Reactive Oxygen Species Precede Protein Kinase C-δ Activation Independent of Adenosine Triphosphate-sensitive Mitochondrial Channel Opening in Sevoflurane-induced Cardioprotection

Background: In the current study, the authors investigated the distinct role and relative order of protein kinase C (PKC)-[delta], adenosine triphosphate-sensitive mitochondrial K+ (mito K+ATP) channels, and reactive oxygen species (ROS) in the signal transduction of sevoflurane-induced cardioprotection and specifically addressed their mechanistic link.

Methods: Isolated rat trabeculae were preconditioned with 3.8% sevoflurane and subsequently subjected to an ischemic protocol by superfusion of trabeculae with hypoxic, glucose-free buffer (40 min) followed by 60 min of reperfusion. In addition, the acute affect of sevoflurane on PKC-[delta] and PKC-[epsilon] translocation and nitrotyrosine formation was established with use of immunofluorescent analysis. The inhibitors chelerythrine (6 [mu]m), rottlerin (1 [mu]m), 5-hydroxydecanoic acid sodium (100 [mu]m), and n-(2-mercaptopropionyl)-glycine (300 [mu]m) were used to study the particular role of PKC, PKC-[delta], mito K+ATP, and ROS in sevoflurane-related intracellular signaling.

Results: Preconditioning of trabeculae with sevoflurane preserved contractile function after ischemia. This contractile preservation was dependent on PKC-[delta] activation, mito K+ATP channel opening, and ROS production. In addition, on acute stimulation by sevoflurane, PKC-[delta] but not PKC-[epsilon] translocated to the sarcolemmal membrane. This translocation was inhibited by PKC inhibitors and ROS scavenging but not by inhibition of mito K+ATP channels. Furthermore, sevoflurane directly induced nitrosylation of sarcolemmal proteins, suggesting the formation of peroxynitrite.     

Nuclear Factor κB and Anesthetic Preconditioning during Myocardial Ischemia-Reperfusion

Background: Volatile anesthetic preconditioning (APC) protects against myocardial ischemia-reperfusion (IR) injury, but the precise mechanisms underlying this phenomenon remain undefined. To investigate the molecular mechanism of APC in myocardial protection, the activation of nuclear factor (NF) [kappa]B and its regulated inflammatory mediators expression were examined in the current study.

Methods: Hearts from male rats were isolated, Langendorff perfused, and randomly assigned to one of three groups: (1) the control group: hearts were continuously perfused for 130 min; (2) the IR group: 30 min of equilibration, 15 min of baseline, 25 min of ischemia, 60 min of reperfusion; and (3) the APC + IR group: 30 min of equilibration, 10 min of sevoflurane exposure and a 5-min washout, 25 min of global ischemia, 60 min of reperfusion. Tissue samples were acquired at the end of reperfusion. NF-[kappa]B activity was determined by electrophoretic mobility shift assay. The NF-[kappa]B inhibitor, I[kappa]B-[alpha], was determined by Western blot analysis. Myocardial inflammatory mediators, including tumor necrosis factor [alpha], interleukin 1, intercellular adhesion molecule 1, and inducible nitric oxide synthase, were also assessed by Western blot analysis.

Results: Nuclear factor [kappa]B-DNA binding activity was significantly increased at the end of reperfusion in rat myocardium, and cytosolic I[kappa]B-[alpha] was decreased. Supershift assay revealed the involvement of NF-[kappa]B p65 and p50 subunits. APC with sevoflurane attenuated NF-[kappa]B activation and reduced the expression of tumor necrosis factor [alpha], interleukin 1, intercellular adhesion molecule 1, and inducible nitric oxide synthase. APC also reduced infarct size and creatine kinase release and improved myocardial left ventricular developed pressure during IR.     

Translocation of Protein Kinase C Isoforms to Subcellular Targets in Ischemic and Anesthetic Preconditioning

Background: Translocation of protein kinase C (PKC) to subcellular targets is a pivotal signaling step in ischemic preconditioning (IPC). However, to date, it is unknown whether PKC isoforms translocate in anesthetic preconditioning (APC).

Methods: The PKC blockers chelerythrine and rottlerin and the adenosine triphosphate-dependent potassium (KATP) channel blockers HMR-1098 and 5-hydroxydecanoate were used to assess the role of PKC and KATP channels in isolated perfused rat hearts subjected to IPC or APC (1.5 minimum alveolar concentration isoflurane) followed by 40 min of ischemia and 30 min of reperfusion. Immunohistochemical techniques were used to visualize PKC translocation after preconditioning. In addition, the phosphorylation status of PKC isoforms was assessed.

Results: Chelerythrine, rottlerin, and 5-hydroxydecanoate blocked IPC and APC with respect to functional recovery, albeit IPC at higher concentrations. HMR-1098 did not affect IPC or APC. PKC[delta] and PKC[epsilon] translocated to nuclei in both IPC and APC, which was inhibited by chelerythrine and rottlerin. PKC[delta] translocated to mitochondria but not to the sarcolemma, and PKC[epsilon] translocated to the sarcolemma and intercalated disks but not to mitochondria. Interestingly, PKC[epsilon] was accumulated at the intercalated disks in control and preconditioned hearts. Phosphorylation of PKC[delta] on serine643 was increased in IPC and APC and blocked by chelerythrine and rottlerin, whereas phosphorylation of PKC[delta] on threonine505 was increased only in IPC and not blocked by chelerythrine or rottlerin. PKC[epsilon] on serine729 did not change its phosphorylation status.     

Protein Kinase C Translocation and Src Protein Tyrosine Kinase Activation Mediate Isoflurane-induced Preconditioning In Vivo: Potential Downstream Targets of Mitochondrial Adenosine Triphosphate-sensitive Potassium Channels and Reactive Oxygen Species

Background: The authors tested the hypotheses that protein kinase C (PKC)-specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo.

Methods: Rats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-[epsilon]V1-2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate-sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats.

Results: Isoflurane significantly (P < 0.05) reduced infarct size (40 +/- 3% [n = 13]) as compared with control experiments (58 +/- 2% [n = 12]). Chelerythrine, rottlerin, PKC-[epsilon]V1-2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 +/- 2% [n = 8], 50 +/- 3% [n = 9], 53 +/- 2% [n = 9], 59 +/- 3% [n = 6], 57 +/- 3% [n = 7], 60 +/- 3% [n = 7], and 53 +/- 3% [n = 6], respectively). Isoflurane stimulated translocation of the [delta] and [epsilon] isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively.     

The Common Inhalation Anesthetic Isoflurane Induces Apoptosis and Increases Amyloid β Protein Levels

Background: The common inhalation anesthetic isoflurane has previously been reported to enhance the aggregation and cytotoxicity of the Alzheimer disease-associated amyloid [beta] protein (A[beta]), the principal peptide component of cerebral [beta]-amyloid deposits.

Methods: H4 human neuroglioma cells stably transfected to express human full-length wild-type amyloid precursor protein (APP) were exposed to 2% isoflurane for 6 h. The cells and conditioned media were harvested at the end of the treatment. Caspase-3 activation, processing of APP, cell viability, and A[beta] levels were measured with quantitative Western blotting, cell viability kit, and enzyme-linked immunosorbent assay sandwich. The control condition consisted of 5% CO2 plus 21% O2 and balanced nitrogen, which did not affect caspase-3 activation, cell viability, APP processing, or A[beta] generation.

Results: Two percent isoflurane caused apoptosis, altered processing of APP, and increased production of A[beta] in H4 human neuroglioma cell lines. Isoflurane-induced apoptosis was independent of changes in A[beta] and APP holoprotein levels. However, isoflurane-induced apoptosis was potentiated by increased levels of APP C-terminal fragments.     

Anesthetic Preconditioning Improves Adenosine Triphosphate Synthesis and Reduces Reactive Oxygen Species Formation in Mitochondria after Ischemia by a Redox Dependent Mechanism

Background: Mitochondrial changes that characterize the heart after anesthetic preconditioning (APC) or the mechanisms by which mitochondrial triggering factors lead to protection are unknown. This study hypothesized that generation of reactive oxygen species (ROS) during APC is required to initiate the mitochondrial protective effects, and that APC leads to improved mitochondrial electron transport chain function and cardiac function during reperfusion.

Methods: Isolated guinea pig hearts were subject to 30 min ischemia and 120 min reperfusion. Prior to ischemia hearts were either untreated (I/R), or treated with sevoflurane (APC), in the presence or absence of the ROS scavenger tiron (TIR), or the superoxide dismutase mimetic MnTBAP (TBAP). Intracellular ROS were measured by spectrofluorometry using the fluorescent probe dihydroethidium (DHE). In another series of experiments, using the same protocol, hearts were reperfused for only 5 min and removed for measurement of adenosine triphosphate (ATP) synthesis by luciferin-luciferase luminometry and ROS generation by dichlorohydro-fluorescein (DCF) fluorescence in isolated mitochondria.

Results: The APC improved cardiac function and reduced infarction. Tiron or MnTBAP abrogated the protection afforded by APC. Mitochondrial ATP synthesis was decreased by 70 +/- 3% after IR alone, by only 7 +/- 3% after APC, by 69 +/- 2% after APC+TIR, and by 71 +/- 3% after APC + TBAP. Mitochondrial ROS formation (DCF) increased by 48 +/- 3% after IR alone, by 0 +/- 2% after APC, by 43 +/- 4% after APC + TIR, and by 46 +/- 3% after APC + TBAP. ROS generation (DHE) was increased in I/R group at 5 and 120 min reperfusion. This was attenuated by APC but this protective effect was abrogated in APC + TIR and APC + TBAP groups.     

蛋白激酶C在未成熟心肌预处理中的作用

目的为进一步加强未成熟心肌保护和临床应用的可行性,探讨蛋白激酶C(PKC)在未成熟心肌预处理保护中的作用。方法建立兔Langendorff灌注模型,将24只幼兔随机分为4组:缺血再灌注组(I/R组)、心脏缺血预处理组(MIP组)、PKC阻滞组(CLT组)和PKC激活剂组(PKC组),观察4组幼兔血流动力学、生化、心肌超微结构等指标。结果 MIP组和PKC组心功能恢复、心肌含水量优于I/R组和CLT组(P0.01),三磷酸腺苷(ATP)含量、超氧化物歧化酶活性、心肌线粒体Ca2+-ATPase活性、心肌线粒体合成ATP的能力优于I/R组和CLT组(P0.01),丙二醛含量、血清肌酸激酶和乳酸脱氢酶漏出率、心肌细胞内Ca2+含量、心肌线粒体Ca2+含量低于I/R组和CLT组(P0.01),心肌超微结构损伤较I/R组和CLT组明显减轻。结论心肌缺血预处理对未成熟心肌具有明显的保护作用,其机制可能是通过PKC的激活起作用。     

Protein Kinase C-ε Primes the Cardiac Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channel to Modulation by Isoflurane

Background: Cardioprotection by volatile anesthetic-induced preconditioning is known to involve intracellular signaling pathways. Recent studies have shown that protein kinase C (PKC) plays an important role in anesthetic-induced preconditioning. In this study, the effects of the activation of specific isozymes of PKC, specifically PKC-[epsilon] and -[delta], on the modulation of the sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channel by isoflurane were investigated.

Methods: The sarcKATP current was measured in ventricular myocytes isolated from guinea pig hearts using the whole cell configuration of the patch clamp technique. Peptides that induced the translocation of specific PKC isozymes were used to activate PKC-[epsilon] and PKC-[delta].

Results: Under whole cell conditions, isoflurane alone was unable to elicit the opening of the sarcKATP channel. Pretreatment with the specific PKC-[epsilon] activator, PP106, primed the sarcKATP channel to open in the presence of isoflurane. The resulting sarcKATP current densities in the presence of 0.88 mm isoflurane were 6.5 +/- 6.0 pA/pF (n = 7) and 40.4 +/- 18.2 pA/pF (n = 7) after pretreatment with 100 and 200 nm PP106, respectively. The PKC-[epsilon] antagonist PP93 abolished this effect. A scrambled peptide of the PKC-[epsilon] activator PP105 did not prime the sarcKATP channel. The PKC-[delta] activator PP114 was significantly less effective in priming the sarcKATP channel. 5-Hydroxydecanoate significantly attenuated the effect of the PKC-[epsilon] activator on the sarcKATP channel. In addition, immunohistochemical analysis showed that the PKC-[epsilon] isoform translocated to both the mitochondria and sarcolemma after anesthetic-induced preconditioning, whereas the PKC-[delta] isoform translocated to the mitochondria.     

Acetylcholine Activates Protein Kinase C-α in Pulmonary Venous Smooth Muscle

Background: The authors investigated whether acetylcholine-induced contraction in pulmonary venous smooth muscle (PVSM) is associated with the activation of specific protein kinase C (PKC) isoforms.

Methods: Isolated canine pulmonary venous rings without endothelium were suspended in modified Krebs-Ringer's buffer for measurement of isometric tension. The effects of nonspecific PKC inhibition (bisindolylmaleimide I; 3 x 10-6 m) and conventional PKC isoform inhibition (Go7936 10-6 m) on the acetylcholine dose-response relation were assessed. The expression of conventional PKC isoforms ([alpha], [beta], [gamma]), novel PKC isoforms ([delta], [varepsilon], [theta]), and atypical PKC isoforms ([zeta], [iota], [mu]) was measured in PVSM cells by Western blot analysis. The immunofluorescence technique and confocal microscopy were used to localize the cellular distribution of PKC isoforms before and after the addition of acetylcholine.

Results: Acetylcholine caused dose-dependent contraction in E-pulmonary veins. Pretreatment with bisindolylmaleimide I or Go7936 attenuated acetylcholine contraction. PKC-[alpha], -[iota], -[mu], and -[zeta] were expressed, whereas PKC-[beta], -[gamma], -[delta], -[varepsilon], and -[theta] were not expressed in PVSM cells. Immunofluorescence staining for PKC isoforms showed that in unstimulated cells, PKC-[alpha] and PKC-[mu] were detected only in the cytoplasm. PKC-[iota] and PKC-[zeta] also exhibited a cytoplasmic immunofluorescence pattern, which was especially abundant in the perinuclear zone. Activation with acetylcholine induced translocation of PKC-[alpha] from cytoplasm to membrane, whereas acetylcholine had no effect on the other PKC isoforms. Translocation of PKC-[alpha] in response to acetylcholine was blocked by the muscarinic receptor antagonist, atropine.     

Intrathecally Administered cGMP-dependent Protein Kinase Iα Inhibitor Significantly Reduced the Threshold for Isoflurane Anesthesia : Implication for a Novel Role of cGMP-dependent Protein Kinase Iα

Background: Inhalational anesthetics have been shown to inhibit the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Previous studies indicated that inhibition of the NO-cGMP pathway decreased the level of consciousness and augmented anesthesia, analgesia, or sedation. The current study investigated the possible involvement of cGMP-dependent protein kinases (PKGs) as major effectors for the NO-cGMP pathway in the anesthetic state.

Methods: After initial baseline determination of the minimum alveolar concentration (MAC), a selective cGMP-dependent protein kinase I[alpha] inhibitor, Rp-8-p-CPT-cGMPS, or an NO donor, (NOC-12), were injected intrathecally. Ten minutes later, MAC measurement was repeated. The rats also were evaluated for the presence of locomotor dysfunction by intrathecal administration of Rp-8-p-CPT-cGMPS and NOC-12 in conscious rats.

Results: Rp-8-p-CPT-cGMPS at 25, 50, 100, and 200 [mu]g/10 [mu]l produced a significant decrease from isoflurane control MAC of -4 +/- 3.1%, 16 +/- 4.5%, 30 +/- 5.0%, and 21 +/- 2.2%, respectively, which was not accompanied by significant changes in either blood pressure or heart rate. In contrast, NOC-12 at 100 [mu]g/10 [mu]l caused an increase from isoflurane control MAC of 23 +/- 5.8%, which was accompanied by significant decrease in blood pressure but not in heart rate. Rp-8-p-CPT-cGMPS (100 [mu]g/10 [mu]l) produced a significant reversal of isoflurane MAC increase induced by NOC-12 (100 [mu]g/10 [mu]l), which was accompanied by significant reversal of the reduction of blood pressure induced by NOC-12. Locomotor activity was not changed.     

Preconditioning by Isoflurane Induces Lasting Sensitization of the Cardiac Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channel by a Protein Kinase C-δ-mediated Mechanism

Background: Cardioprotective effects of volatile anesthetics in anesthetic-induced preconditioning involve activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels. This study addressed the memory phase of anesthetic preconditioning by investigating whether brief exposure to isoflurane produces lasting sensitization of the sarcKATP channel and whether protein kinase C mediates this effect.

Methods: Whole cell sarcKATP channel current (IKATP) was monitored from single isolated rat ventricular cardiomyocytes. Pinacidil was used to open the channel, and the magnitude of activated IKATP was an indicator of channel's ability to open. Involvement of protein kinase C was investigated using chelerythrine and isoform-specific peptide inhibitors and activators of protein kinase C-[delta] and protein kinase C-[varepsilon].

Results: The mean density of IKATP elicited by pinacidil (5 [mu]m) in anesthetic-free conditions was 3.8 +/- 3.7 pA/pF (n = 11). After 10 min of exposure to isoflurane (0.56 mm) and 10 or 30 min of anesthetic washout, pinacidil-elicited IKATP was increased to 15.6 +/- 11.3 pA/pF (n = 12; P < 0.05) and 11.8 +/- 3.9 pA/pF (n = 6; P < 0.05), respectively. In the presence of chelerythrine (5 [mu]m), isoflurane did not potentiate channel opening, and IKATP was 6.6 +/- 4.6 pA/pF (n = 11). Application of protein kinase C-[delta] peptide inhibitor also abolished isoflurane-induced sensitization of sarcKATP channel, and IKATP was 7.7 +/- 5.4 pA/pF (n = 12). In contrast, protein kinase C-[varepsilon] peptide inhibitor did not affect channel sensitization, and pinacidil-elicited current was 14.8 +/- 9.6 pA/pF (n = 12). Interestingly, when both protein kinase C-[delta] and protein kinase C-[varepsilon] activators were applied instead of isoflurane, they sensitized the channel to the same extent as isoflurane (18.9 +/- 7.2 pA/pF, n = 11, and 18.6 +/- 11.1 pA/pF, n = 10, respectively).     

Mechanism of Preconditioning by Isoflurane in Rabbits: A Direct Role for Reactive Oxygen Species

Background: Reactive oxygen species (ROS) contribute to myocardial protection during ischemic preconditioning, but the role of the ROS in protection against ischemic injury produced by volatile anesthetics has only recently been explored. We tested the hypothesis that ROS mediate isoflurane-induced preconditioning in vivo.

Methods: Pentobarbital-anesthetized rabbits were instrumented for measurement of hemodynamics and were subjected to a 30 min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline), or the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptopropionyl glycine (2-MPG; 1 mg [middle dot] kg-1[middle dot] min-1), in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. Isoflurane was administered for 30 min and then discontinued 15 min before coronary artery occlusion. A fluorescent probe for superoxide anion production (dihydroethidium, 2 mg) was administered in the absence of the volatile anesthetic or 5 min before exposure to isoflurane in 2 additional groups (n = 8). Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively.

Results: Isoflurane (P < 0.05) decreased infarct size to 24 +/- 4% (mean +/- SEM; n = 10) of the left ventricular area at risk compared with control experiments (43 +/- 3%; n = 8). NAC (43 +/- 3%; n = 7) and 2-MPG (42 +/- 5%; n = 8) abolished this beneficial effect, but had no effect on myocardial infarct size (47 +/- 3%; n = 8 and 46 +/- 3; n = 7, respectively) when administered alone. Isoflurane increased superoxide anion production as compared with control experiments (28 +/- 12 vs. -6 +/- 9 fluorescence units;P < 0.05).     

Mycophenolic Acid Inhibits Platelet-Derived Growth Factor-Induced Reactive Oxygen Species and Mitogen-Activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells

Vascular smooth muscle cell (VSMC) proliferation is the major pathologic feature associated with chronic allograft nephropathy, and mycophenolic acid (MPA) inhibits VSMC proliferation. Since the role of inosine monophosphate dehydrogenase (IMPDH)-dependent de novo guanosine synthesis is limited in VSMCs, we examined the effects of MPA on platelet-derived growth factor (PDGF)-induced cellular ROS and mitogen-activated protein kinases (MAPK) activation in VSMCs. Primary cultured rat VSMCs were stimulated with PDGF-BB in the presence or absence of MPA. Cell proliferation was assessed by [3H]-thymidine incorporation, ROS by flow cytometry and MAPK activation by Western blot analysis. PDGF increased cell proliferation, cellular ROS and extracellular-regulated protein kinase (ERK) 1/2 and p38 MAPK activation by 3.4-, 1.6-, 3.3- and 3.9-fold, respectively. MPA at above 1 muM inhibited PDGF-induced cellular ROS and ERK 1/2 and p38 MAPK activation, as well as proliferation. Structurally different anti-oxidants and inhibitor of ERK or p38 MAPK blocked PDGF-induced proliferation. Anti-oxidants also inhibited ERK 1/2 and p38 MAPK activation. Exogenous guanosine partially recovered the inhibitory effect of MPA on VSMC proliferation. These results suggest that MPA may inhibit PDGF-induced VSMC proliferation partially through inhibiting cellular ROS, and subsequent ERK 1/2 and p38 MAPK activation in addition to inhibiting IMPDH.     

Interaction of Protamine with α- and β-Adrenoceptor Stimulations in Rat Myocardium

Background : Protamine alters the inotropic responses to [beta]-adrenoceptor stimulation, but its mechanism of action is not well-understood. Moreover, its interaction with [alpha]-adrenoceptor stimulation and the lusitropic (relaxation) response to [beta]-adrenoceptor stimulation remain unknown.

Methods : The effects of protamine (10 or 100 [mu]g/ml) on the responses induced by phenylephrine and isoproterenol were studied in rat left ventricular papillary muscles. Inotropic and lusitropic effects were studied under low and high loads. The authors also studied the interaction of protamine with forskolin (50 [mu]m) and dibutyryl 3',5'-cAMP (0.5 mm). Data are mean percentage of baseline active force +/- SD.

Results : In control groups, phenylephrine (135 +/- 17%, P < 0.05) and isoproterenol (185 +/- 44%, P < 0.05) induced a positive inotropic effect. Isoproterenol induced positive lusitropic effects under low and high loads. Protamine abolished the inotropic responses to [alpha]- (102 +/- 23%, not significant) and [beta]-adrenoceptor stimulations (99 +/- 17%, not significant) but did not modify the lusitropic responses to isoproterenol. Protamine abolished the inotropic responses to forskolin (89 +/- 6 vs. 154 +/- 20%, P < 0.05) and markedly decreased that of dibutyryl 3',5'-cAMP (132 +/- 31 vs. 167 +/- 30%, P < 0.05) but did not modify their lusitropic responses.     

The Actions of Sevoflurane and Desflurane on the γ-Aminobutyric Acid Receptor Type A: Effects of TM2 Mutations in the α and β Subunits

Background: Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the [gamma]-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the [alpha]1, [alpha]2, or [beta]2 subunits.

Methods: GABAA receptor [alpha]1 or [alpha]2, [beta]2 or [beta]3, and [gamma]2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type [alpha]1[beta]2[gamma]2s and [alpha]2[beta]3[gamma]2s receptors, and in receptors harboring mutations in TM2, such as [alpha]1(S270W)[beta]2[gamma]2s, [alpha]1[beta]2(N265W)[gamma]2s, and [alpha]2(S270I)[beta]3[gamma]2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors.

Results: Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA [alpha]1[beta]2[gamma]2s receptor and [alpha]2[beta]3[gamma]2s receptor. Substitution of Ser270 in TM2 of the [alpha] subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in [alpha]1(S270W)[beta]2[gamma]2s and [alpha]2(S270I)[beta]3[gamma]2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the [beta] subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane.     

Propofol Attenuates β-Adrenoreceptor-mediated Signal Transduction via a Protein Kinase C-dependent Pathway in Cardiomyocytes

Background: Activation of [beta] adrenoreceptors by catecholamines is an important mechanism for increasing the inotropic state of the heart. The objectives of the current study were to investigate the effects of propofol on [beta]-adrenoreceptor-mediated increases in cardiomyocyte intracellular Ca2+ concentration ([Ca2+]i), cell shortening, L-type Ca2+ current (ICa) and cyclic adenosine monophosphate (cAMP) accumulation. The authors also investigated the site of action of propofol in the [beta]-adrenoreceptor signaling pathway, as well as the role of protein kinase C (PKC), and tested the hypothesis that propofol would inhibit the [beta]-adrenoreceptor signaling pathway via a PKC-dependent mechanism.

Methods: Freshly isolated ventricular myocytes were obtained from adult rat and guinea pig hearts. Myocyte shortening (video edge detection) and [Ca2+]i (fura-2, 340/380 ratio) were monitored simultaneously in individual cells. Conventional whole cell patch clamp analysis was used to measure the ICa in individual myocytes. cAMP production was assessed in suspensions of myocytes using an enzyme immunoassay kit.

Results: Propofol (0.1-10 [mu]m) had no effect on steady state [Ca2+]i, cell shortening, ICa, or cAMP production. In contrast, propofol caused dose-dependent decreases in isoproterenol-stimulated increases in [Ca2+]i, shortening, ICa, and cAMP. Forskolin-induced increases in [Ca2+]i, shortening, and cAMP production were not altered by propofol. PKC activation with phorbol myristate acetate attenuated isoproterenol-stimulated cAMP production. Inhibition of PKC with bisindolylmaleimide (broad range inhibitor) or Go 6976 (inhibitor of Ca2+-dependent PKC isoforms) abolished propofol-induced inhibition of isoproterenol-stimulated increases in [Ca2+]i, shortening, and cAMP production.     

Impairment of Cardiac β-Adrenoceptor Cellular Signaling by Decreased Expression of Gsα in Septic Rabbits

Background: Abnormalities in the [beta]-adrenergic control of cardiac function play a role in the pathogenesis of several disease states. Because circulatory failure in patients with septic shock is known to be less responsive to catecholamines, we investigated whether the [beta]-adrenoceptor-linked signal transduction mechanisms are altered in the heart of a septic animal model.

Methods: Rabbits were rendered endotoxemic by an intravenous injection of 100 [mu]g/kg Escherichia coli lipopolysaccharide. Three and 6 h later, the myocardial tissues were used for the experiments.

Results: The positive inotropic response to isoproterenol was significantly impaired in papillary muscles isolated from septic rabbits compared with those from controls. The impaired inotropic responsiveness to isoproterenol was not prevented by the nitric oxide synthase inhibitor NG-nitro-l-arginine, indicating no involvement of nitric oxide overproduction. Adenylate cyclase activity stimulated with isoproterenol and 5'-guanylyl imidodiphosphate was markedly reduced in septic myocardium. The contractile and adenylate cyclase responses to colforsin daropate, a direct adenylate cyclase activator, were unaffected by sepsis. Radioligand binding experiments with (-)[125I]iodocyanopindolol revealed no significant alteration in myocardial [beta]-adrenoceptor density or affinity in sepsis. Determination of cardiac Gs[alpha] level by Western blotting showed a reduction of approximately 50% in sepsis. The relative content of Gs[alpha] messenger RNA in septic myocardium also was reduced from the control level by about 50%, as determined by Northern blot analysis. Little change was found in protein and messenger RNA levels of Gi[alpha] in septic myocardium.     

桃仁对糖尿病大血管纤维化大鼠蛋白激酶B信号通路的影响

目的:观察中药桃仁对2型糖尿病大血管纤维化大鼠模型中蛋白激酶B(AKT)信号通路的影响。方法:雄性SD大鼠200只,随机选取30只为空白对照组,余170只采用高脂高糖饲料喂养-腹腔注射链脲佐菌素(STZ)-高脂高糖饲料持续喂养的方法制备2型糖尿病大血管纤维化大鼠模型。造模成功后分为空白对照组(n=29)、模型对照组(n=34)、早期干预组(n=34)、低剂量组(n=32)和高剂量组(n=32)。空白对照组不予特殊处理,早期干预组和低剂量组予桃仁颗粒剂水溶液10 mL/(kg·d)灌胃,高剂量组予桃仁颗粒剂水溶液20 mL/(kg·d)灌胃,模型对照组给予10 mL/(kg·d)生理盐水灌胃。干预7周后分别从各组随机选取5只大鼠处死、取材。实时荧光定量PCR(QPCR)检测AKT mRNA表达;免疫组织化学、Western blotting检测股动脉组织AKT信号通路关键信号分子AKT、磷酸化AKT(p-AKT)的表达。结果:免疫组化显示,空白对照组大鼠血管内皮细胞及中膜平滑肌细胞中有散在黄色物质,呈弱阳性改变,模型对照组呈强阳性反应,早期干预组和高剂量组呈弱阳性反应,低剂量组呈阳性反应。QPCR检测,与空白对照组(1.05±0.05)比较,模型对照组(5.68±0.61)、药物干预组(4.27±0.32、5.33±0.60、4.72±0.28)AKTmRNA表达上调(P0.05或P0.01);与模型对照组比较,药物干预组AKT mRNA表达均上调,且以早期干预组(4.27±0.32)和高剂量组(4.72±0.28)为著(P0.01);Western blotting检测,与空白对照组(0.16±0.01、0.10±0.03)比较,模型对照组(0.38±0.03、0.21±0.02)、药物干预组(0.27±0.04、0.18±0.01;0.30±0.05、0.17±0.01;0.28±0.03、0.19±0.02)AKT、p-AKT表达均显著上调(P0.01);与模型对照组比较,药物干预组AKT、p-AKT表达均上调(P0.01或P0.05);药物干预组两两比较显示,AKT、p-AKT表达无差异(P0.05)。结论:中药桃仁可以抑制糖尿病大鼠大血管纤维化,其机制可能和抑制AKT信号通路有关。