Abstract
We have investigated the contribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAP kinase) in norepinephrine (NE)-induced arachidonic acid (AA) release in rabbit aortic vascular smooth muscle cells (VSMC). NE enhanced release of AA via activation of cytosolic phospholipase A2 (cPLA2) but not secretory PLA2 in VSMC prelabeled with [3H]AA. NE (10 microM) enhanced CaM kinase II and MAP kinase activity. In cells transiently transfected with antisense oligonucleotides complementary to the translation initiation sites of CaM kinase II and MAP kinase, NE-induced AA release was inhibited by 100 and 35% respectively. Treatment of cells with PD-098059, a MAP kinase kinase inhibitor, or with MAP kinase antisense oligonucleotide reduced NE-induced activation of MAP kinase and cPLA2. NE-induced MAP kinase and cPLA2 activation was also inhibited in cells treated with a CaM kinase II inhibitor, KN-93, or with CaM kinase II antisense oligonucleotide. On the other hand, inhibition of MAP kinase kinase with PD-098059 or of MAP kinase with antisense oligonucleotides did not alter the NE-induced increase in CaM kinase II activity. Phosphorylation of MAP kinase and CaM kinase II by NE, studied by 32P incorporation and immune complex kinase assays, was inhibited by KN-93. Collectively, these data suggest that CaM kinase II can activate MAP kinase, which in turn activates cPLA2 to release AA for prostacyclin synthesis in the rabbit VSMC. This novel pathway for activation of MAP kinase by CaM kinase II appears to be mediated through stimulation of MAP kinase kinase. Activation of adrenergic receptors with NE in VSMC caused translocation of CaM kinase II, MAP kinase, and cPLA2 to the nuclear envelope only in the presence of extracellular Ca2+. Okadaic acid, which increased phosphorylation and activity, did not translocate these enzymes. Therefore, it appears that in rabbit VSMC, NE, by promoting extracellular Ca2+ influx, increases CaM kinase II activity, leading to activation of MAP kinase and cPLA2 and translocation to the nuclear envelope, resulting in release of AA from the nuclear envelope for prostacyclin synthesis.
Highlights
We have investigated the contribution of Ca2؉/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAP kinase) in norepinephrine (NE)-induced arachidonic acid (AA) release in rabbit aortic vascular smooth muscle cells (VSMC)
Our previous findings in the VSMC of rabbit aorta that PGI2 synthesis elicited by activation of ␣1 and ␣2 adrenergic receptor (AR) was attenuated by the calmodulin (CaM) inhibitor W-7 [2] raises the possibility that Ca2ϩ/CaM might stimulate cytosolic phospholipase A2 (cPLA2) directly or indirectly via activation of CaM kinase II or MAP
CaM kinase II stimulates cPLA2 by activation of MAP kinase, most probably via MEK. 3) Upon exposure of VSMC to NE, cPLA2, CaM kinase II, and MAP kinase translocate to the nuclear membrane in a Ca2ϩ-dependent manner
Summary
Materials—[3H]AA (100 Ci/mmol) was purchased from DuPont NEN. Hanks’ balanced salt solution, M-199, phosphate-buffered saline, bovine serum albumin, EGTA, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and norepinephrine were purchased from Sigma; PD-098059 [14] was obtained from Parke-Davis (Ann Arbor, MI); KN-93 and okadaic acid were from Calbiochem; L-[1-14C]phosphatidylcholine (specific activity, 57 mCi/mmol) was from American Radiolabeled Chemicals Inc. 11 l of radiolabeled phospholipid stock was dried under N2 and added to 0.5 ml of reaction mixture (9 M dioleoylglycerol, 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 1 mM DTT, 1 mg/ml BSA) and sonicated for 15 min on ice. The reaction mixture (50 l) containing 25 g of protein from cell lysate was incubated at 37 °C for 1 h. Immune Complex Kinase Assays—Cell lysates containing equal amounts of proteins (500 g) from control and NE-treated samples in the presence and absence of inhibitors were incubated with 5 l of ERK-1 and CaM kinase II antibody or anti-mouse IgG for 1 h at 4 °C. The immune complexes were captured by protein A-agarose beads and washed three times with 1 ml of radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate containing 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 2 mM EGTA, 50 g/ml leupeptin, and 0.5% aprotinin). Slides were viewed by confocal fluorescence microscopy (Bio-Rad MRC-1000 Laser Scanning Confocal Imaging system using an argon/krypton lamp) with a ϫ 100 objective lens
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