RESUMEN
This study was undertaken to examine the effects of dietary supplementation of cysteine and taurine in rats with diabetes induced with streptozotocin (STZ, 65 mg/kg body weight). Experimental animals were treated orally (by gavage) with cysteine (200 mg/kg) and taurine (400 mg/kg), alone or in combination, daily for 8 weeks. In one group, rats were also pretreated 3 weeks before the induction of diabetes (prevention arm) whereas in the other, the treatment was started 3 days after the induction of diabetes (reversal arm). Diabetes increased heart weight/body weight (HW/BW) ratio, plasma glucose, triglyceride and cholesterol levels as well as depressed heart rate (HR), blood pressure, left ventricular systolic pressure (LVSP), rate of contraction (+dP/dt), rate of relaxation (-dP/dt), fractional shortening (FS) and cardiac output (CO). The left ventricular internal diameter in systole (LViDs) was increased whereas that in diastole (LViDd) was decreased. In the prevention arm, treatment of the diabetic animals with cysteine or taurine decreased HW/BW ratio and improved HR, FS, +dP/dt and -dP/dt, as well as normalized LViDs, without altering the increase in glucose level. Cysteine decreased plasma triglyceride and cholesterol levels and improved LVSP whereas CO was improved by taurine. In the reversal arm, cysteine alone or with taurine did not correct the changes in hemodynamic parameters, FS and plasma triglycerides. Diabetes-induced cardiac dysfunction and increases in plasma triglycerides can be prevented, but not reversed, by dietary cysteine alone or in combination with taurine.
Asunto(s)
Cardiotónicos , Cisteína/uso terapéutico , Diabetes Mellitus Experimental/complicaciones , Cardiomiopatías Diabéticas/prevención & control , Taurina/uso terapéutico , Animales , Gasto Cardíaco/efectos de los fármacos , Colesterol/sangre , Cardiomiopatías Diabéticas/diagnóstico por imagen , Quimioterapia Combinada , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Insulina/sangre , Masculino , Tamaño de los Órganos , Ratas , Ratas Sprague-Dawley , Triglicéridos/sangre , Ultrasonografía , Función Ventricular Izquierda/efectos de los fármacos , Función Ventricular Derecha/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: We have previously shown that a maternal low protein (LP) diet during pregnancy in the rat results in adverse ventricular remodeling and contractile deficiencies of the neonatal rat heart. Since pathological cardiac hypertrophy is associated with increased expression of genes involved in glucose handling, this study was undertaken to examine if maternal LP diet alters the expression of genes encoding for some key components of glucose metabolism and uptake, and of the insulin receptor (IR) signal transduction in the heart of male offspring. METHODS AND RESULTS: We determined the effect of maternal LP and normal diet (90 and 180 g/casein/kg respectively) on IR ß-subunit, insulin receptor substrate (IRS)-1, phosphotyrosyl protein phosphatase (PTP) 1B, GLUT4 and phosphatidylinositol (PI) 3-kinase in male rat offspring at 24 h and at 1, 4 and 8 wks post-partum. Quantitative real-time RT-PCR revealed significant age-dependent increases in the expression of IR ß-subunit, IRS-1, PTP1B, GLUT4 and PI3-kinase in the LP group with concomitant increases in corresponding protein abundance at 4 wks of age. These changes were associated with increases in left ventricular (LV) internal diameters as well as increases in LV wall thickness. CONCLUSION: A maternal LP diet can induce increases in the gene expression and protein levels of key components of glucose metabolism and the IR signal transduction pathway in the neonatal rat heart, which may be related to accelerated energy supply, demand and utilization for ventricular remodeling due to compromised contractile performance during early life.
Asunto(s)
Dieta con Restricción de Proteínas/efectos adversos , Glucosa/metabolismo , Corazón/fisiopatología , Fenómenos Fisiologicos Nutricionales Maternos , Animales , Western Blotting , Cardiomegalia/fisiopatología , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Expresión Génica , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Desnutrición/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Embarazo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
The accumulation of lipids within arteries remains to be the initial impulse for the pathogenesis of atherosclerosis; however, both inflammation and oxidative stress are considered to play a critical role in this process. Several lipid lowering drugs are used as the first line therapy in atherosclerosis; however, different agents have been found to exhibit beneficial effects which are independent of their lipid lowering activity. Both statins and fibrates have been reported to exert anti-inflammatory and anti-oxidative effects in addition to their anti-atherosclerotic actions. Furthermore, anti-hypertensive, anti-diabetic and anti-platelet drugs, which reduce oxidative stress and inflammation, have been shown to attenuate atherosclerosis. In addition, novel substances such as HDL-related agents, cyclopentenone prostaglandins, lipoprotein-associated phospholipase A(2) inhibitors, 5-lipoxygenase pathway inhibitors, acyl CoA: cholesterol acyltransferase inhibitors, analogues of probucol and lysophosphatidic acid antagonists have been developed for the treatment of atherosclerosis as a consequence of their actions on oxidative stress and inflammation. The present article reviews the involvement of inflammation and oxidative stress in the pathogenesis of atherosclerosis and focuses on the mechanisms of some clinically used as well as potential anti-atherosclerotic substances with anti-inflammatory and anti-oxidative properties.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Aterosclerosis/tratamiento farmacológico , Hipolipemiantes/farmacología , Inflamación/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Animales , Antiinflamatorios/química , Antioxidantes/química , Aterosclerosis/metabolismo , Aterosclerosis/patología , Diseño de Fármacos , Humanos , Hipolipemiantes/química , Inflamación/metabolismo , Inflamación/patología , Metabolismo de los Lípidos/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: We have previously shown that a maternal low protein (LP) diet during pregnancy results in severe depression of neonatal heart contractility due, in part, to an increase in apoptotic loss of cardiomyocytes. The aim of this study was to examine if maternal LP diet would alter replicative potential of neonatal myocardial cells. METHODS AND RESULTS: We determined the effect of maternal LP and normal diet (90 and 180 g/casein/kg respectively) on relative numbers of mitotic myocardial cells in male offspring at birth and at 7-28 days post-partum. Myocardial cells undergoing mitosis were identified by dual-immunofluorescence of cardiac sections for cardiac muscle myosin and phosphorylated histone 3, whereas cells within the cell cycle were identified by immunoreactivity for Ki67 at 14-28 days post-partum. Neonates from control dams displayed the expected gradual decline in mitotic cells from birth to 28 days post-partum. Hearts from LP offspring had lower numbers of mitotic cells at birth, compared to controls, suggestive of subnormal muscle cell numbers at that stage. When placed in normal diet, LP offspring developed increased myocardial mitosis at 7 days compared to controls, which normalized to control levels at 21-28 days post-partum. An increase in Ki67-positive myocardial cells was also observed in the LP exposed group at 28 days of age. CONCLUSION: Maternal LP diet suppresses myocardial replicative potential and this likely contributes to reduced cell numbers at birth. This suppression is lifted by a protein-replete diet which stimulates post-natal replication of myocardial cells and likely results in a catching-up in cell numbers.
Asunto(s)
Dieta con Restricción de Proteínas/efectos adversos , Fenómenos Fisiologicos Nutricionales Maternos , Mitosis/fisiología , Miocitos Cardíacos/metabolismo , Envejecimiento , Animales , Animales Recién Nacidos , Peso Corporal , Ciclo Celular , Proliferación Celular , Femenino , Corazón/crecimiento & desarrollo , Masculino , Miocardio/metabolismo , Miocardio/patología , Tamaño de los Órganos , Embarazo , RatasRESUMEN
Because the left ventricular (LV) hypertrophy due to volume overload induced by arteriovenous (AV) shunt was associated with an increase in phospholipase C (PLC) isozyme mRNA levels, PLC is considered to be involved in the development of cardiac hypertrophy. Since the renin-angiotensin system (RAS) is activated in cardiac hypertrophy, the role of RAS in the stimulation of PLC isozyme gene expression in hypertrophied heart was investigated by inducing AV shunt in Sprague-Dawley rats. The animals were treated with or without losartan (20 mg/kg, daily) for 3 days as well as 1, 2 and 4 weeks, and atria, right ventricle (RV) and LV were used for analysis. The increased muscle mass as well as the mRNA levels for PLC beta1 and beta3 in atria and RV, unlike PLC beta3 gene expression in LV, at 3 days of AVshunt were attenuated by losartan. The increased gene expression for PLC beta1 at 2 weeks in atria, at 1 and 4 weeks in RV, and at 2 and 4 weeks in LV was also depressed by losartan treatment. Likewise, the elevated mRNA levels for PLC beta3 in RV at 1 week and in LVat 4 weeks of cardiac hypertrophy were decreased by losartan. On the other hand, the increased levels of mRNA for PLC gamma1 in RV and LV at 2 and 4 weeks of inducing hypertrophy, unlike in atria at 4 weeks were not attenuated by losartan treatment. While the increased mRNA level for PLC delta1 in LV was reduced by losartan, gene expression for PLC delta1 was unaltered in atria and decreased in RV at 3 days of inducing AV shunt. These results suggest that changes in PLC isozyme gene expression were chamber specific and time-dependent upon inducing cardiac hypertrophy due to AV shunt. Furthermore, partial attenuation of the increased gene expression for some of the PLC isozymes and no effect of losartan on others indicate that both RAS dependent and independent mechanisms may be involved in hypertrophied hearts due to volume overload.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Cardiomegalia/fisiopatología , Losartán/farmacología , Fosfolipasas de Tipo C/metabolismo , Animales , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Expresión Génica , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Fosfolipasas de Tipo C/genéticaRESUMEN
Evidence indicates that, in addition to the L-type Ca2+ channel blockade, Ca2+-antagonists target other functions including the Ca2+-pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca2+-pumps by verapamil and diltiazem could be due to drug-induced depression of phosphatidylethanolamine (PE) N-methylation which modulates these Ca2+-transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of S-adenosyl-L-methionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10(-6)-10(-3) M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10(-3) M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10(-5) M) was associated with a depression of the Vmax value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE N-methylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [3H-methyl]-methionine following the treatment of animals with verapamil, reduced the synthesis of PC by N-methyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca2+-pump and although diltiazem also inhibited the SR Ca2+-pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca2+-release. These results suggest that verapamil and diltiazem alter the cardiac PE N-methyltransferase system. This action is apparently additional to the drugs' effect on L-type Ca2+ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca2+-pumps and altered cardiac function.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Diltiazem/farmacología , Metiltransferasas/antagonistas & inhibidores , Miocardio/metabolismo , Fosfatidilcolinas/biosíntesis , Verapamilo/farmacología , Animales , Sitios de Unión , Calcio/antagonistas & inhibidores , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , Masculino , Metilación , Metiltransferasas/química , Mitocondrias Cardíacas/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , S-Adenosilmetionina/metabolismo , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
Although an excessive amount of circulating catecholamines is known to induce cardiomyopathy, the mechanisms are poorly understood. This study was undertaken to investigate the role of oxidative stress in catecholamine-induced heart dysfunction. Treatment of rats for 24 h with a high dose (40 mg/kg) of a synthetic catecholamine, isoproterenol, resulted in increased left ventricular end diastolic pressure, depressed rates of pressure development, and pressure decay as well as increased myocardial Ca2+ content. The increased malondialdehyde content, as well as increased formation of conjugated dienes and low glutathione redox ratio were also observed in hearts from animals injected with isoproterenol. Furthermore, depressed cardiac sarcolemmal (SL) ATP-dependent Ca2+ uptake, Ca2+-stimulated ATPase activity, and Na+-dependent Ca2+ accumulation were detected in experimental hearts. All these catecholamine-induced changes in the heart were attenuated by pretreatment of animals with vitamin E, a well-known antioxidant (25 mg/kg/day for 2 days). Depressed cardiac performance, increased myocardial Ca2+ content, and decreased SL ATP-dependent, and Na+-dependent Ca2+ uptake activities were also seen in the isolated rat hearts perfused with adrenochrome, a catecholamine oxidation product (10 to 25 microg/ml). Incubation of SL membrane with different concentrations of adrenochrome also decreased the ATP-dependent and Na+-dependent Ca2+ uptake activities. These findings suggest the occurrence of oxidative stress, which may depress the SL Ca2+ transport and result in the development intracellular Ca2+ overload and heart dysfunction in catecholamine-induced cardiomyopathy.
Asunto(s)
Calcio/metabolismo , Isoproterenol/farmacología , Miocardio/metabolismo , Estrés Oxidativo , Sarcolema/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , ATPasas Transportadoras de Calcio/efectos de los fármacos , Corazón/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Sarcolema/efectos de los fármacos , Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Vitamina E/farmacologíaRESUMEN
The cardiac sarcolemmal membrane cis -unsaturated fatty acid-sensitive phospholipase D hydrolyzes phosphatidylcholine to form phosphatidic acid. The functional significance of phosphatidic acid is indicated by its ability to increase [Ca(2+)](i)and augment cardiac contractile performance via the activation of phospholipase C. Accordingly, we tested the hypothesis that a defect occurs in the membrane level of phosphatidic acid and/or the responsiveness of cardiomyocytes to phosphatidic acid in congestive heart failure due to myocardial infarction. Myocardial infarction was produced in rats by ligation of the left coronary artery while sham-operated animals served as control. At 8 weeks after surgery, the experimental animals were at a stage of moderate congestive heart failure. Compared to sham controls, phosphatidic acid-mediated increase in [Ca(2+)](i), as determined by the fura 2-AM technique, was significantly reduced in failing cardiomyocytes. Immunoprecipitation of sarcolemmal phospholipase C isoenzymes using specific monoclonal antibodies revealed that the stimulation of phospholipase C gamma(1)and delta(1)phosphatidylinositol 4,5-bisphosphate hydrolyzing activities by phosphatidic acid was decreased in the failing heart. Although the activity of phospholipase C beta(1)in the failing heart was higher than the control, phosphatidic acid did not stimulate this isoform in control sarcolemma, and produced an inhibitory action in the failing heart preparation. Furthermore, the specific binding of phosphatidic acid to phospholipase C gamma(1)and delta(1)isoenzymes was decreased, whereas binding to phospholipase beta(1)was absent in the failing heart. A reduction in the intramembranal level of phosphatidic acid derived via cis -unsaturated fatty acid-sensitive phospholipase D was also seen in the failing heart. These findings suggest that a defect in phosphatidic acid-mediated signal pathway in sarcolemma may represent a novel mechanism of heart dysfunction in congestive heart failure.
Asunto(s)
Insuficiencia Cardíaca/enzimología , Isoenzimas/metabolismo , Infarto del Miocardio/enzimología , Ácidos Fosfatidicos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Cationes Bivalentes , Ácidos Grasos Insaturados/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Masculino , Infarto del Miocardio/metabolismo , Fosfolipasa C beta , Fosfolipasa C delta , Fosfolipasa C gamma , Fosfolipasa D/metabolismo , Ratas , Ratas Sprague-Dawley , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Cardiac sarcolemmal (SL) cis-unsaturated fatty acid sensitive phospholipase D (cis-UFA PLD) is modulated by SL Ca(2+)-independent phospholipase A(2) (iPLA(2)) activity via intramembrane release of cis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca(2+) concentration and contractile performance of the cardiomyocyte, changes in iPLA(2) activity may contribute to abnormal function of the failing heart. We examined PLA(2) immunoprotein expression and activity in the SL and cytosol from noninfarcted left ventricular (LV) tissue of rats in an overt stage of congestive heart failure (CHF). Hemodynamic assessment of CHF animals showed an increase of the LV end-diastolic pressure with loss of contractile function. In normal hearts, immunoblot analysis revealed the presence of cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)) in the cytosol, with cPLA(2) and iPLA(2) in the SL. Intracellular PLA(2) activity was predominantly Ca(2+) independent, with minimal sPLA(2) activity. CHF increased cPLA(2) immunoprotein and PLA(2) activity in the cytosol and decreased SL iPLA(2) and cPLA(2) immunoprotein and SL PLA(2) activity. sPLA(2) activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA(2) isoenzymes, suggesting that PLA(2)-mediated bioprocesses are altered in CHF.
Asunto(s)
Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/etiología , Infarto del Miocardio/complicaciones , Fosfolipasas A/metabolismo , Animales , Calcio/fisiología , Citosol/enzimología , Ventrículos Cardíacos , Masculino , Miocardio/enzimología , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Sarcolema/enzimología , Distribución TisularAsunto(s)
Cardiomiopatías/metabolismo , Diabetes Mellitus Experimental/metabolismo , Angiopatías Diabéticas/metabolismo , Miocardio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sarcolema/metabolismo , Animales , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
OBJECTIVE: Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P(2)) is not only a precursor to inositol 1,4,5-trisphosphate (Ins 1,4, 5-P(3)) and sn-1,2 diacylglycerol, but also essential for the function of several membrane proteins. The aim of this study was to evaluate the changes in the level of this phospholipid in the cell plasma membrane (sarcolemma, SL) of cardiomyopathic hamster (CMPH) heart. METHODS: We examined the cardiac SL PtdIns 4,5-P(2) mass and the activities of the enzymes responsible for its synthesis and hydrolysis in 250-day-old UM-X7.1 CMPH at a severe stage of congestive heart failure (CHF) and in age-matched controls (Syrian Golden hamsters). RESULTS: The SL PtdIns 4,5-P(2) mass in CMPH was reduced by 72% of the control value. The activities of PtdIns 4 kinase and PtdIns 4-P 5 kinase were depressed by 69 and 50% of control values, respectively. Although, the total phospholipase C (PLC) activity was moderately, although significantly, decreased (by 18% of control), PLCdelta(1) isoenzyme activity in the SL membrane was elevated, with a concomitant increase in its protein content, whereas PLCbeta(1) and gamma(1) isoenzyme activities were depressed despite the increase in their protein levels. A 2-fold increase in the Ins 1,4,5-P(3) concentration in the cytosol of the failing heart of CMPH was also observed. CONCLUSIONS: Reduced SL level of PtdIns 4, 5-P(2) may severely jeopardize cardiac cell function in this hamster model of CHF. In addition, the profound changes in the profile of heart SL PLC isoenzyme could alter the complex second messenger responses of these isoenzymes, and elevated Ins 1,4,5-P(3) levels may contribute to intracellular Ca(2+) overload in the failing cardiomyocyte.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sarcolema/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Western Blotting , Cricetinae , Citosol/metabolismo , Insuficiencia Cardíaca/enzimología , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/metabolismo , Masculino , Mesocricetus , Sarcolema/enzimología , Sistemas de Mensajero Secundario/fisiología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is the substrate for phosphoinositide-phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. PtdIns 4,5-P2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P2. Exposure to the xanthine-xanthine oxidase-ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine-xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an *OH scavenger) did not modify the H2O2-induced depression of the kinases, eliminating any role of *OH. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 microM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutathione induced a significant depression. Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the redox ratio was observed. Thiol modifiers (N-ethylmaleimide, methyl methanethiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction.
Asunto(s)
Miocardio/metabolismo , Oxidantes/metabolismo , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Sarcolema/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Glutatión/farmacología , Disulfuro de Glutatión/farmacología , Corazón/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Ácido Hipocloroso/farmacología , Concentración 50 Inhibidora , Cinética , Oxidación-Reducción , Estrés Oxidativo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ratas , Sarcolema/efectos de los fármacos , Factores de TiempoRESUMEN
We have examined the changes in quantity and activity of cardiac sarcolemmal (SL) phosphoinositide-phospholipase C (PLC)-beta(1), -gamma(1), and -delta(1) in a model of congestive heart failure (CHF) secondary to large transmural myocardial infarction (MI). We also instituted a late in vivo monotherapy with imidapril, an ANG-converting enzyme (ACE) inhibitor, to test the hypothesis that its therapeutic action is associated with the functional correction of PLC isoenzymes. SL membranes were purified from the surviving left ventricle of rats in a moderate stage of CHF at 8 wk after occlusion of the left anterior descending coronary artery. SL PLC isoenzymes were examined in terms of protein mass and hydrolytic activity. CHF resulted in a striking reduction (to 6-17% of controls) of the mass and activity of gamma(1)- and delta(1)-isoforms in combination with a significant increase of both PLC beta(1) parameters. In vivo treatment with imidapril (1 mg/kg body wt, daily, initiated 4 wk after coronary occlusion) improved the contractile function and induced a partial correction of PLCs. The mass of SL phosphatidylinositol 4,5-bisphosphate and the activities of the enzymes responsible for its synthesis were significantly reduced in post-MI CHF and partially corrected by imidapril. The results indicate that profound changes in the profile of heart SL PLC-beta(1), -gamma(1), and -delta(1) occur in CHF, which could alter the complex second messenger responses of these isoforms, whereas their partial correction by imidapril may be related to the mechanism of action of this ACE inhibitor.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Imidazoles/uso terapéutico , Imidazolidinas , Infarto del Miocardio/complicaciones , Sarcolema/efectos de los fármacos , Sarcolema/enzimología , Fosfolipasas de Tipo C/efectos de los fármacos , Animales , Citosol/efectos de los fármacos , Citosol/enzimología , Insuficiencia Cardíaca/etiología , Isoenzimas , Masculino , Ratas , Ratas Sprague-DawleyAsunto(s)
Insuficiencia Cardíaca/enzimología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/enzimología , Miocardio/enzimología , Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Sarcolema/enzimología , Animales , Insuficiencia Cardíaca/etiología , Humanos , Isoenzimas/metabolismo , Fosfolipasa C beta , Fosfolipasa C delta , Fosfolipasa C gamma , Fosfolipasas de Tipo C/metabolismoRESUMEN
Although fibroblast growth factor-2 (FGF-2) plays an important role in cardioprotection and growth, little is known about the signals triggered by it in the adult heart. We therefore examined FGF-2-induced effects on phosphoinositide-specific phospholipase C (PI-PLC) isozymes, which produce second messengers linked to the inotropic and hypertrophic response of the myocardium. FGF-2, administered by retrograde perfusion to the isolated heart, induced an increase in inositol-1,4,5-trisphosphate levels in the cytosol, as well as an increase in total PI-PLC activity associated with sarcolemmal and cytosolic fractions. Furthermore FGF-2 induced a time-dependent elevation in cardiomyocyte membrane-associated PLC gamma1 and PLC beta1 activities, assayed in immunoprecipitated fractions, and moreover, increased the membrane levels of PLC beta1 and PLC beta3. Activation of PLC beta is suggestive of FGF-2-induced cross-talk between FGF-receptor tyrosine kinase and G-protein-coupled signaling in adult cardiomyocytes and underscores the importance of FGF-2 in cardiac physiology.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Isoenzimas/metabolismo , Miocardio/enzimología , Fosfolipasas de Tipo C/metabolismo , Animales , Células Cultivadas , Masculino , Miocardio/citología , Miocardio/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Fosfolipasa C beta , Fosfolipasa C gamma , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
The effect of phospholipase A2 (PLA2)-dependent release of unsaturated fatty acids (FA) on phospholipase D (PLD) function was examined in purified sarcolemmal (SL) membranes isolated from rat heart. PLD hydrolytic activity was determined by measuring either [14C] phosphatidic acid formation from exogenous [14C] phosphatidylcholine (PtdCho) or [3H] choline release from prelabelled SL Ptd[3H]choline. SL membranes with endogenous [3H] PtdCho that were prelabelled with [3H] myristic acid were used for testing PLD transphosphatidylation activity. Exogenous cis-unsaturated FA, arachidonate and oleate, significantly enhanced the [3H] choline formation at 50 and 100 microM, respectively; their effect was maximal at 250 microM and declined at higher concentrations. Use of melittin (which stimulates membrane-bound PLA2, thus releasing FA) or exogenous PLA2 reproduced the stimulatory effect of added arachidonate and oleate. Under melittin, PLA2-dependent FA release was strongly correlated (r = 0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphatidylation activity confirmed the augmented catalytic rate of PLD by these agents. Melittin-evoked PLD activation was completely blocked by 1 microM E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective inhibitor of Ca(2+)-independent v Ca(2-)-dependent PLA2, thus indicating that PLD stimulation under melittin occurred via PLA2. Activity measurement and Western blotting studies revealed the presence of a Ca(2+)-independent, high molecular weight (110 kDa) PLA2 in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These results suggest that Ca(2+)-independent PLA2 and subsequent endogenous mobilization of sn-2 unsaturated FA modulate PLD activity in heart SL membranes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA2 as well as in heart diseases characterized by PLA2 pathological dysfunction.
Asunto(s)
Miocardio/metabolismo , Fosfolipasa D/metabolismo , Fosfolipasas A/metabolismo , Sarcolema/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Ácido Araquidónico/farmacología , Calcio/metabolismo , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Meliteno/farmacología , Naftalenos/farmacología , Ácido Oléico/metabolismo , Ácido Oléico/farmacocinética , Ácido Oléico/farmacología , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacocinética , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasa D/efectos de los fármacos , Fosfolipasas A/inmunología , Fosfolipasas A/farmacología , Fosfolipasas A2 , Pironas/farmacología , Ratas , Sarcolema/efectos de los fármacosRESUMEN
A method for the isolation of highly purified peroxisomes from guinea pig small intestine was developed. This two-stage process involved a rate-dependent banding of a light-mitochondria lambda-fraction followed by a density-dependent banding of the catalase enriched fractions obtained from the first step, using a horizontal rotor. Furthermore, the subcellular localization of glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in guinea pig small intestine was examined. Analysis of density-gradient fractions indicated that approximately 3-4% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and that it is localized to the matrix of peroxisomes. It is therefore suggested that a peroxisomal source of NADPH may be utilized by enzyme systems that use NADPH specifically as a reductant.
Asunto(s)
Glucosafosfato Deshidrogenasa/metabolismo , Intestino Delgado/metabolismo , Microcuerpos/enzimología , Animales , Biomarcadores/química , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad , Cobayas , Masculino , Metrizamida/metabolismo , Microcuerpos/ultraestructura , Microscopía Electrónica , NADP/metabolismo , Octoxinol/farmacologíaRESUMEN
BACKGROUND: Large transmural myocardial infarction (MI) leads to maladaptive cardiac remodeling and places patients at increased risk of congestive heart failure. Angiotensin II, endothelin, and alpha1-adrenergic receptor agonists are implicated in the development of cardiac hypertrophy, interstitial fibrosis, and heart failure after MI. Because these agonists are coupled to and activate Gq alpha protein in the heart, the aim of the present study was to investigate Gq alpha expression and function in cardiac remodeling and heart failure after MI. METHODS AND RESULTS: MI was produced in rats by ligation of the left coronary artery, and Gq alpha protein concentration, localization, and mRNA abundance were noted in surviving left ventricle remote from the infarct and in border and scar tissues from 8-week post-MI hearts with moderate heart failure. Immunohistochemical staining localized elevated Gq alpha expression in the scar and border tissues. Western analysis confirmed significant upregulation of Gq alpha proteins in these regions versus controls. Furthermore, Northern analysis revealed that the ratios of Gq alpha/GAPDH mRNA abundance in both scar and viable tissues from experimental hearts were significantly increased versus controls. Increased expression of phospholipase C (PLC)-beta1 and PLC-beta3 proteins was apparent in the scar and viable tissues after MI versus controls and is associated with increased PLC-beta1 activity in experimental hearts. Furthermore, inositol 1,4,5-tris-phosphate is significantly increased in the border and scar tissues compared with control values. CONCLUSIONS: Upregulation of the Gq alpha/PLC-beta pathway was observed in the viable, border, and scar tissues in post-MI hearts. Gq alpha and PLC-beta may play important roles in scar remodeling as well as cardiac hypertrophy and fibrosis of the surviving tissue in post-MI rat heart. It is suggested that the Gq alpha/PLC-beta pathway may provide a possible novel target for altering postinfarct remodeling.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Insuficiencia Cardíaca/metabolismo , Isoenzimas/metabolismo , Infarto del Miocardio/complicaciones , Miocardio/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Cicatriz/metabolismo , Colágeno/análisis , Fibrosis/metabolismo , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/metabolismo , Inmunohistoquímica , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Fosfolipasa C beta , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
The production of pro-inflammatory cytokines, such as interleukins 1 and 6 and tumour necrosis factors, occurs rapidly following trauma or invasion of the body by pathogenic organisms. The cytokines mediate the wide range of symptoms associated with trauma and infection, such as fever, anorexia, tissue wasting, acute phase protein production and immunomodulation. In part, the symptoms result from a co-ordinated response, in which the immune system is activated and nutrients released, from endogenous sources, to provide substrate for the immune system. Although the cytokine mediated response is an essential part of the response to trauma and infection, excessive production of pro-inflammatory cytokines, or production of cytokines in the wrong biological context, are associated with mortality and pathology in a wide range of diseases, such as malaria, sepsis, rheumatoid arthritis, inflammatory bowel disease, cancer and AIDS. Cytokine biology can be modulated by antiinflammatory drugs, recombinant cytokine receptor antagonists and nutrients. Among the nutrients, fats have a large potential for modulating cytokine biology. A number of trials have demonstrated the anti-inflammatory effects of fish oils, which are rich in n-3 polyunsaturated fatty acids, in rheumatoid arthritis, inflammatory bowel disease, psoriasis and asthma. Animal studies, conducted by ourselves and others, indicate that a range of fats can modulate pro-inflammatory cytokine production and actions. In summary fats rich in n-6 polyunsaturated fatty acids enhance IL1 production and tissue responsiveness to cytokines, fats rich in n-3 polyunsaturated fatty acids have the opposite effect, monounsaturated fatty acids decrease tissue responsiveness to cytokines and IL6 production is enhanced by total unsaturated fatty acid intake. There are a large number of potential cellular mechanisms which may mediate the effects observed. The majority relate to the ability of fats to alter the composition of membrane phospholipids. As a consequence of alterations in phospholipid composition, membrane fluidity may change, altering binding of cytokines to receptors and G protein activity. The nature of substrate for various signalling pathways associated with cytokine production and actions may also be changed. Consequently, alterations in eicosanoid production and activation of protein kinase C may occur. We have examined a number of these potential mechanisms in peritoneal macrophages of rats fed fats with a wide range of fatty acid composition. We have found that the total C18:2 and 20:4 diacyl species of phosphatidylethanolamine in peritoneal macrophages relates in a positive curvilinear fashion with dietary linoleic acid intake; that TNF induced IL1 and IL6 production relate in a positive curvilinear fashion to linoleic acid intake; that leukotriene B4 production relates positively with dietary linoleic acid intake over a range of moderate intakes and is suppressed at high intakes, while PGE2 production is enhanced. There was no clear relationship between linoleic acid intake and membrane fluidity, however fluidity was influenced in a complex manner by the type of fat in the diet, the period over which the fat was fed and the presence of absence of TNF stimulation. None of the proposed mechanisms, acting alone, can explain the positive effect of dietary linoleic acid intake on pro-inflammatory cytokine production. However each may be involved, in part, in the modulatory effects observed.
Asunto(s)
Citocinas/fisiología , Grasas Insaturadas en la Dieta , Ácidos Grasos Insaturados/farmacología , Animales , Artritis Reumatoide/dietoterapia , Asma/dietoterapia , Membrana Celular/fisiología , Citocinas/biosíntesis , Humanos , Inflamación , Enfermedades Inflamatorias del Intestino/dietoterapia , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Psoriasis/dietoterapia , Ratas , Receptores de Citocinas/fisiologíaRESUMEN
Experimental evidence suggests that the myocardial phospholipase D (PLD)-phosphatidate phosphohydrolase (PAP) signalling pathway may regulate Ca2+ movements and contractile performance of the heart. As abnormal Ca2+ homeostasis is associated with diabetic cardiomyopathy, we examined the functional status of the PLD/PAP pathway in sarcolemmal (SL) membranes isolated from insulin-dependent diabetic rat hearts at 8 weeks after a single i.v. injection of streptozotocin (65 mh/kg b.w.). Compared to age-matched controls, SL PLD hydrolytic (producing phosphatidic acid, PtdOH) and transphosphatidylation activities were significantly depressed in diabetic animals, while SL PAP was significantly augmented. The net effect of the altered enzyme activities in diabetic animals was a severely diminished (by 67% of controls) membrane level of PLD-derived PtdOH. Two weeks of insulin therapy to the 6 week diabetic animals normalized PLD, while PAP activity and PtdOH level were significantly modified, but had not completely reverted to control values. The observed changes were not due to hypothyroidism associated to the diabetic model as the induction of hypothyroidism in healthy non-diabetic animals did not affect SL PLD and PAP. The results suggest that the severe reduction of PLD-derived PtdOH and increased production of sn-1,2-diacylglycerol by phosphatidate phosphohydrolase may lead to an impairment of the bioprocesses mediated by these signalling lipids.