力升高(P＜0.01),DMSO+Sevo组PS恢复程度和细胞活力差异无统计学意义(P＞0.05).结论 异氟醚预处理和七氟醚预处理可通过抑制JNK信号通路,减轻大鼠海马脑片缺氧无糖损伤.

Abstract：

Objective To evaluate the role of c-Jun N-terminal kinase (JNK) signaling pathway in the protective effect of isoflurane preconditioning and sevoflurane preconditioning against oxygen-glucose deprivation (OGD) injury in rat hippocampal slices. Methods Male adult SD rats weighing 270-290 g were anesthetized with ether and decapitated. The hippocampi were removed and sagittally sliced (400 μm thick) and placed in artificial cerebral spinal fluid aerated with 95% O2-5% CO2 . Ninety-six hippocampal slices were randomly divided into 8 groups (n = 12 each): control group (group C), OGD group, isoflurane preconditioning group (group Iso),sevoflurane preconditioning group (group Sevo) , SP600125 + isoflurane preconditioning group (group SP + Iso),SP600125 +sevoflurane preconditioning group (group SP + Sevo), DMSO + isoflurane preconditioning group (group DMSO + Iso) and DMSO + sevoflurane preconditioning group (group DMSO + Sevo). Electrophysiological technique was used to record the amplitude of population spike ( PS) in the stratum pyramidale of CA1 region and the degree of recovery of PS was calculated. The cell viability was determined by propidium iodide staining. Results Compared with group C, the degree of recovery of PS and cell viability were significantly decreased in the other groups ( P ＜ 0.01) . Compared with group OGD, the degree of recovery of PS and cell viability were significantly increased in groups Iso, Sevo, SP+Iso, SP+Sevo, DMSO+ Iso and DMSO + Sevo (P＜ 0.01). Compared with group Iso, the degree of recovery of PS and cell viability were significantly increased in group SP+Iso ( P ＜ 0.01) , while no significant change was found in group DMSO + Iso ( P ＞ 0.05) . Compared with group Sevo, the degree of recovery of PS and cell viability were significantly increased in group SP + Sevo ( P ＜ 0.01) , while no significant change was found in group DMSO + Sevo ( P ＞ 0.05). Conclusion Isoflurane preconditioning and sevoflurane preconditioning can attenuate the OGD injury to rat hippocampal slices through inhibiting JNK signaling pathway.     

The Inhibitory Effects of Isoflurane on Protein Tyrosine Phosphorylation-modulated Contraction of Rat Aortic Smooth Muscle

Background: Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in initiating and modulating vascular smooth muscle contraction. The aim of the current study was to examine the effects of isoflurane on sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor-induced, tyrosine phosphorylation-mediated contraction of rat aortic smooth muscle.

Methods: The Na3VO4-induced contraction of rat aortic smooth muscle and tyrosine phosphorylation of proteins including phospholipase C[gamma]-1 (PLC[gamma]-1) and p44/p42 mitogen-activated protein kinase (MAPK) were assessed in the presence of different concentrations of isoflurane, using isometric force measurement and Western blotting methods, respectively.

Results: Na3VO4 (10-4 m) induced a gradually sustained contraction and significant increase in protein tyrosine phosphorylation of a set of substrates including PLC[gamma]-1 and p42MAPK, all of which were markedly inhibited by genistein (5 x 10-5 m), a tyrosine kinase inhibitor. Isoflurane (1.2-3.5%) dose-dependently depressed the Na3VO4-induced contraction (P < 0.05-0.005; n = 8). Isoflurane also attenuated the total density of the Na3VO4-induced, tyrosine-phosphorylated substrate bands and the density of tyrosine-phosphorylated PLC[gamma]-1 band and p42MAPK band (P < 0.05-0.005; n = 4) in a concentration-dependent manner.     

Volatile Anesthetics Increase Intracellular Calcium in Cerebrocortical and Hippocampal Neurons

Background: An increase in intracellular calcium concentration ([Ca2+]i) in neurons has been proposed as an important effect of volatile anesthetics, because they alter signaling pathways that influence neurotransmission. However, the existing data for anesthetic-induced increases in [Ca2+]i conflict.

Methods: Changes in [Ca2+]i were measured using fura-2 fluorescence spectroscopy in rat cortical brain slices at 90, 185, 370, and 705 [micro sign]M isoflurane. To define the causes of an increase in [Ca2+]i, slices were studied in Ca2+-free medium, in the presence of Ca2+-channel blockers, and in the presence of the Ca2+-release inhibitor azumolene. The authors compared the effect of the volatile anesthetic with that of the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane. Single-dose experiments in CA1 neurons in hippocampal slices with halothane (360 [micro sign]M) and in acutely dissociated CA1 neurons with halothane (360 [micro sign]M) and isoflurane (445 [micro sign]M) also were performed.

Results: Isoflurane at 0.5, 1, and 2 minimum alveolar concentrations increased basal [Ca2+]i in cortical slices in a dose-dependent manner (P < 0.05). This increase was not altered by Ca2+-channel blockers or Ca2+-free medium but was reduced 85% by azumolene. The nonanesthetic 1,2-dichlorohexafluorocyclobutane did not increase [Ca2+] (i). In dissociated CA1 neurons, isoflurane reversibly increased basal [Ca (2+)]i by 15 nM (P < 0.05). Halothane increased [Ca2+]i in dissociated CA1 neurons and CA1 neurons in hippocampal slices by approximately 30 nM (P < 0.05).     

地氟醚、七氟醚和异氟醚预处理对缺氧-复氧心肌细胞内游离Ca2+的影响

目的 观察地氟醚，七氟醚和异氟醚预处理对缺氧-复氧心肌细胞内Ca^2 的影响。方法 将原代培养乳鼠心肌细胞随机分为对照，缺氧-复氧及1.5MAC地氧醚，七氟醚和异氟醚预处理后缺氧-复氧五组，用Fra-2标记细胞内Ca^2 ，荧光光度计进行测定。结果 与对照组比，复氧20分钟时，其余四组细胞内Ca^2 均显著升高；而地氟醚，七氟醚和异氟醚预处理组的升高幅度明显低于缺氧-复氧组；三个吸入麻醉药组间无差异，复氧60分钟，缺氧-复氧组虽有明显下降，但仍显著高于对照组，地氟醚，七氟醚烽异氟醚预处理组则降至对照组水平。结论 地氟醚，七氟醚和异氟醚预处理，可明显减轻缺氧-复氧心肌引起的细胞内游离Ca^2 升高。     

Basic fibroblast growth factor stimulates vascular endothelial growth factor release in osteoblasts: divergent regulation by p42/p44 mitogen-activated protein kinase and p38 mitogen-activated protein kinase.

We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O-aminophinoxy)-ethane-N,N,N,N-tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase.     

Role of Protein Kinase C, Ca2+/Calmodulin-dependent Protein Kinase II, and Mitogen-activated Protein Kinases in Volatile Anesthetic-induced Relaxation in Newborn Rabbit Pulmonary Artery

Background: This study examined the responsiveness of skinned pulmonary arteries from newborn rabbit to volatile anesthetics and the role of protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the downstream effectors, mitogen-activated protein kinases (ERK1/2 and p38).

Methods: Pulmonary arterial strips from 9- to 12-day-old rabbits were mounted on force transducers and treated with saponin ("skinned" strips). The skinned strips were activated by pCa 6.3 until force reached a steady state (control). Isoflurane or halothane was then administered. The result (test) was expressed as a percentage of the control. Inhibitors included bisindolylmaleimide (Ca2+-dependent and -independent PKC), Go6976 (Ca2+-dependent PKC), CKIINtide (CaMKII), KN-93 (CaMKII), PD98059 (MEK/ERK1/2), and SB203580 (p38).

Results: The anesthetics dose-dependently decreased pCa-induced force (4-32% for 1-5% isoflurane; 17-76% for 1-3% halothane). The inhibitors of PKC (bisindolylmaleimide and Go6976) and MEK/ERK1/2 (PD98059) completely prevented the relaxation induced by 3% isoflurane and partially prevented that induced by 2% and 3% halothane with the same effective inhibitor concentrations. In contrast, the effective concentration of CaMKII inhibitors was a direct function of the anesthetic concentration for different inhibitors (KN-93 for isoflurane and CKIINtide for halothane), and that of the p38 inhibitor (SB20358) was a direct function of both anesthetics.     

Selective γ-Aminobutyric Acid Type A Receptor Antagonism Reverses Isoflurane Ischemic Neuroprotection

Background: Isoflurane provides protection against severe forebrain ischemia in the rat. The authors hypothesized that this is attributable to interaction with the [gamma]-aminobutyric acid type A (GABAA) receptor resulting in altered time to onset of ischemic hippocampal depolarization.

Methods: Organotypic hippocampal slices were subjected to oxygen-glucose deprivation in the presence of isoflurane and combinations of GABAA (bicuculline) and GABAB (phaclofen) receptor antagonists. Cell death was measured. Rats were subjected to severe forebrain ischemia while anesthetized with fentanyl-nitrous oxide or 1.4% isoflurane. In the isoflurane group, rats also received intravenous bicuculline (0, 1, or 2 mg/kg). Neurologic and histologic outcomes and time to depolarization were assessed.

Results: In slices, 2% isoflurane caused near-complete protection against oxygen-glucose deprivation. This was unaffected by coadministration of phaclofen but largely reversed by bicuculline. The GABAA agonist muscimol was also protective, having an effect equivalent to 1% isoflurane. In rats, isoflurane (0 mg bicuculline) improved neurologic and histologic outcome versus fentanyl-nitrous oxide (CA1 percentage of alive neurons: fentanyl-nitrous oxide, 15 +/- 7; isoflurane, 61 +/- 24). The isoflurane effect was reversed in a dose-dependent manner by bicuculline (CA1 percentage alive: 1 mg/kg, 44 +/- 22; 2 mg/kg, 21 +/- 15). Time to depolarization was delayed with isoflurane versus fentanyl-nitrous oxide (137 vs. 80 s) but was not affected by bicuculline (149 s). In contrast, postischemic time to repolarization was more rapid with fentanyl-nitrous oxide or isoflurane plus bicuculline versus isoflurane alone.     

Isoflurane Activates PKC and Ca2+-Calmodulin-dependent Protein Kinase II via MAP Kinase Signaling in Cultured Vascular Smooth Muscle Cells

Background: Protein kinase C (PKC) and Ca2+-calmodulin-dependent protein kinase II (CaMKII) have been implicated in isoflurane-increased force in skinned femoral arterial strips. The extracellular signal-regulated kinases (ERK1/2) of mitogen-activated protein kinase have been shown to be target effectors of PKC and CaMKII. This study examined the role of the ERK1/2 signaling pathway in isoflurane activation of PKC and CaMKII using cultured vascular smooth muscle cells.

Methods: Vascular smooth muscle cells were prepared by cell migration from isolated rabbit femoral arterial segments. Growth of passage of vascular smooth muscle cells (80-90% confluence, passage 5-10) was arrested for 48 h before experiments, during which time phorbol 1,3-diaceylester treatment was used to down-regulate PKC. Cells were treated for 30 min with one of the inhibitors of mitogen-activated protein kinase kinase (PD98059), PKC (Go6976 and bisindolylmaleimide), or CaMKII (KN-93 and KN-62) at 10 [mu]m. After administration of isoflurane, vascular smooth muscle cells were frozen rapidly, homogenized, and centrifuged. The homogenates were used for identification of phosphorylated ERK1/2 or for further centrifugation to separate the membrane from the cytosol for identification of PKC isoforms ([alpha] and ) by Western blotting.

Results: Isoflurane increased ERK1/2 phosphorylation in a dose-dependent manner and reached a plateau at 10 min. PD98059 or down-regulated PKC blocked the increase of phosphorylated ERK1/2 levels by isoflurane, and bisindolylmaleimide, KN-93, or KN-62, but not by Go6976 reduced levels of phosphorylated ERK1/2. The membrane fraction of PKC but not of PKC[alpha] was increased by isoflurane.     

Mechanisms of Isoflurane-increased Submaximum Ca2+-activated Force in Rabbit Skinned Femoral Arterial Strips

Background: Isoflurane enhances contraction in isolated intact arterial rings by a protein kinase C (PKC) activator and also causes contracture in skinned arterial strips. This study investigated the mechanisms of this isoflurane activation of the contractile proteins of skinned strips.

Methods: The skinned strips, mounted on photodiode force transducers, were prepared from rabbit femoral arteries treated with saponin. The strips were activated by 1 [micro sign]M Ca2+ (buffered with 7 mM EGTA) with or without inhibitors for PKC and calmodulin-dependent protein kinase II (CaM kinase II). When force reached steady state, isoflurane was administered and changes in force were observed. Another group of the strips was frozen to assay myosin light chain phosphorylation (MLC-p) using two-dimensional electrophoresis and immunoblotting. Analysis of variance was used to compare the results from test and control groups. Probability values <0.05 were significant.

Results: Isoflurane (1-5%) dose dependently increased (24-81%) the Ca (2+-activated) force. At 1% and 5% isoflurane, MLC-p did not change either as the force increased or reached a new steady state level. However, with 3% isoflurane, MLC-p transiently decreased (29.1% and 17.1% of total MLC for 0% and 3% isoflurane, respectively). The 3% isoflurane-increased force was blocked by 10 [micro sign]M bisindolymaleidmide, an inhibitor of PKC, but not by 10 [micro sign]M Go-6976, an inhibitor of Ca2+-dependent PKC, and was enhanced 50% by 0.1 mM KN-62, an inhibitor of CaM kinase II.