Masanobu Koide

Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells.

In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.

Cytokine-induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells

In unstimulated cultured vascular smooth muscle cells (VSMC), mRNA of an inducible macrophage‐type of nitric oxide synthase (iNOS) was barely detectable. Interferon γ (IFNγ) and tumor necrosis factor α (TNFα) markedly increased iNOS mRNA levels in time‐ and dose‐dependent manners. The induction of iNOS mRNA paralleled the cytokine‐induced nitrite production. Actinomycin D abolished the IFNγ‐ and TNFγ‐induced increases in iNOS mRNA and nitrite production. Cycloheximide, which abolished both the IFNγ‐ and TNFα‐induced increases in nitrite production, had no effect on the IFNγ‐induced increase in iNOS mRNA but markedly inhibited the TNFα‐induced one. These results suggest that IFNγ directly induces the expression of the iNOS gene whereas TNFα mainly induces it via the induction of an intermediary protein in cultured VSMC.

Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time‐ and dose‐dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5‐hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C‐activating phorbol ester, phorbol 12 myristate, 13‐acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.

Involvement of MAP kinase activators in angiotensin II-induced activation of MAP kinases in cultured vascular smooth muscle cells

In cultured vascular smooth muscle cells (VSMC) angiotensin II (ang II) induces tyrosine and serine/threonine phosphorylation and activation of two mitogen‐activated protein (MAP) kinases, When extracts of ang II‐stimulated VSMC were fractionated by Mono Q anion‐exchange column chromatography, three peaks of the activities which in vitro activate inactive MAP kinases were detected. These MAP kinase activator activities were not detected in extracts of unstimulated VSMC. In vitro activation of MAP kinases by the MAP kinase activators was accompanied by tyrosine and serine/threonine phosphorylation of MAP kinases. These results suggest that the MAP kinase activators are involved in the ang II‐induced phosphorylation and activation of MAP kinases in VSMC.

Stimulation of protein-tyrosine phosphorylation by endothelin-1 in cultured vascular smooth muscle cells.

In cultured rat aortic smooth cells, endothelin-1 induced tyrosine phosphorylation of at least five proteins with molecular masses of about 79, 77, 73, 45 and 40 kDa in dose- and time-dependent manners. Platelet-derived growth factor also induced tyrosine phosphorylation of the same set of proteins in addition to other proteins including platelet-derived growth factor receptors. This growth factor markedly stimulated DNA synthesis and an increase in cell number in this cell type, but endothelin-1 failed to stimulate these responses under the same conditions. These results demonstrate for the first time that endothelin-1 induces tyrosine phosphorylation of some proteins but suggest that these reactions are not enough to stimulate proliferation of vascular smooth muscle cells.

Cyclic AMP Elevating Agents Synergize with Inflammatory Cytokines to Induce an Inducible Type of Nitric Oxide Synthase in Cultured Vascular Smooth Muscle Cells

In vascular smooth muscle cells (VSMC), inflammatory cytokines such as interleukin 1 (ILl), tumor necrosis factor a (TNFa), and interferon y (IFNy) stimulate nitric oxide (NO) production via the expression of an inducible type of nitric oxide synthase (iNOS). NO thus produced could modulate the development of vascular inflammatory lesions including atherosclerosis and arterial injury following balloon angioplasty. Since the activity of iNOS is regulated mainly at the transcriptional level, it is very important to identify the extracellular stimuli and the intracellular signaling mechanisms that regulate the iNOS expression. In the present study, we examined the effect of CAMP-elevating agents on iNOS expression in cultured VSMC.