House SJ, Ginnan RG, Armstrong SE, Singer HA. Calcium/ calmodulin-dependent protein kinase II-␦ isoform regulation of vascular smooth muscle cell proliferation.

In vascular smooth muscle (VSM) and many other cells, G protein receptor-coupled activation of mitogen-activated protein kinases has been linked, in part, to increases in free intracellular Ca(2+). Previously, we demonstrated that ionomycin-, angiotensin II-, and thrombin-induced activation of extracellular signal-regulated kinase (ERK)1/2 in VSM cells was attenuated by pretreatment with KN-93, a selective inhibitor of the multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaM kinase II). In the present study, we show that the Ca(2+)-dependent pathway leading to activation of ERK1/2 is preceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation and is attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibits Ca(2+)-dependent PYK2 activation and EGF receptor tyrosine phosphorylation in response to ionomycin, ATP, and platelet-derived growth factor but has no effect on phorbol 12,13-dibutyrate- or EGF-induced responses. The results implicate CaM kinase II as an intermediate in the Ca(2+)/calmodulin-dependent activation of PYK2.

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCɛ activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCɛ protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression, prevented hypoxia-induced increases in total PKC and PKCɛ activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCɛ translocation peptide inhibitor or PKCɛ gene deletion decreased hypoxic increase in [Ca 2+ ] i in PASMCs, whereas the conventional PKC inhibitor GÖ6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCɛ, contributing to hypoxia-induced increase in [Ca 2+ ] i and contraction in PASMCs. KeywordsHypoxia; protein kinase C; reactive oxygen species; mitochondria; intracellular calcium; pulmonary artery smooth muscle cells Hypoxic pulmonary vasoconstriction (HPV) is observed in isolated lungs, pulmonary arteries, and pulmonary artery smooth muscle cells (PASMCs). The pulmonary circulation differs from the systemic circulation in response to oxygen tension; pulmonary arteries constrict to physiological hypoxia (~ 20-60 mmHg PO 2 ), whereas systemic arteries vasodilate. The mechanisms for these opposing responses to hypoxia appear to lie within the vascular SMCs. Hypoxia increases intracellular Ca 2+ concentration ([Ca 2+ ] i ) and contracts PASMCs. In contrast, SMCs from systemic arteries display decreased [Ca 2+ ] i and relax in response to hypoxia. The response of PASMCs to acute hypoxia involves calcium entry through voltagedependent and store-operated Ca 2+ channels, as well as Ca 2+ release from the sarcoplasmic reticulum [1][2][3][4][5][6][7][8]. Hypoxia-dependent changes in reactive oxygen species (ROS) concentration have been proposed to mediate HPV by several laboratories, although the details of this hypothesis differ greatly [9; 10]. However, the signaling pathways underlying artery-specific, acute hypoxic vasoconstriction remain to be fully elucidated. AnimalsPKCɛ −/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME); Swiss-Webster mice from Taconic (Germantown, NY). Glutathione peroxidase-1 (Gpx1) overexpression mice were generated and maintained as described previously [18]. All animal experiments were approved by the Institutional Animal Care and Use Committee of Albany Medical College. To examine the effects of pharmacological reagents, control experiments were carried out in cells or tissues from the same mice. For experimen...

Signaling cascades initiated or regulated by calcium (Ca(2+)), reactive oxygen (ROS), and nitrogen (RNS) species are essential to diverse physiological and pathological processes in vascular smooth muscle. Stimuli-induced changes in intracellular Ca(2+) regulate the activity of primary ROS and RNS, producing enzymes including NADPH oxidases (Nox) and nitric oxide synthases (NOS). At the same time, alteration in intracellular ROS and RNS production reciprocates through redox-based post-translational modifications altering Ca(2+) signaling networks. These may include Ca(2+) pumps such as sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase (SERCA), voltage-gated channels, transient receptor potential canonical (TRPC), melastatin2 (TRPM2), and ankyrin1 (TRPA1) channels, store operated Ca(2+) channels such as Orai1/stromal interaction molecule 1 (STIM1), and Ca(2+) effectors such as Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In this review, we summarize and highlight current experimental evidence supporting the idea that cross-talk between Ca(2+) and ROS/RNS may represent a well-integrated signaling network in vascular smooth muscle.

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.