Ca2+-dependent modulation of single human cardiac L-type calcium channels by the calcineurin inhibitor cyclosporine

OBJECTIVE: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. METHODS: Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. RESULTS: A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. CONCLUSIONS: We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.     

Dephosphorylation of WR-2721 with mouse tissue homogenates

Mouse liver homogenate had an optimum pH of 8.6 to 8.8 for dephosphorylation of WR-2721 in the analyzed pH range from 5.2 to 10.0. At this optimum pH condition, the dephosphorylation activities of six mouse tissue homogenates were analyzed. Kidney, liver and small intestine homogenates showed higher dephosphorylation activities (935, 336 and 314 nmoles/mg protein/hr, respectively) than spleen and lung homogenates (86, 49 nmoles/mg protein/hr, respectively). Furthermore, serum did not show any dephosphorylation activity. The high activity found in liver homogenate agrees well with our previous data with mouse L cells. However, optimum pH from 8.6 to 8.8 in liver homogenate is quite different from the data reported by using Ehrlich ascites tumor cells (optimum pH was 5.6). Therefore, it is suggested that WR-2721 administered into mouse is efficiently dephosphorylated in certain tissues such as liver to its active form with the enzyme(s) different from that found in Ehrlich ascites tumor cells.     

Src is dephosphorylated at tyrosine 530 in human colon carcinomas

Objective:Src is a protein tyrosine kinase that plays important roles in cancer development,and Src kinase activity has been found to be elevated in several types of cancers.However,the cause of the elevation of Src kinase activity in the majority of human colon carcinomas is still largely unknown.We aim at finding the cause of elevated Src kinase activity in human colon carcinomas.Methods:We employed normal colon epithelial FHC cells and examined Src activation in human colon carcinoma specimens from 8 patients.Protein expression levels were determined by Western blotting,and the activity of Src kinase by kinase assay.Results:Actin levels were different between tumor and normal tissues,demonstrating the complexities and inhomogeneities of the tissue samples.Src kinase activities were increased in the majority of the colon carcinomas as compared with normal colon epithelial cells (range 13-29).Src protein levels were reduced in the colon carcinomas.Src Y530 phosphorylation levels were reduced to a higher extent than protein levels in the carcinomas.Conclusion:The results suggest that Src specific activities were highly increased in human colon carcinomas;phosphorylation at Src Y530 was reduced,contributing to the highly elevated Src specific activity and Src kinase activity.     

Immunochemical characterization of a monoclonal antibody specific for Alzheimer''s disease associated protein

In this study, the monoclonal antibody PHF-1 which recognizes epitopes unique to Alzheimer's disease associated proteins (ADAP) has been characterized. Crossed affinity immunoelectrophoresis was used to estimate the binding constant for the interaction of PHF-1 with ADAP and to estimate the fraction of PHF-1 reactive protein. The binding constant of PHF-1 was determined to be 1.3 x 10(-8) M. Furthermore, the effect of dephosphorylation on the electrophoretic pattern of the PHF-1 reactive protein and the ensuing changes in its immunoreactivity were demonstrated.     

siRNA介导的E2F-1基因沉默对胃癌细胞MGC803中Rb蛋白表达水平的影响

目的 应用RNA干扰技术研究E2F-1基因沉默在人胃癌MGC803细胞中对Rb蛋白表达的影响.方法 将重组质粒E2F-1-siRNA转染MGC803细胞,筛选稳定株,利用RT-PCR检测转染前后细胞E2F-1 mRNA表达水平,并通过蛋白免疫印记法（Western blot）检测各组细胞E2F-1蛋白的表达水平来验证质粒转入胃癌细胞的情况.采用Western blot检测三组细胞中总Rb蛋白和磷酸化Rb蛋白的表达情况,计算出非磷酸化Rb蛋白表达情况,统计各组间的差异.结果 重组质粒E2F-1-siRNA成功转入胃癌细胞中;有效抑制了E2F-1 mRNA的表达,E2F-1蛋白水平显著下降,与阴性对照组及未转染组相比分别降低了83.2%和84.6%,去磷酸化Rb蛋白分别增加了61.22%（P〈0.05）和66.60%（P〈0.05）,差异有统计学意义.结论 沉默表达E2F-1基因后,可以有效降低Rb蛋白的水平,促进其活化形式去磷酸化Rb蛋白的产生.     

Dephosphorylation of JC virus agnoprotein by protein phosphatase 2A: inhibition by small t antigen

Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein-protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82-124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of Sm t-Ag with respect to the regulation of its phosphorylation. Collectively, these results suggest that there is an interplay between agnoprotein, Sm t-Ag and PP2A with respect to the regulation of JCV life cycle and this could be important for the progression of the JCV-induced disease, PML.     

酪氨酸的磷酸化及去磷酸化对心脏钾离子通道的调控

心脏钾离子通道是人体内广泛存在的、种类最多、作用最复杂的一类离子通道,并在人类心肌细胞动作电位的各个时程中发挥着重要的作用。近年来由于新兴技术如膜片钳技术、基因突变技术、分子克隆技术等诸多技术的运用和发展,人们对钾离子通道的基因表达、分子结构、生理病理作用及调控机制等方面都有了很深的认识,然而对蛋白磷酸化与去磷酸化对心脏钾离子通道的调控尤其是酪氨酸磷酸化和去磷酸化调控方面的研究却比较少,为此做一简单综述。     

Cyclosporin A, an inhibitor of calcineurin, impairs memory formation in day-old chicks

Considerable evidence exists that changes in the phosphorylation state of neuronal proteins are correlated with learning and that inhibition of various protein kinases disrupts memory formation. Given the reversible nature of protein phosphorylation, a role for protein phosphatases in memory processing also seems likely. It has been shown recently that administration of the phosphatase inhibitor, okadaic acid, disrupts memory formation in day-old chicks, with retention deficits first appearing at approximately 40 min post-training [93]. In the present study the intracranial administration of the immunosuppressant cyclosporin A was also found to produce retention deficits in day-old chicks trained on a single-trial, passive-avoidance task, but the deficits were not significant until 85 min post-training. The difference could not be attributed to differences in the pharmacokinetics of the drugs. Since okadaic acid preferentially inhibits protein phosphatases 1 and 2A, while cyclosporin A is reported to inhibit only the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, it is possible that different phosphatases may be involved in distinct stages of memory formation, as has been reported previously for protein kinases. The possibility that cyclosporin A may, in addition, act through inhibition of cyclophilin's peptidyl-prolyl-cis/trans- isomerase activity is also canvassed.     

Dephosphorylation of myofibrillar proteins in actomyosin preparations and in isolated perfused rat hearts after β-agonist withdrawal

The in vitro dephosphorylation of myosin P light chains and troponin-I was studied in preparations of natural actomyosin isolated after isoproterenol treatment of perfused rat hearts. These preparations contained endogenous phosphatase(s) which on incubation removed incorporated ²²P from myosin P light chains more rapidly than from troponin-I. The in situ effects of isoproterenol pulse-treatment were also studied in perfused rat hearts, with respect to myofibrillar protein phosphorylation and the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ and Ca²⁺-ATPase activities of natural actomyosin. Abrupt removal of the β-adrenergic stimulus from perfused rat hearts caused the (incomplete) dephosphorylation of both myosin P light chains and troponin-I; the former was dephosphorylated in apparent synchrony with the slowing of the beating rates and falling cyclic AMP contents (definitely decreased after 1 min), whereas troponin-I dephosphorylation only began after 2 min and was correlated with changes in force development. The dephosphorylation of P light chains was accompanied by a decrease in the V_max |ATP| of the myosin Ca²⁺-ATPase activities, but the latter reached pre-stimulation values before basal phosphorylation levels of the protein were attained. An increase in the V_max |Ca²⁺| of $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ activities and an increase in their sensitivity to Ca²⁺ was correlatable with the removal of phosphate from troponin-I. The results suggest that the phosphorylations of troponin-I and myosin light chains in adrenergically stimulated hearts serve separate functions in the context of positive inotropism.     

阿尔茨海默病患者脑中双螺旋丝的体外去磷酸化作用

目的探讨阿尔茨海默病（ＡＤ）脑损伤逆转的可能性及其途径。方法用去磷酸化和免疫印迹法研究ＡＤ脑损伤可逆性。结果蛋白磷酸酯酶（ＰＰ）２Ａ和ＰＰ２Ｂ可使ＡＤ神经原纤维缠结中的Ｉ型双螺旋丝（ＰＨＦＩｔａｕ）在Ｓｅｒ１９９／Ｓｅｒ２０２去磷酸化，Ｓｅｒ３９６／Ｓｅｒ４０４部分去磷酸化；此外，ＰＰ２Ａ和ＰＰ２Ｂ可分别使ＰＨＦＩＩｔａｕ的Ｓｅｒ４６和Ｓｅｒ２３５去磷酸化；去磷酸化后ＰＨＦＩＩｔａｕ的相对电泳迁移率加快。Ｍｎ２＋和Ｍｇ２＋可增加上述酶对ＰＨＦＩＩｔａｕ的去磷酸化作用。酶的去磷酸化作用具有浓度依赖性，随着酶浓度的增加，去磷酸化作用增强。结论因为ｔａｕ蛋白异常过度磷酸化并形成ＰＨＦ被认为是ＡＤ神经原纤维退化的基础，ＰＨＦ可在体外被蛋白磷酸酯酶去磷酸化的结果提示，ＡＤ脑损伤可能是可逆的。