Apoptosis of cardiac myocytes is one of the causes of heart failure. Here we examine the mechanism by which the activation of ␤-adrenergic receptor induces cardiomyocyte apoptosis. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling and DNA ladder analyses revealed that isoproterenol (Iso) induced the apoptosis of cardiac myocytes of neonatal rats through an increase in intracellular Ca 2؉ levels. The Iso-induced cardiomyocyte apoptosis was strongly inhibited by the L-type Ca 2؉ channel antagonist nifedipine and by the calcineurin inhibitors cyclosporin A and FK506. Iso reduced the phosphorylation levels of the proapoptotic Bcl-2 family protein Bad and induced cytochrome c release from mitochondria to the cytosol through calcineurin activation. Infusion of Iso increased calcineurin activity by ϳ3-fold in the hearts of wild-type mice but not in the hearts of transgenic mice that overexpress dominant negative mutants of calcineurin. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling analysis revealed that infusion of Iso induced apoptosis of cardiac myocytes and that the number of apoptotic cardiomyocytes was significantly less in the hearts of the transgenic mice compared with the wild-type mice. These results suggest that calcineurin plays a critical role in Iso-induced apoptosis of cardiac myocytes, possibly through dephosphorylating Bad.

Background-Rho and its effector Rho-kinase/ROCK mediate cytoskeletal reorganization as well as smooth muscle contraction. Recent studies indicate that Rho and ROCK are critically involved in vascular remodeling. Here, we tested the hypothesis that Rho/ROCK are critically involved in angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs) by mediating a specific signal cross-talk. Methods and Results-Immunoblotting demonstrated that Ang II stimulated phosphorylation of a ROCK substrate, regulatory myosin phosphatase targeting subunit (MYPT)-1. Phosphorylation of MYPT-1 as well as migration of VSMCs induced by Ang II was inhibited by dominant-negative Rho (dnRho) or ROCK inhibitor, Y27632. Ang II-induced c-Jun NH 2 -terminal kinase (JNK) activation, but extracellular signal-regulated kinase (ERK) activation was not mediated through Rho/ROCK. Thus, infection of adenovirus encoding dnJNK inhibited VSMC migration by Ang II. We have further demonstrated that the Rho/ROCK activation by Ang II requires protein kinase C-␦ (PKC␦) and proline-rich tyrosine kinase 2 (PYK2) activation, but not epidermal growth factor receptor transactivation. Also, VSMCs express PDZ-Rho guanine nucleotide exchange factor (GEF) and Ang II stimulated PYK2 association with tyrosine phosphorylated PDZ-RhoGEF. A ngiotensin II (Ang II) has been implicated in various cardiovascular diseases such as hypertension, atherosclerosis, and restenosis after angioplasty. 1 Therefore, there has been considerable interest in defining the functional significance of signaling pathways of the Ang II type 1 receptor (AT 1 ), which is dominantly expressed in vascular smooth muscle cells (VSMCs). 1-3 Through this receptor, Ang II stimulates hypertrophy and hyperplasia of VSMCs. 2 The AT 1 receptor primarily couples to G q leading to elevation of intracellular Ca 2ϩ and activation of protein kinase C (PKC). 2 In addition, tyrosine kinase activation by Ang II is linked to downstream mitogenactivated protein kinase (MAPK) activation, thereby mediating the growth promoting response in VSMCs. 2,4,5 In this regard, several key tyrosine kinases have been identified that may contribute to the growth-promoting effects of the AT 1 receptor. These kinases include epidermal growth factor receptor (EGFR) and proline-rich tyrosine kinse 2 (PYK2). 4,5 In addition to its growth responses, VSMC migration by Ang II is strongly implicated in various cardiovascular diseases, 6 whereas the detailed signaling mechanisms by which the AT 1 receptor mediates migration are insufficiently characterized. At least, a MAPK, ERK appears to be required for Ang II-induced migration of VSMCs. 7 Recently, Zahn et al showed that 3 major MAPKs, ERK, p38MAPK, and c-Jun NH 2 -terminal kinase (JNK), are all required for VSMC migration induced by platelet-derived growth factor (PDGF), 8 suggesting that these MAPKs coordinately mediate VSMC migration. A small G protein, Rho, and its effector Rho-kinase/ ROCK, are involved in many aspects of cell motility, from smooth muscle...

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To determine whether angiotensin (ANG) II, a vasoconstrictor hormone, activates constitutive nitric oxide synthase (cNOS) in endothelial cells (ECs), we investigated the cellular mechanism by which ANG II induces nitric oxide (NO) formation in cultured bovine ECs. ANG II rapidly (within 1 min) and dose-dependently (10-9-106 M) increased nitrate/nitrite (NOx) production. This effect of ANG II was abolished by a NOS inhibitor, NG-monomethyl-L-arginine.An ANG II type 1 (AT1) receptor antagonist (DuP 753), but not an ANG II type 2 (AT2) receptor antagonist (PD 123177), dose-dependently inhibited ANG II-induced NOx production.A Cat+-channel blocker (barnidipine) failed to affect ANG II-induced NOx production, whereas an intracellular Ca2+ chelator (BAPTA) and a calmodulin inhibitor (W-7) abolished NOx production induced by ANG II. A protein kinase C (PKC) inhibitor (H-7) and down-regulation of endogenous PKC after pretreatment with phorbol ester decreased NOx production stimulated by ANG II. ANG II transiently stimulated inositol 1,4,5-trisphosphate (IP3) formation, and increased cytosolic free Ca2+ concentrations; these effects were blocked by DuP 753. Our data demonstrate that ANG II stimulates NO release by activation of Ca2+/calmodulin-dependent cNOS via AT1 receptors in