Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 25
Filter
Add more filters










Publication year range
1.
Cell Mol Biol (Noisy-le-grand) ; 49(8): 1385-9, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14984014

ABSTRACT

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.


Subject(s)
Endothelium, Vascular/cytology , Molsidomine/pharmacology , Nitric Oxide Synthase/metabolism , Serotonin Antagonists/pharmacology , Serotonin/pharmacology , Animals , Cattle , Dose-Response Relationship, Drug , Endothelium, Vascular/enzymology , Feedback, Physiological , Molsidomine/analogs & derivatives , Nitric Oxide Synthase Type III , Peroxynitrous Acid/metabolism , Phosphorylation/drug effects
2.
Biochem Biophys Res Commun ; 286(4): 692-6, 2001 Aug 31.
Article in English | MEDLINE | ID: mdl-11520052

ABSTRACT

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr(402) phosphorylation. By contrast, Y402F inhibited PYK2 Tyr(402) phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr(402) phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.


Subject(s)
Angiotensin II/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/metabolism , Protein-Tyrosine Kinases/physiology , Adenoviridae/genetics , Animals , Cells, Cultured , Focal Adhesion Kinase 2 , Genetic Vectors , JNK Mitogen-Activated Protein Kinases , Leucine/metabolism , Models, Biological , Muscle, Smooth, Vascular/drug effects , Mutation , Phosphorylation , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/genetics , Rats , Rats, Sprague-Dawley , Transfection
3.
Biochem Biophys Res Commun ; 280(4): 1116-9, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11162642

ABSTRACT

Angiotensin II (Ang II) is known to stimulate reactive oxygen species (ROS) generation and epidermal growth factor (EGF) receptor transactivation to mediate growth-promoting signals such as extracellular signal-regulated kinase (ERK) in vascular smooth muscle cells (VSMCs). However, how ROS and EGF receptor interact to orchestrate these signals in VSMCs remains unclear. Here we found that an antioxidant, N-acetylcysteine, inhibited ERK activation and EGF receptor tyrosine phosphorylation induced by Ang II. Moreover, H(2)O(2) stimulates EGF receptor tyrosine phosphorylation and EGF receptor inhibitors attenuated H(2)O(2)-induced ERK activation. These data indicate that ROS mediate Ang II-induced EGF receptor transactivation, a critical mechanism for ERK-dependent growth in VSMCs.


Subject(s)
Acetylcysteine/pharmacology , Angiotensin II/metabolism , ErbB Receptors/metabolism , Mitogen-Activated Protein Kinases/metabolism , Animals , Antioxidants/pharmacology , Blotting, Western , Calcimycin/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation , Hydrogen Peroxide/pharmacology , Immunoblotting , Ionophores/pharmacology , Male , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Phosphorylation , Precipitin Tests , Pyrimidines/pharmacology , Quinazolines , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species , Time Factors , Transcriptional Activation , Tyrphostins/pharmacology
4.
J Biol Chem ; 276(11): 7957-62, 2001 Mar 16.
Article in English | MEDLINE | ID: mdl-11116149

ABSTRACT

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.


Subject(s)
Angiotensin II/pharmacology , ErbB Receptors/physiology , Metalloendopeptidases/physiology , Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/physiology , Muscle, Smooth, Vascular/drug effects , Animals , Enzyme Activation , JNK Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/enzymology , Rats , Rats, Sprague-Dawley , Transcriptional Activation , p38 Mitogen-Activated Protein Kinases
5.
Cell Mol Biol (Noisy-le-grand) ; 47(6): 1059-62, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11785657

ABSTRACT

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.


Subject(s)
Hypoglycemic Agents/antagonists & inhibitors , Insulin/pharmacology , Muscle, Smooth, Vascular/enzymology , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Animals , Aorta, Thoracic/cytology , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , Insulin Antagonists/metabolism , Insulin Receptor Substrate Proteins , Male , Muscle, Smooth, Vascular/drug effects , Phosphoproteins/metabolism , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Protein Kinase C/antagonists & inhibitors , Proto-Oncogene Proteins c-akt , Rats , Rats, Sprague-Dawley , Receptor, Insulin/metabolism , Tetradecanoylphorbol Acetate/pharmacology
6.
Endocrinology ; 141(9): 3120-6, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10965882

ABSTRACT

Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant, alpha-tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of p120 and p70, and ERK activation in VSMCs. H2O2 also enhanced PTK activity. From these data, we conclude that ROS play a critical role in the Ang II-induced PTK and ERK activation in VSMCs, thereby contributing to vascular growth associated with enhanced Ang II activity.


Subject(s)
Angiotensin II/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Reactive Oxygen Species/physiology , Animals , Blotting, Western , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Enzyme Activators/pharmacology , Hydrogen Peroxide/metabolism , Indicators and Reagents , Male , NADPH Oxidases/metabolism , Oxidants/metabolism , Precipitin Tests , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects
7.
Biochem Biophys Res Commun ; 270(3): 761-5, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10772898

ABSTRACT

In vascular smooth muscle cells (VSMCs), the focal adhesion kinase-related tyrosine kinase PYK2/CAKbeta is activated by vascular mitogens. Because reactive oxygen species (ROS) are assumed to mediate mitogenic signals by these agonists, we examined the possible link between ROS and PYK2 in cultured rat VSMCs. Here we present several lines of evidence showing that PYK2 is activated by ROS in VSMCs. The inhibitory effect of an antioxidant, N-acetyl-cysteine, on PYK2 activation by its specific agonists further suggests the pivotal role of PYK2 in vascular remodeling associated with enhanced ROS production.


Subject(s)
Muscle, Smooth, Vascular/enzymology , Protein-Tyrosine Kinases/metabolism , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/enzymology , Cells, Cultured , Enzyme Activation , Focal Adhesion Kinase 2 , Hydrogen Peroxide/pharmacology , Kinetics , Male , Muscle, Smooth, Vascular/cytology , Oxidation-Reduction , Phosphorylation , Protein-Tyrosine Kinases/drug effects , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism
8.
J Cardiovasc Pharmacol ; 35(3): 398-402, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10710124

ABSTRACT

5-Hydroxytryptamine (5-HT) is sequestered and released by endothelial cells, acts as an endothelial cell mitogen, promotes the release of nitric oxide (NO), and has been associated with the p44/p42 mitogen-activated protein kinase (MAPK) cascade. NO also acts as a cell mitogen and promotes signals that culminate in the phosphorylation of MAPK. The aim of this study was to test whether endothelial 5-HT receptors stimulate dual (tyrosyl- and threonyl-) phosphorylation of MAPK through a mitogen-activated protein kinase kinase-1 (MEK-1) and eNOS-dependent pathway in bovine aortic endothelial cells (BAECs). As shown by Western blot analysis, 5-HT and the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) stimulate time- and concentration-dependent (0.001-10 microM) phosphorylation of MAPK in these cells. The agonist-stimulated phosphorylation of MAPK was blocked by the 5-HT1b-receptor antagonist isamoltane (0.01-10 p3M) and the MEK-1 inhibitor PD 098059 ([2-(2'-amino-3'-methoxy-phenyl)-oxanaphthalen-4-one]; 0.01-10 microM¿. The eNOS inhibitor L-N(omega)-iminoethyl-L-ornithine (L-NIO; 0.01-10 microM) failed to block the 1 microM 5-NOT-stimulated responses. Our findings suggest that the 5-HT receptors (specifically 5-HT1B) mediate signals to MEK-1 and subsequently to MAPK through an eNOS-independent pathway in BAECs.


Subject(s)
Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Ornithine/analogs & derivatives , Serotonin/pharmacology , Animals , Autoradiography , Cattle , Cells, Cultured , Endothelium, Vascular/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/metabolism , Ornithine/pharmacology , Phosphorylation/drug effects , Receptors, Serotonin/drug effects
9.
Hypertension ; 35(1 Pt 2): 313-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10642317

ABSTRACT

Angiotensin II (Ang II) is now believed to play a critical role in the pathogenesis of hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMCs). Several G(i)- and G(q)-coupled receptors, including the Ang II type 1 (AT(1)) receptor, activate Rho and Rho-associated kinase in Swiss 3T3 cells and cardiac myocytes. However, little is known about the role of Rho-kinase in Ang II-induced vascular hypertrophy in VSMCs. In the present study, we explored the role of Rho and Rho-kinase in Ang II-induced protein synthesis in VSMCs. In unstimulated cells, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II (100 nmol/L). This effect was completely blocked by the AT(1) receptor blocker candesartan but not by the Ang II type 2 (AT(2)) receptor antagonist PD123319. Botulinum C(3) exoenzyme, which inactivated RhoA, attenuated Ang II-induced [(3)H]leucine incorporation. The specific Rho-kinase inhibitor, Y-27632, dose-dependently abolished Ang II-induced protein synthesis and also suppressed Ang II-induced c-fos mRNA expression. On the other hand, Y-27632 had no effect on Ang II-stimulated phosphorylation of p70 S6 kinase and extracellular signal-regulated kinase 1/2, which are reported to be involved in Ang II-induced protein synthesis, nor had it any effect on the Ang II-induced phosphorylation of PHAS-I, a heat- and acid-stable eIF-4E-binding protein. The phosphorylation of PHAS-I is regulating for translation initiation. These observations suggest that the Rho, Rho-kinase, and c-fos pathways may play a role in Ang II-induced hypertrophic changes of VSMCs through a novel pathway.


Subject(s)
Angiotensin II/pharmacology , Botulinum Toxins , Carrier Proteins , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Protein Serine-Threonine Kinases/metabolism , ADP Ribose Transferases/pharmacology , Amides/pharmacology , Animals , Aorta, Thoracic/cytology , Biological Transport/drug effects , Cells, Cultured , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Hypertrophy , Intracellular Signaling Peptides and Proteins , Leucine/pharmacokinetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Phosphorylation , Proto-Oncogene Proteins c-fos/genetics , Pyridines/pharmacology , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Ribosomal Protein S6 Kinases/metabolism , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases
10.
J Biol Chem ; 274(52): 36843-51, 1999 Dec 24.
Article in English | MEDLINE | ID: mdl-10601235

ABSTRACT

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.


Subject(s)
Angiotensin II/pharmacology , ErbB Receptors/physiology , Muscle, Smooth, Vascular/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/physiology , Ribosomal Protein S6 Kinases/metabolism , Serine/metabolism , ras Proteins/physiology , Animals , CHO Cells , Calcium/metabolism , Cricetinae , Muscle, Smooth, Vascular/cytology , Phosphatidylinositol 3-Kinases/physiology , Phosphorylation , Proto-Oncogene Proteins c-akt , Quinazolines , Rats , Rats, Sprague-Dawley , Transcriptional Activation , Tyrphostins/pharmacology
11.
Eur J Pharmacol ; 376(1-2): 203-6, 1999 Jul 02.
Article in English | MEDLINE | ID: mdl-10440105

ABSTRACT

We have reported that angiotensin II induces the epidermal growth factor (EGF) receptor transactivation leading to extracellular signal-regulated kinase (ERK) activation in rat vascular smooth muscle cells. Here, we report that the EGF receptor kinase inhibitor AG1478 and the ERK kinase inhibitor PD98059 markedly inhibited angiotensin II-induced c-Fos expression and protein synthesis but not c-Jun expression in these cells. These data suggest that the EGF receptor transactivation and subsequent ERK activation are indispensable for angiotensin II-mediated growth promotion of vascular smooth muscle cells providing a new mechanistic insight whereby angiotensin II contributes abnormal vascular remodeling.


Subject(s)
Epidermal Growth Factor/metabolism , Muscle Proteins/biosynthesis , Muscle, Smooth, Vascular/metabolism , Proto-Oncogene Proteins c-fos/biosynthesis , Animals , Immunoblotting , In Vitro Techniques , Rats , Rats, Sprague-Dawley
12.
Endocrinology ; 140(8): 3562-72, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10433212

ABSTRACT

Lipopolysaccharide (LPS) is responsible for initiating host responses leading to septic shock, and tumor necrosis factor-alpha (TNF alpha) is thought to be its primary mediator. In addition, TNF alpha is one of the major components of the pathogenesis of insulin resistance in various conditions. It has been shown that LPS induced TNF alpha production in rat vascular smooth muscle cells (VSMC). However, little is known about the signaling pathway by which VSMC in culture produce TNF alpha. We investigated the possible signaling components involved in this pathway. LPS elicited phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK, degradation of inhibitor of kappaB (IkappaB), and an increase in nuclear binding activity of activating protein-1 and nuclear factor-kappaB (NF-kappaB). Different types of NF-kappaB inhibitors, pyrrolidine dithiocarbamate and MG132, which specifically abolished IkappaB degradation and subsequent NF-kappaB activation by LPS, suppressed TNF alpha secretion from VSMC. Although PD98059, a specific MAPK kinase inhibitor and SB203580, a specific p38 MAPK inhibitor, had no effect on NF-kappaB activity, SB203580 suppressed TNF alpha secretion; however, PD98059 did not. A cotransfection assay showed that transfection of dominant negative IkappaB or pretreatment with SB203580 suppressed the TNF alpha gene promotor-dependent transcription. TNF alpha messenger RNA expression induced by LPS was inhibited by pyrrolidine dithiocarbamate, MG132, and SB203580, but not by PD98059. These observations indicate that TNF alpha production in VSMC is stimulated by LPS, and its transcription and translation are dependent on NF-kappaB activation through proteasome-mediated IkappaB degradation. It is likely that p38 MAPK may play a critical role in regulating transcription of the TNF alpha gene in VSMC, unlike in other cell lines.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Gene Expression Regulation , Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular/physiology , NF-kappa B/metabolism , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/genetics , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/physiology , Cell Communication/physiology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Imidazoles/pharmacology , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Pyridines/pharmacology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein Kinases
13.
Circ Res ; 85(1): 5-11, 1999 Jul 09.
Article in English | MEDLINE | ID: mdl-10400905

ABSTRACT

The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal-regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch-induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.


Subject(s)
Actins/physiology , Aorta/physiology , Botulinum Toxins , GTP-Binding Proteins/physiology , Muscle, Smooth, Vascular/physiology , Signal Transduction/physiology , ADP Ribose Transferases/pharmacology , Angiotensin II/pharmacology , Animals , Aorta/cytology , Biological Transport/physiology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA/biosynthesis , Enzyme Activation/drug effects , Enzyme Activation/physiology , GTP-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Muscle, Smooth, Vascular/cytology , Platelet-Derived Growth Factor/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Rats , Stress, Mechanical , rho-Associated Kinases , rhoA GTP-Binding Protein
14.
Hypertension ; 33(1 Pt 2): 201-6, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9931105

ABSTRACT

-PYK2, a recently identified Ca2+-sensitive tyrosine kinase, has been implicated in extracellular signal-regulated kinase (ERK) activation via several G protein-coupled receptors. We have reported that angiotensin II (Ang II) induces Ca2+-dependent transactivation of the epidermal growth factor receptor (EGFR) which serves as a scaffold for preactivated c-Src and downstream adaptors (Shc/Grb2), leading to ERK activation in cultured rat vascular smooth muscle cells (VSMC). Herein we demonstrate the involvement of PYK2 in this cascade. Ang II rapidly induced tyrosine phosphorylation of PYK2, whose effect was completely inhibited by an AT1 receptor antagonist and an intracellular Ca2+ chelator. A Ca2+ ionophore also induced PYK2 tyrosine phosphorylation to a level comparable with that by Ang II, whereas phorbol ester-induced phosphorylation was less than that by Ang II. Moreover, PYK2 formed a complex coprecipitable with catalytically active c-Src after Ang II stimulation. Although a selective EGFR kinase inhibitor completely abolished Ang II-induced recruitment of Grb2 to EGFR and markedly attenuated Ang II-induced ERK activation, it had no effect on Ang II-induced PYK2 tyrosine phosphorylation or its association with c-Src and Grb2. These data suggest that the AT1 receptor uses Ca2+-dependent PYK2 to activate c-Src, thereby leading to EGFR transactivation, which preponderantly recruits Grb2 in rat VSMC.


Subject(s)
Angiotensin II/physiology , Aorta, Thoracic/physiology , ErbB Receptors/physiology , Muscle, Smooth, Vascular/physiology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Angiotensin II/pharmacology , Animals , Aorta, Thoracic/drug effects , Calcium/metabolism , Cells, Cultured , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Epidermal Growth Factor/pharmacology , ErbB Receptors/drug effects , Focal Adhesion Kinase 2 , Humans , Models, Biological , Muscle, Smooth, Vascular/drug effects , Phosphorylation , Rats , Rats, Sprague-Dawley , Receptors, Angiotensin/drug effects , Receptors, Angiotensin/physiology , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology
15.
J Am Soc Nephrol ; 10 Suppl 11: S57-61, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9892141

ABSTRACT

In addition to its well known involvement in Gq/11-mediated vasoconstriction and its key roles in the homeostasis of electrolyte balances, the angiotensin II type 1 (AT1) receptor activates mitogen-activated protein kinase (MAPK) and p42/44 extracellular signal-regulated kinase. The extracellular signal-regulated kinase activation is mediated by activation of p21-Ras, Raf-1, and MAPK kinase in rat vascular smooth muscle cells. The mechanism for Gq-mediated activation of the tyrosine kinase pathways has not been clear. It was found that the initial release of intracellular Ca2+ results in the activation of the epidermal growth factor receptor (EGF-R), without autocrine release of epidermal growth factor. EGF-R provides a scaffold needed for the activation of p21-Ras, which leads to the activation of MAPK. MAPK plays pivotal roles in the activation of complex growth-promoting pathways. The pathway from the EGF-R involves protein tyrosine phosphorylation initiated by AT1 receptors. On the other hand, the angiotensin II type 2 (AT2) receptor counteracts the AT1 receptor-mediated tyrosine kinase activation by activating several tyrosine phosphatases and serine/threonine phosphatases, and it suppresses the cell growth process stimulated by various growth factors. The relative importance of AT1 and AT2 receptor actions depends on the levels of AT1 and AT2 receptor expression.


Subject(s)
Phosphoprotein Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Angiotensin/metabolism , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Enzyme Activation , ErbB Receptors/metabolism , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Signal Transduction
16.
J Biol Chem ; 273(15): 8890-6, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9535870

ABSTRACT

We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ ionophore and completely inhibited by an intracellular Ca2+ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.


Subject(s)
Adaptor Proteins, Signal Transducing , Angiotensin II/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium/metabolism , ErbB Receptors/metabolism , Muscle, Smooth, Vascular/enzymology , Proteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Tyrphostins , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Aorta, Thoracic/enzymology , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , GRB2 Adaptor Protein , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Nitriles/pharmacology , Phosphorylation , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , src Homology Domains
17.
Hypertension ; 31(1 Pt 2): 248-53, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9453311

ABSTRACT

Lysophosphatidylcholine (lyso-PC) has been implicated in atherogenesis and the inflammatory process. Although lyso-PC has been reported to contribute to the mitogenic effect of oxidized LDL on rat cultured vascular smooth muscle cells (VSMCs), the signaling mechanisms by which lyso-PC promotes its proliferation are poorly characterized. Mitogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduces extracellular cues to intracellular responses. We therefore examined the effect of lyso-PC on MAP kinase activation, proto-oncogene expression, and AP-1 binding activity using cultured rat VSMC. Marked activation of MAP kinase occurred within 10 minutes of lyso-PC treatment, whereupon rapid inactivation ensued. MAP kinase activation by lyso-PC was concentration-dependent (6.25 to 25 micromol/L). Pertussis toxin treatment did not affect lyso-PC-induced MAP kinase phosphorylation. Lyso-PC (25 micromol/L) also increased the mRNA expression of c-fos and c-jun genes. An electrophoretic mobility shift assay showed that AP-1 binding activity was enhanced by lyso-PC. To examine the upstream signaling of MAP kinase, we used several inhibitors on MAP kinase activation induced by lyso-PC. Although lyso-PC induced sustained increase in intracellular Ca2+ concentration, EGTA had no effect on MAP kinase activation induced by lyso-PC. However, protein kinase C inhibitor GF109203X and downregulation of protein kinase C activity by prolonged treatment with phorbol ester inhibited lyso-PC-induced MAP kinase activation. These data suggest that lyso-PC transmits its mitogenic activity through a MAP kinase-AP-1 pathway, which exists downstream of its protein kinase C activation in VSMCs.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Lysophosphatidylcholines/pharmacology , Muscle, Smooth, Vascular/metabolism , Proto-Oncogene Proteins c-fos/biosynthesis , Proto-Oncogene Proteins c-jun/biosynthesis , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , Cell Nucleus/metabolism , Cells, Cultured , Enzyme Activation , Genes, fos , Genes, jun , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Transcription Factor AP-1/metabolism , Transcription, Genetic/drug effects
19.
J Biol Chem ; 271(24): 14169-75, 1996 Jun 14.
Article in English | MEDLINE | ID: mdl-8662912

ABSTRACT

In cultured rat vascular smooth muscle cells, angiotensin II (Ang II) induced a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C inhibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca2+ chelation by TMB-8, but not extracellular Ca2+ chelation or inhibition of Ca2+ influx, abolished Ang II-induced MAPK activation. The calmodulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activation by Ang II as well as by the Ca2+ ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21(ras), and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be mediated by p21(ras)activation through a currently unidentified tyrosine kinase that lies downstream of Gq-coupled Ca2+/calmodulin signals.


Subject(s)
Angiotensin II/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/metabolism , Muscle, Smooth, Vascular/physiology , Proto-Oncogene Proteins p21(ras)/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Aorta, Thoracic , Calcimycin/pharmacology , Calcium/metabolism , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Gallic Acid/analogs & derivatives , Gallic Acid/pharmacology , Genistein , Imidazoles/pharmacology , Indoles/pharmacology , Isoflavones/pharmacology , Kinetics , Maleimides/pharmacology , Molecular Sequence Data , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/enzymology , Peptides/chemical synthesis , Peptides/metabolism , Pertussis Toxin , Protein Kinase C/antagonists & inhibitors , Pyrrolidinones/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction , Substrate Specificity , Type C Phospholipases/antagonists & inhibitors , Virulence Factors, Bordetella/pharmacology
20.
Am J Physiol ; 264(4 Pt 2): H1028-40, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8386477

ABSTRACT

To determine the role of the Na(+)-Ca2+ exchange systems of nerve terminal and sarcolemmal membrane on development of tension in rabbit aortic rings, internal or external Na+ concentration was changed with either ouabain or Na(+)-free solution, respectively. Ouabain produced a verapamil-insensitive but external Na(+)- and Ca(2+)-dependent biphasic tension with distinct lag periods both of which were shortened by depolarization with KCl. The first phase of tension was inhibited by prazosin, phentolamine, in vitro neurolysis with 6-hydroxydopamine and in vivo treatment with reserpine to deplete catecholamines in nerve terminals. Therefore, first phase of tension was attributed to catecholamines released from nerve terminals induced by increased axoplasmic Ca2+ concentration mediated by the neural Na(+)-Ca2+ exchanger due to the increased axoplasmic Na+ concentration resulting from inhibition of the Na(+)-Ka+ pump with ouabain. In the absence of the first phase of tension, the second phase of tension was enhanced by caffeine, presumably by preventing sequestration of the sarcolemmal Na(+)-Ca2+ exchanger-mediated increase in cytosolic Ca2+ concentration in vascular smooth muscle cells. The prazosin-insensitive tension was dependent on the external Na+ concentration and was also attributed to the sarcolemmal Na(+)-Ca2+ exchanger of vascular smooth muscle. The magnitude of the increase in tension with ouabain or Na(+)-free solution attributed to the sarcolemmal Na(+)-Ca2+ exchanger of vascular smooth muscle was larger than that mediated by the exchanger of the nerve terminal. It was concluded that the Na(+)-Ca2+ exchange systems of both the nerve terminal and the vascular smooth muscle sarcolemma contribute to the development of tension by different mechanisms and to different extents when internal or external Na+ concentration was changed.


Subject(s)
Carrier Proteins/physiology , Muscle Contraction/physiology , Muscle, Smooth, Vascular/physiology , Animals , Caffeine/pharmacology , Calcium/pharmacology , Dose-Response Relationship, Drug , Female , Hydrogen-Ion Concentration , Intracellular Membranes/enzymology , Intracellular Membranes/physiology , Intracellular Membranes/ultrastructure , Isometric Contraction/drug effects , Isometric Contraction/physiology , Male , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Nerve Endings/physiology , Ouabain/pharmacology , Phentolamine/pharmacology , Prazosin/pharmacology , Rabbits , Sarcolemma/enzymology , Sarcolemma/physiology , Sarcolemma/ultrastructure , Sodium/pharmacology , Sodium-Calcium Exchanger , Sodium-Potassium-Exchanging ATPase/analysis , Vascular Resistance/drug effects , Vascular Resistance/physiology , Verapamil/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...