ABSTRACT
There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate, PKC activity using an assay kit and assay of Na+/K+ATPase activity spectrophotometrically. We purified m-calpain and PKCα by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCα by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCα (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCα in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCα increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2+]i-mCalpain-PKCα signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.
Subject(s)
Amino Acid Sequence , Animals , Calpain/metabolism , Cattle , Caveolae/drug effects , Caveolae/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Molecular Sequence Data , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Ouabain/pharmacology , Protein Kinase C-alpha/chemistry , Protein Kinase C-alpha/metabolism , Proteolysis/drug effects , Pulmonary Artery/cytology , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
We investigated the effect of phenylephrine (PE)- and isoproterenol (ISO)-induced cardiac hypertrophy on subcellular localization and expression of caveolin-3 and STAT3 in H9c2 cardiomyoblast cells. Caveolin-3 localization to plasma membrane was attenuated and localization of caveolin-3 to caveolae in the plasma membrane was 24.3% reduced by the catecholamine-induced hypertrophy. STAT3 and phospho-STAT3 were up-regulated but verapamil and cyclosporin A synergistically decreased the STAT3 and phospho-STAT3 levels in PE- and ISO-induced hypertrophic cells. Both expression and activation of STAT3 were increased in the nucleus by the hypertrophy. Immunofluorescence analysis revealed that the catecholamine-induced hypertrophy promoted nuclear localization of pY705-STAT3. Of interest, phosphorylation of pS727-STAT3 in mitochondria was significantly reduced by catecholamine-induced hypertrophy. In addition, mitochondrial complexes II and III were greatly down-regulated in the hypertrophic cells. Our data suggest that the alterations in nuclear and mitochondrial activation of STAT3 and caveolae localization of caveolin-3 are related to the development of the catecholamine-induced cardiac hypertrophy.
Subject(s)
Animals , Rats , Catecholamines/pharmacology , Caveolae/metabolism , Caveolin 3/metabolism , Cell Line , Hypertrophy/metabolism , Mitochondria/metabolism , Myocardium/cytology , Myocytes, Cardiac/cytology , STAT3 Transcription Factor/metabolismABSTRACT
Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, bAPP secretion was up-regulated by caveolin-1 over-expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by beta-secretase via PKC downregulation.