The function of the recently discovered angiotensin II type 2 (AT2) receptor remains elusive. This receptor is expressed abundantly in fetus, but scantily in adult tissues except brain, adrenal medulla, and atretic ovary. In this study, we demonstrated that this receptor mediates programmed cell death (apoptosis). We observed this effect in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line), which express abundant AT2 receptor but not AT1 receptor. The cellular mechanism appears to involve the dephosphorylation of mitogen-activated protein kinase (MAP kinase (3,4) which is abundantly expressed in fetal tissues and immature brain (5) but present only at low levels in certain adult tissues (6-8). Recently, we and others have reported that the AT2 receptor mediates anti-growth effects on vascular smooth muscle (9) and endothelial (10) cells. To examine further the mechanism of the anti-growth action of the AT2 receptor, we studied its effect on programmed cell death (apoptosis). We also elucidated the biochemical signal transduction mechanism of the AT2 receptor mediating apoptosis.We first studied PC12W cells, a subline of the PC12 rat pheochromocytoma cell line (11), which express high levels of the AT2 but not the AT1 receptor. These cells differentiate into neuronal-like cells in the presence of nerve growth factor (NGF) and provide a good model for investigating programmed neural cell death (12). Second we studied R3T3 cells, a mouse fibroblast cell line whose expression of AT2 receptor is modulated by the growth state of the cells-i.e., very low in actively growing cells but increasing markedly in the confluent, quiescent state (13). This cell line is a good model for studying the growth-regulatory function of AT2 receptor. MATERIALS AND METHODSCells and Treatment. PC12W cells were grown and underwent differentiation in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) plus 10% horse serum, 5% fetal bovine serum (FBS), and NGF (10 ng/ml). R3T3 cells were maintained in DMEM plus 10% FBS.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. FIG. 1. Ang 1I-enhanced apoptosis in PC12W cells. Hoechst 33342(blue) stains chromatin in cells with an intact cell membrane and propidium iodide (pink) stains chromatin in cells without cell membrane integrity. After cells differentiated in medium containing 10% horse serum, 5% FBS, and NGF at 10 ng/ml for 1 week, the medium was exchanged with serum-free medium containing NGF at 1 ng/ml for 2 days. No apoptotic changes were observed in this condition (A). Morphological apoptotic changes appeared 2 days after addition of Ang 11 (0.1 ,uM), showing nuclear condensation (C), fragmentation (D), and margination (E). (X400.)Staining with Chromatin-Binding Dyes. PC12W cells were seeded into six-well plates (Falcon) at 2.0 x 105 cells per well. The chromatin binding dyes Hoechst 3334...

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Objectives-To elucidate the potential role of cytokines in the pathogenesis of cardiomyopathy and myocarditis. Background-Experimental studies show that certain cytokines depress myocardial contractility and that tumour necrosis factor-a plays an important part in the pathogenesis of myocardial injury in animal models of viral and autoimmune myocarditis. Methods-Plasma interleukin 1-a, interleukin I-fl, interleukin-2, interleukin-6, tumour necrosis factor-a, tumour necrosis factor-fl, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, interferon-a and interferon-y were measured in 13 patients with acute myocarditis, 23 patients with dilated cardiomyopathy, 51 patients with hypertrophic cardiomyopathy, nine patients with acute myocardial infarction, 18 patients with angina pectoris, 12 patients with essential hypertension and 17 healthy controls. Results-Increased concentrations of cytokines were not detected in the controls. In patients with acute myocarditis, interleukin 1-a was detected in 23% (mean (SD) 25 (11) pglml), interleukin l-pl in 31% (56 (34) pglml), tumour necrosis factor-a in 46% (61 (31) pglml), and macrophage colony stimulating factor was 2-5 (1X8) nglml (normal 1*9 (0*4)). In patients with dilated cardiomyopathy, tumour necrosis factor-a was detected in 35% (402 (555) (Br HeartJ_ 1994;72:561-566)

Studies on the effects of proinflammatory cytokines on the heart suggest that they play some roles in the pathogenesis of congestive heart failure (CHF). To determine the involvement of proinflammatory cytokine in cardiac hypertrophy and CHF induced by mechanical overload, we investigated the expression of interleukin (IL)-1 beta and monocyte chemotactic and activating factor (MCAF)/monocyte chemoattractant protein-1 (MCP-1) in the left ventricle (LV) of Dahl salt-sensitive (DS) rats that showed hypertrophy of the LV induced by hypertension and subsequently developed CHF. The IL-1 beta mRNA content in the LV of DS rats increased 3.9-fold when LV hypertrophy developed, and the increase reached 6.2-fold at the CHF stage compared with that of age-matched Dahl salt-resistant (DR) rats. The amount of IL-1 beta in the LV was positively correlated with the LV weight/body weight ratio. Most of the IL-1 beta immunoreactivity was localized in the endothelial cells and interstitial macrophages. The mRNA levels of MCAF in the LV increased 3.6-fold at 11 weeks and reached 4.8-fold at the CHF stage relative to the age-matched DR rats. MCAF protein was localized to the endothelial cells and interstitial macrophages. In DS rats, the number of interstitial macrophages increased diffusely throughout the LV. We suggest that increased chemokine expression, macrophage infiltration, and proinflammatory cytokine expression play some role in the pathogenesis of cardiac hypertrophy and failure induced by chronic mechanical overload.