When cultured in low serum-containing growth medium, the mouse C 2 C 12 cells exit cell cycle and undergo a welldefined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane-and nuclear-enriched proteins, and phospho-proteins from the proliferating C 2 C 12 cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C 2 C 12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C 2 C 12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phosphoproteins also occurred during conversion of C 2 C 12 myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESIquadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate interand intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation. Molecular & Cellular

Abstract-We reported that norepinephrine and angiotensin II (Ang II) activate the Ras/mitogen-activated protein (MAP) kinase pathway primarily through the generation of cytochrome P450 (CYP450) metabolites. The purpose of the present study was to determine the contribution of Ras and CYP450 to Ang II-dependent hypertension in rats. Infusion of Ang II (350 ng/min for 6 days) elevated mean arterial blood pressure (MABP) (171Ϯ3 mm Hg for Ang II versus 94Ϯ5 for vehicle group, PϽ0.05). Ras is activated on farnesylation by farnesyl protein transferase (FPT). When Ang II was infused in combination with FPT inhibitor FPT III (232 ng/min) or BMS-191563 (578 ng/min), the development of hypertension was attenuated (171Ϯ3 mm Hg for Ang II plus vehicle versus 134Ϯ5 mm Hg for Ang II plus FPT III and 116Ϯ6 mm Hg for Ang II plus BMS-191563, PϽ0.05). Treatment with the MAP kinase kinase inhibitor PD-98059 (5 mg SC) reduced MABP. The CYP450 inhibitor aminobenzotriazole (50 mg/kg) also diminished the development of Ang II-induced hypertension to 113Ϯ8 mm Hg. The activities of Ras, MAP kinase, and CYP450 measured in the kidney were elevated in hypertensive animals. The infusion of FPT III, BMS-191563, or aminobenzotriazole reduced the elevation in Ras and MAP kinase activity. Morphological studies of the kidney showed that FPT III treatment ameliorated the arterial injury, vascular lesions, fibrinoid necrosis, focal hemorrhage, and hypertrophy of muscle walls observed in hypertensive animals. These data suggest that the activation of Ras and CYP450 contributes to the development of Ang II-dependent hypertension and associated vascular pathology. (Hypertension. 2000;36:604-609.)Key Words: angiotensin II Ⅲ Ras Ⅲ kinases Ⅲ hypertension, experimental Ⅲ cytochrome P450 Ⅲ kidney A ngiotensin II (Ang II), the major biologically active component of the renin-angiotensin system, contributes to the regulation of vascular tone, salt and water balance, and blood pressure. 1,2 It promotes vascular smooth muscle cell (VSMC) migration, hypertrophy, and delayed hyperplasia. [3][4][5] Ang II also stimulates NADPH oxidase, p 21 Ras, and phospholipase (PL)A 2 , PLC, and PLD. 6 -9 The activation of PLA 2 and PLD by Ang II leads to the release of arachidonic acid, 9 -11 which is metabolized by cyclooxygenase to prostaglandins and thromboxane A 2 and by lipoxygenase to hydroperoxyeicosatetraenoic acid (HPETE) and hydroxyeicosatetraenoic acid (HETE). The cyclooxygenase products prostaglandin (PG)E 2 and PGI 2 attenuate the vascular and renal actions of Ang II and contribute to the antihypertensive mechanisms. 12 On the other hand, the cyclooxygenase product thromboxane A 2 and the lipoxygenase product 12-HPETE, which inhibits PGI 2 synthase and thereby promotes the vasoconstrictor effect of endoperoxide PGH 2 , contribute to the prohypertensive mechanisms. 13 The level of blood pressure appears to be determined by the balance between the antihypertensive and prohypertensive eicosanoids. 13 Recently, it was reported that Ang II also promotes the metabol...

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.

A previous study conducted in rat-1 cells expressing ␣ 1A -adrenergic receptors showed that phenylephrine (PHE) stimulates phospholipase D (PLD) activity. This study was conducted to determine the contribution of protein kinase C (PKC) to PHEinduced PLD activation in these cells. PKC inhibitors bisindolylmaleimide (BIM) I and Ro 31-8220, but not Gö 6976 or a pseudosubstrate peptide inhibitor of PKC␣, decreased PLD activity and arachidonic acid release elicited by PHE. However, antisense oligonucleotides directed against PKC ␣, ␦, ⑀, and reduced PKC isoform levels by about 80% but failed to alter PHE-induced PLD activation, indicating that these PKC isoforms are not involved in PLD activation elicited by ␣ 1A -adrenergic receptor stimulation. Ectopic expression of a kinase-deficient mutant of the PKC-related kinase PKN significantly attenuated PHE-induced PLD activation. On the other hand, BIM I and Ro 31-8220 blocked PHE-mediated increase in intracellular Ca 2ϩ but Gö 6976 and the peptide inhibitor did not. In the absence of extracellular Ca 2ϩ , PHE failed to increase PLD activity. These results indicate that ␣ 1A -adrenergic receptor-stimulated PLD activation is mediated by a mechanism independent of PKC␣, ␦, ⑀, and , but dependent on a PKCrelated kinase, PKN. Moreover, PKC inhibitors BIM I and Ro 31-8220 block PHE-induced PLD activity by inhibiting calcium signal. Caution should be used in interpreting the data obtained with PKC inhibitors in vivo.