RESUMEN
In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNFalpha induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNFalpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNFalpha-mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNFalpha. These findings suggest that the elevated TNFalpha levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNFalpha-induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors.
Asunto(s)
Apoptosis/efectos de los fármacos , Miocardio/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Análisis de Varianza , Animales , Benzoxazoles/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Ceramidas/farmacología , Daño del ADN/genética , Electroforesis en Gel de Agar , Peróxido de Hidrógeno/farmacología , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente , Isquemia Miocárdica/metabolismo , Compuestos de Quinolinio/metabolismo , Ratas , Transducción de Señal/fisiología , Esfingolípidos/metabolismo , Esfingolípidos/farmacología , Esfingosina/metabolismo , Esfingosina/farmacologíaRESUMEN
We have previously demonstrated that PRL acts as an autocrine growth factor in the pituitary tumor GH3 cell line. In this study we present evidence that the 235-1 cell line also uses PRL as an autocrine growth factor, and that in this case interaction between PRL and its receptor occurs intracellularly. First, the PRL produced by 235-1 cells was shown to be capable of initiating a proliferative response by placement in the Nb2 bioassay. Second, PRL receptors were demonstrated to be present, but, unusually, primarily in the Golgi complex by light and electron microscope immunocytochemistry. Incubation of the cells in anti-PRL had no effect on 235-1 cell proliferation until interleukin-1 treatment caused the PRL receptors to move to the cell surface, whereupon cell proliferation was inhibited. Specific interference with PRL biosynthesis in noninterleukin-1-treated cells also inhibited cell proliferation. Thus, the essential elements of an autocrine loop are present and can be demonstrated to be functional by antibody inhibition of growth after movement of the receptors to the cell surface and by antisense inhibition of growth with the receptors in their normal intracellular location.
Asunto(s)
Hipófisis/metabolismo , Prolactina/metabolismo , Receptores de Prolactina/metabolismo , Animales , Secuencia de Bases , División Celular , Línea Celular , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Interleucina-1/farmacología , Datos de Secuencia Molecular , Hipófisis/citología , Ratas , Receptores de Prolactina/análisisRESUMEN
Because PRL has growth factor activities in several tissues, we have asked whether it also has autocrine growth factor activity in pituitary GH3 cells. GH3 cells were grown at increasing densities in the presence or absence of antirat PRL (polyclonal and monoclonal) or nonspecific antibodies. Cell proliferation increased with increasing cell density, as did the concentration of PRL in the medium. Antirat PRL, but not control antibody, markedly inhibited but did not eliminate cell proliferation, and this effect was diminished with increasing PRL concentration in the medium. PRL receptors were demonstrated on 40-50% of the cells by indirect immunofluorescence using a specific antirat PRL receptor monoclonal antibody. Cell surface PRL was colocalized to the same 40-50% of the cells and copatched or cocapped along with the receptors. Absence or presence of PRL receptors did not correlate with stage of the cell cycle, as judged by ethidium bromide dual labeling. Cell surface PRL was found to be on PRL-containing cells. These data have fulfilled four criteria necessary for establishment of a substance as a secreted autocrine growth factor: 1) the factor must be secreted; 2) in log growth phase, increased cell proliferation should occur at increased cell densities; 3) the cells must display a receptor for the factor; and 4) there must be a growth response to the factor. Thus we have established that PRL is an autocrine growth factor for at least 40-50% of the GH3 cell population. This, to our knowledge, is the first example of autocrine growth factor activity of a major hormone normotopically expressed.
Asunto(s)
Neoplasias Hipofisarias/patología , Prolactina/fisiología , Animales , Anticuerpos , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Neoplasias Hipofisarias/metabolismo , Prolactina/inmunología , Ratas , Receptores de Prolactina/metabolismo , Células Tumorales CultivadasRESUMEN
Tumor necrosis factor-alpha (TNF alpha) is a potentially powerful anti-neoplastic agent; however, its therapeutic usefulness is limited by its cardiotoxic and negative inotropic effects. Accordingly, studies were undertaken to gain a better understanding of the mechanisms of TNF alpha-mediated cardiodepression. Single cell RT-PCR, [125I]TNF alpha ligand binding and Western immunoblotting experiments demonstrated that rat cardiac cells predominantly express type I TNF alpha receptors (TNFRI or p60). TNF alpha inhibited cardiac L-type Ca2+ channel current (ICa) and contractile Ca2+ transients. Thus, it is possible that the negative inotropic effects of TNF alpha are the result of TNFRI-mediated blockade of cardiac excitation-contraction coupling.
Asunto(s)
Miocardio/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Secuencia de Bases , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Canales de Calcio/metabolismo , Cartilla de ADN/química , Colorantes Fluorescentes/metabolismo , Expresión Génica/genética , Immunoblotting , Técnicas In Vitro , Indoles/metabolismo , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/genéticaRESUMEN
This chapter describes the use of cultured adult primary ventricular cardiomyocytes as a model for cytokine-mediated septic shock. We describe the methods for preparation of adult cardiomyocytes from adult rat hearts for culture. We also describe techniques designed to assess the responses of single cells in culture to the proinflammatory cytokine, TNFα. We describe in detail the use of this cell-culture model to evaluate TNFα-induced voltage-dependent calcium fluxes as well as the expression of genes that mediate the TNFα response. Because cardiomyocyte cultures may be heterogeneous and may include a variety of other cell types (fibroblasts, endothelial cells, etc.), it is sometimes prudent to examine cells on a single-cell basis to ensure that the physiological or molecular process of interest is characteristic of the cardiomyocyte. The issue of heterogeneity is particularly problematic when attempting to examine the expression of low-abundant transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In our laboratory, we have employed single-cell RT-PCR as a tool for examining the expression of genes coding for TNFα receptors in isolated myocardial cells. The procedures can be applied to acutely isolated cells as well as those cultured and subjected to chronic treatments. By using these techniques, it is possible to implicate molecular mechanisms/pathways in the physiological responsiveness of the adult ventricular cardiomyocyte to cytokines.
RESUMEN
The protease, alpha-thrombin (alpha Th), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess alpha Th receptors (alpha Th-R), or if long term treatment with alpha Th can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat alpha Th-R-specific probe. After 48 h, alpha Th induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the alpha Th-selective protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. The alpha Th-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by alpha Th to be transcriptional. The abilities of alpha Th to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell alpha Th-Rs are stimulated by the specific proteolytic actions of alpha Th, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth.
Asunto(s)
Factor Natriurético Atrial/biosíntesis , Cardiomegalia/patología , Expresión Génica/efectos de los fármacos , Miocardio/metabolismo , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/fisiología , Trombina/farmacología , Tirfostinos , Alcaloides , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/análisis , Benzofenantridinas , Northern Blotting , Cardiomegalia/inducido químicamente , Catecoles/farmacología , Células Cultivadas , Ventrículos Cardíacos , Cinética , Datos de Secuencia Molecular , Miocardio/patología , Nitrilos/farmacología , Oligopéptidos/síntesis química , Fenantridinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de Proteasas/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/análisis , Radioinmunoensayo , Ratas , Receptores de Superficie Celular/efectos de los fármacos , Receptores de TrombinaRESUMEN
The bacterial endotoxin lipopolysaccharide (LPS) contributes to the cardiovascular collapse and death observed in patients with sepsis. Because LPS has such profound effects on cardiac performance, we speculate that direct effects of LPS could be demonstrated on cardiomyocytes in culture, and that these direct effects are mediated by the LPS receptor, CD14. Accordingly, in this study, we provide evidence for CD14-dependent cardiotoxic effects of LPS including the LPS-stimulated secretion of tumor necrosis factor alpha (TNF-alpha) from cardiomyocytes. TNF-alpha is an inflammatory cytokine which is known for its negative inotropic effects on cardiac performance, but has not until recently been shown to be produced by cardiac cells. In this study, LPS was found to stimulate strongly in a dose-dependent manner the secretion of TNF-alpha from cultured adult rat cardiomyocytes. Further, LPS-induced TNF-alpha secretion was blocked by an inhibitor of TNF-alpha processing, metallomatrix protease inhibitor (TAPI). Molecular and immunological evidence demonstrated the presence of LPS receptors (CD14) on cardiomyocytes. Attenuated TNF-alpha secretion following PI-PLC treatment confirmed the functional importance of CD14 for LPS-mediated myocardial effects. Importantly, LPS also triggered apoptosis in cultured cardiomyocytes as quantified by single-cell gel electrophoresis of nuclei exhibiting DNA fragmentation patterns characteristic of apoptosis (i.e. cardiac comets). Apoptotic cell death was blocked by pre-incubation with the soluble TNF-alpha receptor fragment (TNFRII:Fc), suggesting that LPS-induced apoptosis was TNF-alpha-dependent and probably involved an autocrine function for the TNF-alpha whose secretion was under LPS control. The results of this study suggest that the cardiodepressant effects of LPS are dependent on CD14 signaling and may not only be due to acute negative inotropic effects of TNF-alpha but also may be complicated by TNF-alpha-induced apoptotic cell death which effectively reduces the number of working myocardial cells.