RESUMEN
Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.
Asunto(s)
Arginina/metabolismo , Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/metabolismo , Caveolas/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Aorta , Arginina/farmacología , Western Blotting , Bradiquinina/farmacología , Bovinos , Caveolas/efectos de los fármacos , Caveolas/metabolismo , Cromatografía Líquida de Alta Presión , Citrulina/farmacología , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Óxido Nítrico Sintasa de Tipo IIIRESUMEN
Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore- (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to approximately 125 kDa and 2 minor bands corresponding to approximately 105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular , Óxido Nítrico/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Vanadatos/farmacología , Animales , Proteínas Portadoras/farmacología , Bovinos , Compartimento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/fisiología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Receptores de Superficie Celular/metabolismo , Tirosina/metabolismoRESUMEN
Insulin resistance in the liver and peripheral tissues together with a pancreatic cell defect are the common causes of type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibiton of PTPases may be an effective method in the treatment of type 2 diabetes. A series of azolidinediones has been prepared as protein tyrosine phosphatase 1B (PTP1B) inhibitors. Several compounds were potent inhibitors against the recombinant rat and human PTP1B enzymes with submicromolar IC(50) values. Elongated spacers between the azolidinedione moiety and the central aromatic portion of the molecule as well as hydrophobic groups at the vicinity of this aromatic region were very important to the inhibitory activity. Oxadiazolidinediones 87 and 88 and the corresponding acetic acid analogues 119 and 120 were the best h-PTP1B inhibitors with IC(50) values in the range of 0.12-0.3 microM. Several compounds normalized plasma glucose and insulin levels in the ob/ob and db/db diabetic mouse models.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Hipoglucemiantes/síntesis química , Proteínas de la Membrana/antagonistas & inhibidores , Oxazoles/síntesis química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , 4-Nitrofenilfosfatasa/antagonistas & inhibidores , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Técnicas In Vitro , Insulina/sangre , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Ratones Obesos , Oxazoles/química , Oxazoles/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Insulin resistance in the ob/ob mouse model is associated with a reduction in insulin-induced protein-tyrosine phosphorylation in tissues such as liver. To ascertain whether this decrease in phosphorylation may be due to increased phosphatase activity, protein-tyrosine phosphatase (PTPase) activity was determined in particulate and soluble fractions from livers of 5- to 23-week-old ob/ob mice and age-matched lean littermates. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate, corresponding to residues 1142 to 1153 of the insulin receptor and containing the major autophosphorylation sites of the regulatory domain. The ob/ob mice were hyperinsulinemic across all age groups, but only the youngest mice (aged 5 to 7 weeks) were hyperglycemic. Most PTPase activity was present in the liver particulate fraction and was 19% to 114% greater in ob/ob mice as compared with controls. PTPase activity in the liver soluble fraction was 26% less than control values in the youngest ob/ob mice (5 to 7 weeks), but increased with age and was 41% and 131% above control values at 21 to 23 and 25 to 27 weeks of age, respectively. Oral administration of the PTPase inhibitor sodium orthovanadate (0.6 mg/mL in drinking water for 2 weeks) to young ob/ob mice caused a significant reduction in the elevated particulate PTPase activity, with concomitant decreases in plasma insulin and plasma glucose. Assessment of PTPase activity with a monophosphate form of the same synthetic peptide, TRDIY(P)ETDYYRK, showed lower PTPase activities as compared with the triphosphate form and no significant differences between ob/ob and control preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Resistencia a la Insulina/fisiología , Obesidad/metabolismo , Fosfopéptidos/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Glucemia/análisis , Modelos Animales de Enfermedad , Insulina/sangre , Hígado/enzimología , Estudios Longitudinales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosforilación , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Vanadatos/farmacologíaRESUMEN
The ability of aldose reductase inhibitors to prevent the decline in neural Na+,K(+)-ATPase activity in diabetic rats has not been confirmed by all laboratories. In this study, the efficacy of two structurally different aldose reductase inhibitors was evaluated under different experimental conditions. Na+,K(+)-ATPase activity was measured in sciatic nerves from streptozocin-induced diabetic rats fed normal rodent chow or a chow supplemented with 68% sucrose. Nerve homogenates from chow-fed rats were prepared with a Dounce tissue grinder, whereas homogenates from the sucrose-fed rats were prepared with an Ultra-Turrax disperser. In the chow-fed rats, 4 weeks of untreated diabetes resulted in an increase in neural sorbitol and fructose, a decrease in myoinositol, and a 54% decline in Na+,K(+)-ATPase activity. Sorbinil administration (20 mg/kg/day) completely prevented the rise in sorbitol and fructose and the depletion of myoinositol, but did not prevent the decline in Na+,K(+)-ATPase activity. In diabetic rats fed the sucrose diet for 4, 6, and 8 weeks, the neural sorbitol and fructose levels were elevated, the myoinositol concentration declined, and the Na+,K(+)-ATPase activity was 26 to 28% below the control. Prevention or intervention treatment with sorbinil (20 mg/kg/day) or tolrestat (50 mg/kg/day) for 4 to 6 weeks prevented the alterations in sorbitol, fructose, and myoinositol, and also prevented the decline in Na+,K(+)-ATPase activity. In conclusion, prevention and intervention therapy with aldose reductase inhibitors prevented the decline in Na+,K(+)-ATPase in sciatic nerves of sucrose-fed streptozocin-diabetic rats that were homogenized with an Ultra-Turrax disperser, but not in sciatic nerves from streptozocin-diabetic rats fed normal rodent chow that were homogenized with a Dounce tissue grinder. These findings indicate that the assessment of aldose reductase inhibitor efficacy is dramatically affected by the type of nerve preparation assayed and/or the diet.