RESUMO
Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.
Assuntos
Coração/efeitos dos fármacos , Miocárdio/metabolismo , Citrato de Sildenafila/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acidose/enzimologia , Acidose/metabolismo , Acidose/patologia , Animais , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/citologia , Miocárdio/patologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio/metabolismoRESUMO
Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. However, the molecular mechanisms underlying these arrhythmias are still unclear. To better understand this process, acidosis produced by CO2 increase from 5% to 30%, resulting in intracellular pH (pHi) change from 7.15 to 6.7, was incorporated into a myocyte model of excitation-contraction coupling and contractility, including acidotic inhibition of L-type Ca(2+) channel (I(CaL)), Na(+)-Ca(2+) exchanger, Ca(2+) release through the SR ryanodine receptor (RyR2) (I(rel)), Ca(2+) reuptake by the SR Ca(2+) ATPase2a (I(up)), Na(+)-K(+) pump, K(+) efflux through the inward rectifier K(+) channel and the transient outward K(+) flow (I(to)) together with increased activity of the Na(+)-H(+) exchanger (I(NHE)). Simulated CaMKII regulation affecting I(rel), I(up), I(CaL), I(NHE) and I(to) was introduced in the model to partially compensate the acidosis outcome. Late Na(+) current increase by CaMKII was also incorporated. Using this scheme and assuming that diastolic Ca(2+) leak through the RyR2 was modulated by the resting state of this channel and the difference between SR and dyadic cleft [Ca(2+)], postacidotic delayed after depolarizations (DADs) were triggered upon returning to normal pHi after 6 min acidosis. The model showed that DADs depend on SR Ca(2+) load and on increased Ca(2+) leak through RyR2. This postacidotic arrhythmogenic pattern relies mainly on CaMKII effect on I(CaL) and I(up), since its individual elimination produced the highest DAD reduction. The model further revealed that during the return to normal pHi, DADs are fully determined by SR Ca(2+) load at the end of acidosis. Thereafter, DADs are maintained by SR Ca(2+) reloading by Ca(2+) influx through the reverse NCX mode during the time period in which [Na(+)]i is elevated.
Assuntos
Acidose/enzimologia , Arritmias Cardíacas/enzimologia , Simulação por Computador , Potenciais da Membrana , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Acidose/complicações , Acidose/patologia , Acidose/fisiopatologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Humanos , Canais Iônicos/metabolismo , Transporte de Íons , Proteínas Musculares/metabolismo , Miócitos Cardíacos/patologiaRESUMO
Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca(2+)-ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin-induced diabetes. We have also examined the influence of the acidosis state on this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH(4)Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca(2+)-ATPase (total, independent, and dependent) was determined in the homogenate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be higher in the diabetic animals. Ca(2+)-ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Diabetes Mellitus Experimental/enzimologia , Microssomos/enzimologia , Glândulas Salivares/citologia , Glândulas Salivares/enzimologia , Acidose/complicações , Acidose/enzimologia , Animais , Cálcio/metabolismo , Diabetes Mellitus Experimental/complicações , Masculino , Glândula Parótida/enzimologia , Ratos , Ratos Wistar , Frações Subcelulares/enzimologia , Glândula Submandibular/enzimologiaRESUMO
Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.
Assuntos
Acidose/complicações , Arritmias Cardíacas/etiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Miócitos Cardíacos/enzimologia , Retículo Sarcoplasmático/metabolismo , Acidose/enzimologia , Acidose/fisiopatologia , Potenciais de Ação , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/fisiopatologia , Benzilaminas/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Dantroleno/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Ratos , Ratos Wistar , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sulfonamidas/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , Função Ventricular Esquerda , Pressão VentricularRESUMO
The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effect were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.
Assuntos
Acidose/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Isoenzimas/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Fosfatos/metabolismo , Fosfatos/fisiologia , CoelhosRESUMO
Antecedentes: La cocarboxilasa no degradable (CND), es una coenzima importante para la síntesis de la deshidrogenasa pirúvica, enzima que regula el catabolismo del piruvato y posiblemente la clave para la recuperación de la acidosis metabólica. Objetivo: Evaluar la eficacia de la cocarboxilasa no degradable para manejar la acidosis metabólica del recién nacido. Material y métodos: Se realizó un estudio doble ciego controlado, seleccionado aleatoriamente 30 neonatos durante un año, con diagnóstico gasométrico de acidosis metabólica (pH de 7.30 o menos, bicarbonato de 18 o menos, y exceso de base de -9 o mayor). Se administró solución isotónica de cloruro de sodio (grupo I) 1ml/kg, bicarbonato de sodio (grupo II) 2mEq/kg y CND (grupo III) a razón de 40 mg/kg. El volumen en todos los casos fue de 1 mL/kg. La primera dosis se administró en cuanto se realizó el diagnóstico y las dos siguientes con espacio de 8 horas. Se determinó el efecto de las sustancias en el pH y la corrección de la acidosis metabólica. Resultados: Los resultados mostraron un discreto aumento del pH con la CND sin diferencias para ninguna de las sustancias sobre la acidosis metabólica
Assuntos
Recém-Nascido , Humanos , Acidose/enzimologia , Carboxiliases/farmacocinética , Carboxiliases/uso terapêutico , Bicarbonato de Sódio/uso terapêutico , Cloreto de Sódio/uso terapêuticoRESUMO
Two patients with biotin-responsive multiple carboxylase deficiency, both presenting with predominant lactic acidosis, are reported. One with disease of early neonatal onset had considerable acute neurologic and persistent dermatologic abnormalities. The other, with late juvenile-onset disease, had chronic neurologic abnormalities without dermatologic findings. Early-onset cases generally have been associated with holocarboxylase synthetase deficiency, whereas those of juvenile onset have been characterized as representing defects in intestinal biotin absorption. However, enzyme analyses of fibroblasts from both patients, grown in biotin-deficient medium, revealed markedly diminished activities of pyruvate, propionyl-CoA, and beta-methylcrotonyl-CoA carboxylases, and all three enzymes showed normal activities after growth in biotin-rich medium. Furthermore, lymphoblast enzyme analysis in the patient with disease of early onset had previously revealed a defect in holocarboxylase synthetase, and fibroblast complementation studies showed that both patients belong to the bio complementation group. These findings indicate that considerable clinical heterogeneity exists among patients with holocarboxylase synthetase deficiency, an observation which does not permit differentiation of the biochemical forms of multiple carboxylase deficiency on the basis of age at onset and clinical presentation.
Assuntos
Acidose/genética , Biotina/deficiência , Carbono-Carbono Ligases , Carbono-Nitrogênio Ligases , Lactatos/metabolismo , Ligases/deficiência , Acidose/enzimologia , Fatores Etários , Carboxiliases/deficiência , Células Cultivadas , Pré-Escolar , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Absorção Intestinal , Ácido Láctico , Linfócitos/enzimologia , Masculino , Metilmalonil-CoA Descarboxilase , Doença da Deficiência de Piruvato CarboxilaseRESUMO
We have studied a patient with 5-oxoprolinuria who presented with hemolysis and metabolic acidosis as a neonate; he has had normal growth and development to one year of age. Compensated hemolytic anemia persists, and he requires alkalinizing agents for correction of acidosis. Biochemical studies have confirmed that a deficiency of glutathione synthetase is responsible for the 5-oxoprolinuria. Genetic heterogeneity was apparent on comparative study of glutathione synthetase kinetics in cells from two patients with this disorder. The consequences of the deficiency of glutathione synthetase, decreased intracellular glutathione, and overproduction of 5-oxoproline are discussed with reference to the possible cellular roles of these compounds.
Assuntos
Glutationa Sintase/deficiência , Peptídeo Sintases/deficiência , Pirrolidinonas/urina , Ácido Pirrolidonocarboxílico/urina , Acidose/enzimologia , Aberrações Cromossômicas/enzimologia , Transtornos Cromossômicos , Eritrócitos/enzimologia , Fibroblastos/enzimologia , Glutationa/metabolismo , Humanos , Lactente , Recém-Nascido , Icterícia Neonatal/enzimologia , Leucócitos/enzimologia , Masculino , MutaçãoRESUMO
Slices of duodenum and jejunum produce ammonia from glutamine in vitro. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. Gut contains glutamine 1 as well as ç-glutamyl transpeptidase. These enzymes do not show any increase during starvation (SUMMARY)
Assuntos
21003 , Ratos , Técnicas In Vitro , Amônia , Glutamina/metabolismo , Intestino Delgado/metabolismo , Acidose/enzimologia , Doença Aguda , Doença Crônica , Duodeno/metabolismo , gama-Glutamiltransferase/metabolismo , Glutaminase/metabolismo , Jejuno/metabolismo , Especificidade de Órgãos , InaniçãoRESUMO
The kinetics of the induction of rat kidney phosphoenolpyruvate carboxykinase activity after triamcinolone and ammonium chloride administration have been investigated with a view to the further differentiation of the two processes. The half-life of kidney phosphoenolpyruvate carboxykinase activity, as measured from the decay curve after a single dose of triamcinolone, is approximately 1.4 hr. This compares with a half-life for the enzyme from acidotic kidney of approximately 3.4 hr. Analysis of the data indicates that the induction of phosphoenolpyruvate carboxykinase activity by triamcinolone may be attributed to an increase in de novo protein synthesis. Induction by acidosis is qualitatively distinct and is partly attributed to a reduction in the rate of decay of phosphoenolpyruvate carboxykinase activity. The activities of the gluconeogenic enzymes glucose-6-phosphotase, fructose-1,6-diphosphotase and phosphoenolpyruvate carboxykinase in both liver and kidney have been measured in animals separately treated with triamcinolone and ammonium chloride. Triamcinolone significantly increases the activities of liver phosphoenolpyruvate carboxykinase, kidney glucose-6-phosphatase, and kidney phosphoenolpyruvate carboxykinase only; ammonium chloride stimulates a 200 percent increase in kidney phosphoenolpyruvate carboxykinase, but has no effect on the other enzymes. The induction processes whereby triamcinolone increases phosphoenolpyruvate carboxykinase activities in liver and kidney differ qauntitatively. (AU
Assuntos
Ratos , 21003 , Masculino , Acidose/enzimologia , Cloreto de Amônio , Carboxiliases/metabolismo , Rim/enzimologia , Triancinolona Acetonida/farmacologia , Acidose/classificação , Adrenalectomia , Cloreto de Amônio/administração & dosagem , Cloreto de Amônio/farmacologia , Indução Enzimática , Frutose-Bifosfatase/análise , Glucose-6-Fosfatase/análise , Injeções Intramusculares , Injeções Intraperitoneais , Córtex Renal/enzimologia , Fígado/enzimologia , /metabolismo , Fatores de Tempo , Triancinolona Acetonida/administração & dosagemRESUMO
A method for the assay of phosphoenolpyruvate carboxykinase is presented, based on the enzymic determination of the phosphoenolpyruvate produced by the enzyme reaction. The subcellular distribution of phosphoenolpyruvate carboxykinase in the kidney of several animal species resembled the distribution in the liver. The rise in enzyme activity in the kidney cortex of rats made acidotic by feeding with ammonium chloride was not prevented by the administration of ethionine or actinomycin. The possibility is suggested that in the kidney acidosis causes activation of an inactive form of the enzyme already present. (AU)
Assuntos
Humanos , Cães , Cobaias , Coelhos , Ratos , 21003 , Acidose/enzimologia , Carboxiliases/análise , Rim/enzimologia , Acidose/induzido quimicamente , Cloreto de Amônio , Fracionamento Celular , Citoplasma/enzimologia , Dactinomicina/farmacocinética , Ativação Enzimática , Etionina/farmacocinética , Rim/efeitos dos fármacos , Mitocôndrias/enzimologia , Fosfoenolpiruvato/metabolismo , UltracentrifugaçãoRESUMO
Experiments were done on rats to investigate the nature of the renal response to metabolic acidosis and the changes in enzyme activity associated with increased ammoniagenesis. When metabolic acidosis was induced with oral feeding of ammonium chloride for 48 hr, there was an increase of activity of the enzyme phosphoenolpyruvate carboxykinase (PEPCK) in whole kidneys as well as in the kidney cortex. There was no change in PEPCK in liver, and glucose-6-phosphatase showed no change in kidney or liver in response to metabolic acidosis. The increase in PEPCK activity in kidney cortex varied with the degree of acidosis and there was a close correlation between cortical PEPCK activity and urinary ammonia. Kidney cortex mitochondrial PEPCK did not change in response to metabolic acidosis. An increase in PEPCK occurred as early as 6 hr after NH4Cl feeding, before there was any increase in kidney glutaminase I activity. Rats fed sodium phosphate, or given triamcinolone intramuscularly, developed a metabolic alkalosis, but there was increased urinary ammonia and an increase in activity of renal cortical PEPCK. Triamcinolone plus ammonium chloride induced a greater increase of PEPCK activity than triamcinolone by itself; on the contrary, the rise of glucose-6-phosphatase induced by triamcinolone was not enhanced by acidosis. Glucose-6-phosphatase from control and acidotic rats had identical kinetic characteristics. The results indicate that increased PEPCK activity is constantly related to increases of urinary ammonia. It is proposed that the increase of PEPCK activity is the key event in the ammoniagenesis and gluconeogenesis which follow on metabolic acidosis. (AU)