Mechanism of oleic acid-induced gap junctional disassembly in rat cardiomyocytes

This study investigated the mechanism of oleic acid (OA) on gap junctions and identified the protein kinase C (PKC) isoforms involved in OA-mediated gap junction disassembly in cardiomyocytes. Control cardiomyocytes showed continuous staining of the plasma membrane at cell-cell contact areas using antibodies reacting with connexin 43 (Cx43). The spontaneous contraction rate of cultured cardiomyocytes was reduced in a time-dependent manner by OA. In addition, Cx43 expression at cell-cell junction decreased, suggesting the disassembly of gap junction. Staining for PKC and PKCalpha, which were shown to colocalize with Cx43, also decreased with increased duration of OA treatment. The effects of OA on these distributional changes at cell junctions were reversed by 24 h incubation in fresh culture medium devoid of OA. Immunoprecipitation assays confirmed the biochemical binding between Cx43 and PKC/PKCalpha, and this protein interaction was not affected by OA. This may provide the basis for simultaneous detachment of Cx and PKC/PKCalpha from the cell-cell junction to the cytosol upon OA stimulation. Western blot analysis showed that OA-induced Cx43 Ser368 phosphorylation, and that this effect could be blocked by cotreatment with the general PKC inhibitor, calphostin C, the PKC inhibitor, eV1-2, or the Src kinase inhibitor, PP1, but not by the PKCalpha inhibitor, G?6976. eV1-2 also prevented the OA-induced disassembly of gap junctions. Taken together, these data suggest that OA-induced Cx43 Ser368 phosphorylation is mediated by activation of PKC and Src kinase and might be responsible for OA-induced gap junctional disassembly.     

条件培养液预处理对乳鼠心肌细胞缺氧复氧损伤的影响及其机理探讨

目的观察短暂缺氧条件培养液对乳鼠心肌细胞缺氧复氧损伤的影响，并探讨其作用机理。方法采用乳鼠心肌细胞缺氧／复氧（A／R）损伤模型。用短暂缺氧条件培养液进行预处理。结果条件培养液预处理能够提高A／R损伤后心肌细胞存活率（7638±4．12vsA／R组56．87±3．17％，P＜0．01），减少细胞MDA产生（0．91±0．05VSA／R组1．61±0．06nmol／mg·pr，P＜0．01），减少LDH漏出（35．12±845vsA／R组56．47±9．15U／L，P＜0．01）及细胞内蛋白漏出（0．32±0．04vsA／R组0．43±0．07，P＜0．01）。PKC抑制剂（H_7）完全阻断条件培养液预处理的上述保护作用。结论条件培养液对乳鼠心肌细胞的缺氧复氧损伤具有保护作用，其机理是通过PKC介导的。     

细胞外信号调节激酶在糖尿病大鼠肺病变中的作用

目的　观测糖尿病大鼠肺组织的病理改变及蛋白激酶C、胞外调节蛋白激酶活性的变化 ,探讨细胞信号传导系统在糖尿病大鼠肺病变中的作用。方法　制作糖尿病大鼠模型 ,4w后应用透射电镜观察大鼠肺组织病理改变 ,采用改良的Takay法测定蛋白激酶C活性 ,同位素法及蛋白质免疫印迹分析方法检测胞外调节蛋白激酶在糖尿病大鼠肺组织表达的变化。结果　糖尿病大鼠 4w肺组织病理改变为毛细血管基底膜及Ⅱ型肺泡上皮细胞基底膜不同程度增厚 ,肺间质胶原成份增多 ,蛋白激酶C、胞外调节蛋白激酶在肺组织活性增强。结论　链脲菌素糖尿病大鼠肺组织高糖环境下细胞内信号传导系统被激活 ,可能参与了糖尿病肺部并发症的发生和发展     

血管平滑肌细胞钙敏感性调控与休克血管低反应性发生的关系

血管平滑肌细胞钙敏感性调节足血管平滑肌舒缩调节的重要途径,Rho激酶、蛋白激酶C（PKC）和蛋白激酶G（PKG）是参与其中的关键分子。在休克等病理条件下,上述分子的活性发生变化,引起钙敏感性降低,从而导致了血管对缩血管物质的反应性降低,即血管的低反应性。     

Composite microparticles with in vivo reduction of the burst release effect

The aim of this study was to develop microparticles containing nanoparticles (composite microparticles) for prolonged drug delivery with reduced burst effect in vitro and in vivo. Such composite microparticles were prepared with hydrophobic and biodegradable polymers [poly(ε-caprolactone), poly(lactic-co-glycolic) acid]. Ibuprofen was chosen as the model drug, and microparticles were prepared by the extraction technique with ethyl acetate as the solvent. Nanoparticles and microparticles and an ibuprofen solution (Pedea^®) were administered subcutaneously at the dose of 1 mg of ibuprofen per kg to overnight-fasted rats (male Wistar). Composite microparticles showed prolonged ibuprofen release and less burst effect when compared to simple microparticles (without nanoparticles inside) or nanoparticles both in vitro (PBS buffer) and in vivo. Moreover, ibuprofen was still detected in the plasma after 96 h with composite microparticles. Consequently, it has been demonstrated that composite microparticles were able to reduce burst release and prolong the release of ibuprofen for a long period of time.     

Isoproterenol disperses distribution of NADPH oxidase,MMP-9, and pPKCε in the heart,which are mitigated by endothelin receptor antagonist CPU0213

Aim： Spatial dispersion of bioactive substances in the myocardium could serve as pathological basis for arrhythmogenesis and cardiac impairment by β-adrenoceptor stimulation. We hypothesized that dispersed NADPH oxidase, protein kinase Cε （PKCε）, early response gene （ERG）, and matrix metalloproteinase 9 （MMP-9） across the heart by isoproterenol （ISO） medication might be mediated by the endothelin （ET） - ROS pathway. We aimed to verify if ISO induced spatially heterogeneous distribution of pPKCε, NAPDH oxidase, MMP-9 and ERG could be mitigated by either an ET receptor antagonist CPU0213 or iNOS inhibitor aminoguanidine. Methods：Rats were treated with ISO （1 mg/kg sc） for 10 days, and drug interventions （mg/kg） either CPU0213 （30 sc） or aminoguanidine （100 ip） were administered on days 8-10. Expression of NADPH oxidase, MMP-9, ERG, and PKCε in the left and right ventricle （LV, RV） and septum （S） were measured separately. Results： Ventricular hypertrophy was found in the LV, S, and RV, in association with dispersed QTc and oxidative stress in ISO-treated rats. mRNA and protein expression of MMP-9, PKCε, NADPH oxidase and ERG in the LV, S, and RV were obviously dispersed, with augmented expression mainly in the LV and S. Dispersed parameters were re-harmonized by either CPU0213, or aminoguanidine. Conclusion： We found at the first time that ISO-induced dispersed distribution of pPKCε, NADPH oxidase, MMP-9, and ERG in the LV, S, and RV of the heart, which were suppressed by either CPU0213 or aminoguanidine. It indicates that the ET-ROS pathway plays a role in the dispersed distribution of bioactive substances following sustained β-receptor stimulation.     

Ammonia metabolism,the brain and fatigue; revisiting the link

This review addresses the ammonia fatigue theory in light of new evidence from exercise and disease studies and aims to provide a view of the role of ammonia during exercise. Hyperammonemia is a condition common to pathological liver disorders and intense or exhausting exercise. In pathology, hyperammonemia is linked to impairment of normal brain function and the onset of the neurological condition, hepatic encephalopathy. Elevated blood ammonia concentrations arise due to a diminished capacity for removal via the liver and lead to increased exposure of organs, such as the brain, to the toxic effects of ammonia. High levels of brain ammonia can lead to deleterious alterations in astrocyte morphology, cerebral energy metabolism and neurotransmission, which may in turn impact on the functioning of important signalling pathways within the neuron. Such changes are believed to contribute to the disturbances in neuropsychological function, in particular the learning, memory, and motor control deficits observed in animal models of liver disease and also patients with cirrhosis. Hyperammonemia in exercise occurs as a result of an increased production by contracting muscle, through adenosine monophosphate (AMP) deamination (the purine nucleotide cycle) and branched chain amino acid (BCAA) deamination prior to oxidation. Plasma concentrations of ammonia during exercise often achieve or exceed those measured in liver disease patients, resulting in increased cerebral uptake. In this article we propose that exercise-induced hyperammonemia may lead to concomitant disturbances in brain function, potentially through similar mechanisms underpinning pathology, which may impact on performance as fatigue or reduced function, especially during extreme exercise.     

Pharmacologic therapy for diabetic retinopathy

Diabetic retinopathy remains one of the major causes of acquired blindness in developed nations. This is true despite the development of laser treatment, which can prevent blindness in the majority of those who develop macular oedema (ME) or proliferative diabetic retinopathy (PDR). ME is manifest by retinal vascular leakage and thickening of the retina. The hallmark of PDR is neovascularisation (NV) – abnormal angiogenesis that may ultimately cause severe vitreous cavity bleeding and/or retinal detachment. Pharmacologic therapy aimed specifically at preventing vascular leakage and NV would be a welcome addition to the armamentarium. PDR and ME could be prevented by improved metabolic control or by pharmacologically blunting the biochemical consequences of hyperglycaemia (e.g., with aldose reductase inhibitors, inhibitors of non-enzymatic glycation or by protein kinase C [PKC] inhibition). The angiogenesis in PDR could be treated via growth factor (e.g., vascular endothelial growth factor [VEGF], insulin like growth factor-1 [IGF-1]) blockade, integrin (e.g., alpha-v beta-3) blockade, extracellular matrix alteration (e.g., with steroid compounds) or interference with intracellular signal transduction pathways (e.g., PKC and mitogen activated protein kinase [MAPK] pathway proteins). Some of these antiangiogenic agents may also prove useful for treating or preventing ME. Numerous potentially useful antiangiogenic compounds are in development; two drugs are presently in clinical trials for treatment of the preproliferative stage of PDR, while two are in clinical trials for treatment of ME.     

Mice deficient in PKC theta demonstrate impaired in vivo T cell activation and protection from T cell-mediated inflammatory diseases

In the present study we have characterized T cell-driven immune function in mice that are genetically deficient in PKC theta. In response to simple immunologic stimulation invoked by in vivo T cell receptor (TCR) cross-linking, these mice showed significantly depressed plasma cytokine levels for IL-2, IL-4, IFNγ, and TNFα compared to wild-type (WT) mice. In parallel, spleen mRNA levels for these cytokines were reduced, and NF-κB activation was also reduced in PKC theta knockouts (KO). Injection of allogeneic cells into the footpad of PKC theta deficient mice provoked a significantly diminished local T cell response compared to WT mice similarly challenged. Unlike comparable cells from wild type mice, CD45RBhi T cells harvested from PKC theta deficient mice failed to induce colitis in the SCID-CD45RB cell transfer model of IBD. In another T cell-dependent model of inflammatory disease, PKC theta deficient animals developed far less severe neurologic signs and reduced spinal cord inflammatory cell infiltrate compared to WT controls in the MOG-induced EAE model. A fundamental role for PKC theta in T cell activation and in the development of T cell-mediated inflammatory diseases is indicated by these results.