RESUMEN
OBJECTIVES: To determine sarcolemmal Na+/H+ exchanger (NHE) activity and expression in human ventricular myocardium. BACKGROUND: Although the sarcolemmal NHE has been implicated in various physiological and pathophysiological phenomena in animal studies, its activity and expression in human myocardium have not been studied. METHODS: Ventricular myocardium was obtained from unused donor hearts with acute myocardial dysfunction (n = 5) and recipient hearts with chronic end stage heart failure (n = 11) through a transplantation program. Intracellular pH (pHi) was monitored in enzymatically isolated single ventricular myocytes by microepifluorescence. As the index of sarcolemmal NHE activity, the rate of H+ efflux at a pHi of 6.90 J(H6.9)) was determined after the induction of intracellular acidosis in bicarbonate-free medium. Na+/H+ exchanger isoform 1 (NHE1) expression in ventricular myocardium was determined by immunoblot analysis. RESULTS: Human ventricular myocytes exhibited readily detectable sarcolemmal NHE activity after the induction of intracellular acidosis, and this activity was suppressed by the NHE1-selective inhibitor HOE-642 (cariporide) at 1 micromol/L. Sarcolemmal NHE activity of myocytes was significantly greater in recipient hearts (JH6.9 = 1.95+/-0.18 mmol/L/min) than it was in unused donor hearts (J(H6.9 = 1.06+/-0.15 mmol/L/min). In contrast, NHE1 protein was expressed in similar abundance in ventricular myocardium from both recipient and unused donor hearts. CONCLUSIONS: Sarcolemmal NHE activity of human ventricular myocytes arises from the NHE1 isoform and is inhibited by HOE-642. Sarcolemmal NHE activity is significantly greater in recipient hearts with chronic end-stage heart failure than it is in unused donor hearts, and this difference is likely to arise from altered posttranslational regulation.
Asunto(s)
Miocardio/citología , Miocardio/metabolismo , Sarcolema/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Adulto , Antiarrítmicos/farmacología , Femenino , Guanidinas/farmacología , Ventrículos Cardíacos , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Sulfonas/farmacologíaRESUMEN
Increased sarcolemmal Na(+)/H(+) exchanger activity has been implicated as a mediator of the cardiac actions of angiotensin II. We studied the receptor subtypes and signaling pathways involved in the regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes. Cells were loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, and acid efflux rates estimated during recovery from intracellular acidosis were used to quantify exchanger activity. Sarcolemmal Na(+)/H(+) exchanger activity was not affected by angiotensin II alone but was increased by angiotensin II plus PD123319 (AT(2) antagonist). In contrast, angiotensin II plus losartan (AT(1) antagonist) or CGP42112A (AT(2) agonist) did not affect exchanger activity. The increase in Na(+)/H(+) exchanger activity induced by angiotensin II plus PD123319 was blocked by losartan, PD98059 (extracellular signal-regulated kinase inhibitor), GF109203X (protein kinase C inhibitor), and tyrphostin AG1478 (epidermal growth factor receptor kinase inhibitor). Extracellular signal-regulated kinase phosphorylation and activity, measured by immunoblot analysis and an immune-complex kinase assay, respectively, were increased significantly by angiotensin II plus PD123319; these increases were blocked by losartan and PD98059. The increase in extracellular signal-regulated kinase phosphorylation induced by angiotensin II plus PD123319 was blocked also by GF109203X and tyrphostin AG1478. These data show that AT(1) stimulation increases sarcolemmal Na(+)/H(+) exchanger activity in adult rat ventricular myocytes and that this response requires extracellular signal-regulated kinase activation through a protein kinase C- and epidermal growth factor receptor-mediated mechanism. The positive effect of AT(1) stimulation on Na(+)/H(+) exchanger activity is counteracted by simultaneous AT(2) stimulation through a mechanism that does not involve direct inhibition of the exchanger or attenuation of extracellular signal-regulated kinase activation.
Asunto(s)
Angiotensina II/fisiología , Miocardio/metabolismo , Receptores de Angiotensina/fisiología , Sarcolema/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Angiotensina II/farmacología , Animales , Animales Recién Nacidos/metabolismo , Senescencia Celular/fisiología , Ventrículos Cardíacos , Masculino , Proteínas Quinasas Activadas por Mitógenos/fisiología , Miocardio/citología , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/metabolismo , Sarcolema/efectos de los fármacosRESUMEN
CYP3A4 is involved in the metabolism of numerous biologically active compounds, including testosterone. A genetic variant located in the P450NF (nifedipine) specific element (NFSE) has been identified that disrupts a transciptional regulatory element located in the 5' regulatory region of CYP3A4. The CYP3A4 variant (CYP3A4-V) is associated with the clinical presentation of prostate cancer. There are significant differences in CYP3A4 metabolism and rates of prostate cancer across ethnic groups that may be associated with CYP3A4 genotypes. Therefore, we estimated the frequency of the CYP3A4 variant in three ethnic groups with different prostate cancer incidence rates. The frequency (q) of CYP3A4-V was significantly different (p<0.0001) in African Americans (q=0.53), U.S. Caucasians (q=0.09), and Taiwanese (q=0.0). CYP3A4-V segregated in a Mendelian manner in one large African American family, and 7 of 16 (44%) biologically unrelated "marry-ins" carried a CYP3A4 variant allele. Reflecting population-specific prostate cancer incidence rates, our results suggest a high frequency of this variant in African Americans compared with U.S. Caucasians and Taiwanese.
Asunto(s)
Alelos , Sistema Enzimático del Citocromo P-450/genética , Variación Genética/genética , Oxigenasas de Función Mixta/genética , Neoplasias de la Próstata/etnología , Neoplasias de la Próstata/genética , Pueblo Asiatico/genética , Población Negra/genética , Citocromo P-450 CYP3A , Humanos , Masculino , Factores de Riesgo , Población Blanca/genéticaRESUMEN
The purpose of the present study was to devise a technique for the isolation of a relatively homogeneous mononuclear leukocyte (MNL) preparation from rabbit whole blood and determine the density and affinity of beta-adrenoceptors on MNL. A modified method based upon that by Böyum was developed to maximize isolation of MNL from red blood cells (RBC), granulocytes, and platelets. The method involved an initial centrifugation to remove platelets and two centrifugations with a Ficoll solution to eliminate RBC and granulocytes. beta-Adrenoceptor density as determined with [125I]cyanopindolol and MNL membrane or whole cell preparations ranged between 317 and 360 sites per cell. Affinity for the binding sites was dependent upon whether membrane or whole cell preparations were studied, being 56.3 +/- 9.9 and 11.4 +/- 1.4 pM, respectively. Binding sites were found to be saturable and noncooperative. In addition, the binding sites demonstrated selectivity and stereospecificity for beta-adrenoceptor ligands. It is concluded that the modified method of harvesting MNL from rabbit whole blood provides a relatively homogeneous cell suspension that can be used to study the beta-adrenoceptor system.