RESUMEN
First principles calculations based on density functional theory and many body perturbation theory have been employed to study the optical absorption properties of a newly synthesized oligo-thiophene molecule, with a quaterthiophene central unit, that has been designed for solution-processed bulk-heterojunction solar cells. To this aim we have employed the GW approach to obtain quasiparticle energies as a pre-requisite to solve the Bethe-Salpeter equation for the excitonic Hamiltonian. We show that the experimental absorption spectrum can be explained only by taking into account the inter-molecular transitions among the π-stacked poly-conjugated molecules that are typically obtained in solid-state organic samples.
RESUMEN
Low pH (<5.0) can significantly decrease root growth but whether this is a direct effect of H(+) or an active plant response is examined here. Tomato (Solanum lycopersicum cv Micro-Tom) roots were exposed directly or gradually to low pH through step-wise changes in pH over periods ranging from 4 to 24 h. Roots exposed gradually to pH 4.5 grew even less than those exposed directly, indicating a plant-coordinated response. Direct exposure to pH 4.0 suppressed root growth and caused high cell mortality, in contrast to roots exposed gradually, in which growth remained inhibited but cell viability was maintained. Total class III peroxidase activity increased significantly in all low pH treatments, but was not correlated with the observed differential responses. Use of the enzyme inhibitors salicylhydroxamic acid (SHAM) or diphenyleneiodonium chloride (DPI) suggest that peroxidase and, to a lesser extent, NADPH oxidase were required to prevent or reduce injury in all low pH treatments. However, a role for other enzymes, such as the alternative oxidase is also possible. The results with SHAM, but not DPI, were confirmed in tobacco BY-2 cells. Our results indicate that root growth inhibition from low pH can be part of an active plant response, and suggest that peroxidases may have a critical early role in reducing loss of cell viability and in the observed root growth constraint.
Asunto(s)
NADPH Oxidasas/metabolismo , Peroxidasas/metabolismo , Solanum lycopersicum/enzimología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Solanum lycopersicum/efectos de los fármacos , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/fisiología , Proteínas Mitocondriales/metabolismo , Compuestos Onio/farmacología , Oxidorreductasas/metabolismo , Peroxidasas/antagonistas & inhibidores , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Salicilamidas/farmacología , Nicotiana/efectos de los fármacos , Nicotiana/enzimología , Nicotiana/crecimiento & desarrollo , Nicotiana/fisiologíaRESUMEN
The Y1 and Y5 receptors for neuropeptide Y have overlapping functions in regulating anxiety. We previously demonstrated that conditional removal of the Y1 receptor in the Y5 receptor expressing neurons in juvenile Npy1r(Y5R-/-) mice leads to higher anxiety but no changes in hypothalamus-pituitary-adrenocortical axis activity, under basal conditions or after acute restraint stress. In the present study, we used the same conditional system to analyze the specific contribution of limbic neurons coexpressing Y1 and Y5 receptors on the emotional and neuroendocrine responses to social chronic stress, using different housing conditions (isolation vs. group-housing) as a model. We demonstrated that control Npy1r(2lox) male mice housed in groups show increased anxiety and hypothalamus-pituitary-adrenocortical axis activity compared with Npy1r(2lox) mice isolated for six weeks immediately after weaning. Conversely, Npy1r(Y5R-/-) conditional mutants display an anxious-like behavior but no changes in hypothalamus-pituitary-adrenocortical axis activity as compared with their control littermates, independently of housing conditions. These results suggest that group housing constitutes a mild social stress for our B6129S mouse strain and they confirm that the conditional inactivation of Y1 receptors specifically in Y5 receptor containing neurons increases stress-related anxiety without affecting endocrine stress responses.
Asunto(s)
Ansiedad/genética , Receptores de Neuropéptido Y/genética , Conducta Social , Estrés Psicológico/genética , Animales , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Ratones , Mutación , Sistema Hipófiso-Suprarrenal/metabolismoRESUMEN
Some in vivo and ex vivo studies demonstrated a resistance to the vasodilating effects of nitric oxide (NO) in insulin-resistant states and, in particular, obese Zucker rats (OZR). To evaluate the biochemical basis of this phenomenon, we aimed to identify defects of the NO/cGMP/cGMP-dependent protein kinase (PKG) pathway in cultured vascular smooth muscle cells (VSMCs) from OZR and lean Zucker rats (LZR) by measuring: 1) NO donor ability to increase cGMP in the absence and presence of inhibitors of soluble guanylate cyclase (sGC) and phosphodiesterases (PDEs); 2) NO and cGMP ability to induce, via PKG, vasodilator-stimulated phosphoprotein (VASP) phosphorylation at serine 239 and PDE5 activity; 3) protein expression of sGC, PKG, total VASP, and PDE5; 4) superoxide anion concentrations and ability of antioxidants (superoxide dismutase+catalase and amifostine) to influence the NO/cGMP/PKG pathway activation; and 5) hydrogen peroxide influence on PDE5 activity and VASP phosphorylation. VSMCs from OZR vs. LZR showed: 1) baseline cGMP concentrations higher, at least in part owing to reduced catabolism by PDEs; 2) impairment of NO donor ability to increase cGMP, even in the presence of PDE inhibitors, suggesting a defect in the NO-induced sGC activation; 3) reduction of NO and cGMP ability to activate PKG, indicated by the impaired ability to phosphorylate VASP at serine 239 and to increase PDE5 activity via PKG; 4) similar baseline protein expression of sGC, PKG, total VASP, and PDE5; and 5) higher levels of superoxide anion. Antioxidants partially prevented the defects of the NO/cGMP/PKG pathway observed in VSMCs from OZR, which were reproduced by hydrogen peroxide in VSMCs from LZR, suggesting the pivotal role of oxidative stress.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , GMP Cíclico/fisiología , Resistencia a la Insulina , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/fisiología , Estrés Oxidativo , Transducción de Señal/fisiología , Animales , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Modelos Animales de Enfermedad , Masculino , Proteínas de Microfilamentos/metabolismo , Músculo Liso Vascular/citología , Inhibidores de Fosfodiesterasa/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Ratas , Ratas ZuckerRESUMEN
AIMS/HYPOTHESIS: We previously demonstrated that insulin stimulates vascular endothelial growth factor (VEGF) synthesis and secretion via phosphatidylinositol-3 kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways in vascular smooth muscle cells (VSMC) from humans and from insulin-sensitive lean Zucker fa/+ rats. We also showed that this effect is attenuated in VSMC from insulin-resistant obese Zucker fa/fa rats. As it is not known whether the effects of insulin on VEGF involve activation of hypoxia-inducible factor-1 (HIF-1), we aimed to evaluate: (1) whether insulin modulates HIF-1alpha protein synthesis and activity; (2) the insulin signalling pathways involved; and (3) the role of insulin resistance. METHODS: Using aortic VSMC taken from humans and Zucker rats and cultured in normoxia, the following were evaluated: (1) dose-dependent (0.5, 1, 2 nmol/l) and time-dependent (2, 4, 6 h) effects exerted by insulin on HIF-1alpha content in both nucleus and cytosol, measured by Western blots; (2) insulin effects on HIF-1 DNA-binding activity on the VEGF gene, measured by electrophoretic mobility shift assay; and (3) involvement of the insulin signalling molecules in these insulin actions, by using the following inhibitors: LY294002 (PI3-K), PD98059 (extracellular signal regulated kinase [ERK]), SP600125 (Jun N terminal kinase [JNK]), SB203580 (p38 mitogen-activated protein kinase) and rapamycin (mammalian target of rapamycin), and by detecting the insulin signalling molecules by Western blots. RESULTS: In aortic VSMC from humans and Zucker fa/+ rats cultured in normoxia insulin increases the HIF-1alpha content in cytosol and nucleus via dose- and time-dependent mechanisms, and HIF-1 DNA-binding activity on the VEGF gene. The insulin-induced increase of HIF-1alpha is blunted by the translation inhibitor cycloheximide, LY294002, PD98059, SP600125 and rapamycin, but not by SB203580. It is also reduced in Zucker fa/fa rats, which present an impaired ability of insulin to induce Akt, ERK-1/2 and JNK-1/2 phosphorylation. CONCLUSIONS/INTERPRETATION: These results provide a biological mechanism for the impaired collateral vessel formation in obesity.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Resistencia a la Insulina/fisiología , Insulina/farmacología , Insulina/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Cinética , Músculo Liso Vascular/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Zucker , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin-resistant states. MATERIALS AND METHODS: In VSMC derived from human arterioles and from aortas of insulin-sensitive Zucker fa/+rats and insulin-resistant Zucker fa/fa rats incubated with different concentrations of human regular insulin with or without inhibitors of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3-K), mitogen-activated protein kinase (MAPK), nitric oxide synthase (NOS) and guanosine 3',5'cyclic monophosphate(cGMP)-dependent protein kinase (PKG), we measured protein expression (Western blot) and secretion (ELISA) of VEGF. RESULTS: We found that in VSMCs from humans and from insulin-sensitive Zucker fa/+rats, insulin increases VEGF protein expression and secretion, with mechanisms blunted by wortmannin and LY294002 (PI3-K inhibitors), PD98059 (MAPK inhibitor), L-NMMA (NOS inhibitor) and Rp-8pCT-cGMPs (PKG inhibitor). Also the NO donor sodium nitroprusside (SNP) and the cGMP analogue 8-Bromo-cGMP increase VEGF protein expression and secretion, with mechanisms inhibited by wortmannin and PD98059. The insulin effects on VEGF are impaired in VSMCs from Zucker fa/fa rats, which also present a reduced insulin ability to increase NO. CONCLUSIONS: In VSMCs from humans and insulin-sensitive Zucker fa/+rats: (i) insulin increases VEGF protein expression and secretion via both PI3-K and MAPK; (ii) the insulin effects on VEGF are mediated by nitric oxide. The insulin action on both nitric oxide and VEGF is impaired in VSMCs from Zucker fa/fa rats, an animal model of metabolic and vascular insulin-resistance.
Asunto(s)
GMP Cíclico/análogos & derivados , Resistencia a la Insulina/fisiología , Insulina/fisiología , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , Animales , Western Blotting , Células Cultivadas , GMP Cíclico/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Ratas , Ratas Zucker , Factor A de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: Subjects with central obesity exhibit platelet hyperactivity, which is involved in the atherosclerotic process and therefore can account for the increased risk of cardiovascular morbidity and mortality. The aim of the study was to evaluate whether alterations of platelet function in obesity involve synthesis and/or action of the two antiaggregating cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). MATERIALS AND METHODS: In platelets from 16 obese and 15 control subjects we investigated the influence on platelet responses to the Adenosine-5-diphosphate sodium salt (ADP) exerted by (i) prostacyclin analogue Iloprost (0.31-5 nmol L(-1)) and the cAMP analogue 8-bromo-cAMP (10-500 micro mol L(-1)); and by (ii) nitric oxide (NO) donor sodium nitroprusside (SNP) (5-100 micro mol L(-1)) and the cGMP analogue 8-bromo-cGMP (10-500 micro mol L(-1)). IC(50) (minimal concentration of each inhibitor necessary to reduce platelet response to ADP by half) was determined. Iloprost and SNP ability to increase cyclic nucleotides was also measured. RESULTS: Significantly greater IC(50) were observed in obese subjects than in healthy controls (1.59 +/- 0.16 vs. 0.80 +/- 0.08 nmol L(-1), P = 0.0001 for Iloprost, and 27.6 +/- 6.5 vs. 7.0 +/- 1.7 micro mol L(-1), P = 0.006, for SNP); when data from control and obese subjects were pooled together, IC(50) of Iloprost and SNP correlated with the homeostasis model assessment (HOMA IR), which is a parameter used to measure the insulin resistance (r = 0.588, P = 0.029 and r = 0.640, P = 0.006, respectively). Also the antiaggregating effect of 8-Br-cAMP and 8-Br-cGMP was smaller in the obese subjects. Finally, the ability of Iloprost to increase platelet cAMP and the ability of SNP to increase both cGMP and cAMP were reduced in obese subjects. CONCLUSIONS: Platelet resistance to the antiaggregating effects of prostacyclin and NO in obesity is attributable to impairment of cyclic nucleotide synthesis and action. As cyclic nucleotides are the main effectors of platelet antiaggregation, the resistance to them can account for platelet hyperactivity in obesity.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Iloprost/farmacología , Obesidad/sangre , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/fisiología , Adulto , Índice de Masa Corporal , Femenino , Humanos , Masculino , Agregación Plaquetaria/efectos de los fármacosRESUMEN
BACKGROUND: N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability. MATERIALS AND METHODS: 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin). RESULTS: N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets. CONCLUSION: N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.
Asunto(s)
Acetilcisteína/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Acetilcisteína/sangre , Adenosina Difosfato/antagonistas & inhibidores , Adenosina Difosfato/farmacología , Adulto , Calcimicina/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colágeno/farmacología , GMP Cíclico/sangre , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/sangre , Humanos , Ionomicina/farmacología , Masculino , Oxadiazoles/farmacología , Inhibidores de Agregación Plaquetaria/sangre , Purinonas/farmacología , Quinoxalinas/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
In this study, we explored the ability of sodium nitroprusside to inhibit the aggregation of human platelets in platelet-rich plasma (PRP) and whole blood and its effects on intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). The experiments investigated dose-dependent effects of nitroprusside starting from concentrations in the range of circulating levels achievable in vivo during drug administration in humans. Furthermore, we investigated the time-course of both antiaggregating action and the influence on cyclic nucleotide synthesis. Results showed that sodium nitroprusside inhibited the aggregation induced by adenosine 5-diphosphate (ADP) and collagen starting from concentration as low as 2 micromol/l. The IC(50) value for ADP-induced aggregation in PRP was 18.7+/-2.4 micromol/l. The inhibition of platelet aggregation showed a time-dependent behaviour and was not reversible within 90 min. The accumulation of intraplatelet cGMP in the presence of sodium nitroprusside exhibited a comparable time-course characterized by an early increase, a steady state and a late further increase. The time-course of cAMP synthesis was very similar to that of cGMP. Our data evidenced a long-lasting inhibition of platelet responses by sodium nitroprusside and excluded a desensitization of platelet guanylyl cyclase after 3-h exposure to nitric oxide (NO). Furthermore, they indicated a role of cAMP accumulation in the antiaggregating effects of nitroso donor: the simultaneous increase of intracellular content of cAMP and cGMP can synergize in the reduction of the platelet responses.
Asunto(s)
Plaquetas/fisiología , AMP Cíclico/sangre , GMP Cíclico/sangre , Nitroprusiato/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Adenosina Difosfato/farmacología , Adulto , Plaquetas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Cinética , Masculino , Factores de TiempoRESUMEN
AIMS/HYPOTHESIS: Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca2+-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. METHODS: In hVSMC incubated with insulin, NO donors and the Ca2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as L-(3H)-citrulline production from L-(3H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. RESULTS: i) By incubating hVSMC with 2 nmol/l insulin for 0-240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 +/- 0.01 to 2.62 +/- 0.10 pmol/10(6) cells at 30 min (p = 0.0001); cAMP rose from 0.9 +/- 0.09 to 11.65 +/- 0.74 pmol/10(6) cells at 15 min (p=0.0001). ii) Sodium nitroprusside (100 mol/l) and glyceryltrinitrate (100 micromol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 micromol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. CONCLUSION/INTERPRETATION: Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated.
Asunto(s)
AMP Cíclico/análisis , GMP Cíclico/análisis , Expresión Génica , Insulina/farmacología , Músculo Liso Vascular/citología , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/metabolismo , Calcio/farmacología , Células Cultivadas/efectos de los fármacos , Citrulina/metabolismo , Humanos , Ionomicina/farmacología , Músculo Liso Vascular/enzimología , Óxido Nítrico/biosíntesis , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
Platelet nitric oxide is involved in the control of aggregability via cyclic 3',5'-guanosine monophosphate synthesis. Since L-arginine provides a guanidino nitrogen group for nitric oxide synthesis through nitric oxide synthase activity, we tried to clarify whether an increased availability of this amino acid can directly modulate the response of human platelets. In our conditions, L-arginine (at 100-6000 micromol/L) was able to influence the response of human platelets stimulated with adenosine 5-diphosphate and collagen both in PRP and in whole blood. The anti-aggregating effect was not present when D-arginine was used. Permeabilized platelets exhibited an increased sensitivity to L-arginine. Also, an increased availability of Ca2+ enhanced L-arginine effect. L-arginine (at 120-500 micromol/L) increased cyclic 3',5'-guanosine monophosphate levels in resting platelets; the amino acid also determined an increase of cyclic 3',5'-guanosine monophosphate in platelets at the end of adenosine 5-diphosphate-induced aggregation. Nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine prevented L-arginine effects on aggregation and cyclic 3',5'-guanosine monophosphate synthesis. Phosphodiesterase III inhibitor milrinone and antioxidative thiol N-acetyl-L-cysteine enhanced the effect of L-arginine on cyclic 3',5'-guanosine monophosphate. In conclusion, L-arginine exerts inhibitory effects on human platelet response through a nitric oxide-dependent synthesis of cyclic 3',5'-guanosine monophosphate. A positive interplay on platelet response between L-arginine and milrinone or antioxidative thiol N-acetyl-L-cysteine was evidenced.
Asunto(s)
Acetilcisteína/farmacología , Arginina/farmacología , Plaquetas/fisiología , Depuradores de Radicales Libres/farmacología , Guanosina Monofosfato/fisiología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Antioxidantes/farmacología , Plaquetas/efectos de los fármacos , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Humanos , Óxido Nítrico/fisiología , Agregación Plaquetaria/fisiología , omega-N-Metilarginina/farmacologíaRESUMEN
L-Canavanine is a naturally occurring L-amino acid that interferes with L-arginine-utilizing enzymes owing to its structural analogy with this L-amino acid. In macrophages and polymorphonuclear leukocytes, which express inducible nitric oxide synthase (iNOS), L-canavanine is able to prevent the L-arginine-derived synthesis of nitric oxide (NO). Its effects on constitutive NOS (cNOS) are far less clear. Because human platelets synthesize NO from L-arginine through a cNOS and because intracellular NO levels modulate platelet function, we have investigated the effects of L-canavanine on parameters potentially influenced by NO, such as platelet levels of 3',5'-cyclic guanosine monophosphate (cGMP) and responses to different aggregating agents. In our experimental conditions, L-canavanine was able to influence the response of human platelets to different aggregating agents such as catecholamines, 5-hydroxytryptamine, and ADP. Low L-canavanine concentrations (10-100 micromol/l) decreased platelet responses, whereas a high concentration (1 mmol/l) was unable to exert antiaggregating effects. In resting platelets, L-canavanine reduced the levels of cGMP, starting from a concentration of 1 mmol/l; furthermore, at the same concentrations, it was able to reduce cGMP levels at the end of the aggregation induced by collagen. In conclusion, L-canavanine exerts differential effects on human platelets in relation to the concentrations: at low levels, it exerts antiaggregating effects by actions independent of NOS inhibition, whereas, at high levels, it inhibits NO synthesis and does not exert antiaggregating effects.
Asunto(s)
Plaquetas/efectos de los fármacos , Canavanina/farmacología , Adenosina Difosfato/fisiología , Arginina/farmacología , Plaquetas/fisiología , Catecolaminas/fisiología , GMP Cíclico/metabolismo , Interacciones Farmacológicas , Humanos , Técnicas In Vitro , Óxido Nítrico Sintasa/antagonistas & inhibidores , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Pruebas de Función Plaquetaria , Serotonina/fisiologíaRESUMEN
OBJECTIVE: Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM. RESEARCH DESIGN AND METHODS: We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 mumol/l GTN. RESULTS: Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 +/- 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 +/- 0.2 to 21.1 +/- 3.7 pmol/10(9) platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 +/- 5.0 mumol/l in healthy control subjects, 26.0 +/- 6.0 mumol/l in lean NIDDM patients (NS vs. control subjects), 123.6 +/- 24.0 mumol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 +/- 19.2 mumol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 mumol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance. CONCLUSIONS: The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.
Asunto(s)
Plaquetas/fisiología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus/sangre , Insulina/farmacología , Nitroglicerina/farmacología , Obesidad/sangre , Agregación Plaquetaria/efectos de los fármacos , Delgadez/sangre , Adenosina Difosfato/farmacología , Adulto , Análisis de Varianza , Glucemia/análisis , Plaquetas/efectos de los fármacos , Presión Sanguínea , Índice de Masa Corporal , Colesterol/sangre , HDL-Colesterol , GMP Cíclico/sangre , Diabetes Mellitus/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Hemoglobina Glucada/análisis , Humanos , Insulina/sangre , Resistencia a la Insulina , Masculino , Obesidad/fisiopatología , Agregación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/farmacología , Valores de Referencia , Triglicéridos/sangreRESUMEN
The insulin-induced platelet anti-aggregating effect is attributed to a nitric oxide (NO)-mediated increase of cyclic guanosine monophosphate (cGMP). The aim of this work, carried out in human platelets, is to show whether insulin increases NO synthesis in platelets and whether it enhances not only cGMP but also cyclic adenosine monophosphate (cAMP) in these cells. We observed that 1) insulin dose-dependently increases NO production, evaluated as citrulline synthesis from L-arginine (n = 4, P = 0.015); 2) insulin dose-dependently increases not only cGMP but also cAMP: for instance, after 8 min of insulin incubation at 1,920 pmol/l, cAMP increased from 39.8 +/- 1.4 to 121.3 +/- 12.6 pmol/10(9) platelets (n = 16, P = 0.0001); 3) when insulin is incubated for 120 min, the increase of cGMP and cAMP shows a plateau between 2 and 20 min, and while the effect on cGMP is significant until 120 min, the effect on cAMP is no more significant at 60 and 120 min; 4) insulin increases the effects on cAMP of the adenylate cyclase agonists Iloprost and forskolin (n = 5, P = 0.0001) and enhances their platelet anti-aggregating effects (n = 6 and 8, respectively; P = 0.0001); and 5) the inhibition of NO synthase by N(G)-monomethyl-L-arginine blunts both the insulin effects on basal cGMP and cAMP (n = 4) and those on the Iloprost- and forskolin-induced cAMP increase (n = 5). Thus, insulin increases NO synthesis in human platelets, and, through NO, enhances both cGMP and cAMP. The platelet anti-aggregating effect exerted by insulin is, therefore, a NO-mediated phenomenon involving both cGMP and cAMP.
Asunto(s)
Plaquetas/efectos de los fármacos , AMP Cíclico/sangre , GMP Cíclico/sangre , Insulina/farmacología , Óxido Nítrico/biosíntesis , Adulto , Plaquetas/química , Plaquetas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND & AIMS: Oncogenes were activated in experimental models of hepatocyte regeneration. We studied the intrahepatic expression of c-fos, c-myb and c-myc protooncogenes in 117 patients with chronic liver disease: 12 with hepatitis B, 15 HBsAg carriers, 73 with hepatitis C and 17 with non-viral liver damage. METHODS: Oncoproteins were detected by indirect immunofluorescence using high affinity and monoclonal antibodies. Grade and stage of liver damage were measured by numerical score. RESULTS: Nuclear c-fos and/or c-myb were found in 7 (58.3%) hepatitis B patients, in 38 (52%) hepatitis C patients, in 1 (6.6%) HBsAg carrier (p < 0.004) and in none of the non-viral disease patients (p < 0.0001). In no case was c-myc detected. Oncoproteins were correlated with the histological activity index (p < 0.0001) and its components: lobular degeneration and periportal necrosis (p < 0.0001), fibrosis (p < 0.005) and portal inflammation (p < 0.03). Thirty-one chronic hepatitis C patients were treated with alpha-IFN: 9 out of 14 oncoprotein-positive patients (64%) were non-responders, 5 (36%) relapsed and none was a sustained responder. Conversely 9 out of 17 (53%) oncoprotein-negative patients, including 3 patients with histologically active cirrhosis, showed long-term response (p < 0.005). CONCLUSIONS: Intrahepatic c-fos and c-myb were detected in chronic viral hepatitis patients, but not in non-viral liver diseases. Their expression correlated with the grade and stage of liver disease and with poor response to alpha-IFN.
Asunto(s)
Antivirales/uso terapéutico , Expresión Génica , Genes fos , Genes myc , Hepatitis Crónica/tratamiento farmacológico , Hepatitis Crónica/metabolismo , Interferón-alfa/uso terapéutico , Proteínas Oncogénicas de Retroviridae/genética , Adulto , Femenino , Técnica del Anticuerpo Fluorescente , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/patología , Hepatitis Crónica/patología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas v-myb , Proteínas Oncogénicas de Retroviridae/biosíntesisRESUMEN
It has been shown that, in streptozotocin diabetic rats, protamine-retarded insulin administered in vivo stimulates intimal hyperplasia in balloon-injured carotid artery. The aim of this study was to evaluate the influence of protamine on cultured human vascular smooth muscle cells (h VSMC), by observing its effects on adhesion, chemotaxis and proliferation. hVSMC were isolated during abdominal surgery, cultured and utilized at passages 6-10. We observed that protamine stimulates: 1) cell adhesion in the concentration range 0.04-20 micrograms/ml (analysis of variance, ANOVA, p < 0.0001); 2) cell chemotaxis in the absence of fetal calf serum (FCS) in the concentration range 1-200 micrograms/ml (ANOVA, p < 0.0001) and in the presence of 1% FCS in the concentration range 5-200 micrograms/ml (ANOVA, p < 0.0001), further enhancing the chemotaxis induced by 10% FCS in the concentration range 20-200 micrograms/ml (ANOVA, p < 0.0001); 3) cell proliferation and 3H-thymidine incorporation from 1 to 5 micrograms/ml (ANOVA, p < 0.0001); 4) cell c-fos oncoprotein nuclear expression. We also observed that protamine effects on chemotaxis, proliferation and c-fos expression are inhibited by heparin that human insulin stimulates cell proliferation and 3H-thymidine incorporation (ANOVA, p < 0.0001) at concentrations equal to or greater than 480 pmol/l and that these effects of insulin persist in the presence of protamine. In conclusion, protamine influences hVSMC behaviour by interfering with biological functions involved in atherogenesis. The concentrations used in this short-term in vitro study were higher than those probably occurring in vivo in patients chronically treated by protamine-retarded insulin preparations: further studies, therefore, are needed to evaluate the safety of protamine as a retardant of insulin action in vivo.
Asunto(s)
Antagonistas de Heparina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Protaminas/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente Indirecta , Heparina/farmacología , Humanos , Insulina/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/inmunologíaRESUMEN
Insulin increases both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in human vascular smooth muscle cells (hVSMC) and attenuates noradrenaline-induced vasoconstriction. In the present study, we aimed at investigation in hVSMC: 1) the interrelationships between insulin-induced increases of cGMP and cAMP; 2) the insulin effect on the catecholamine modulation of cAMP. Catecholamines cause both vasoconstriction and vasodilation. Vasoconstriction is attributable to the reduced synthesis of cAMP in hVSMC through alpha 2-adrenoceptors and to direct effects on calcium fluxes through alpha 1-adrenoceptors; vasodilation is attributable to the increased synthesis of cAMP through beta-adrenoceptors. In the present study, we determined the influence of insulin on cAMP in hVSMC incubated with or without: a) the inhibitor of guanylate cyclase methylene blue or the inhibitor of nitric oxide synthase NG-monomethyl-L-arginine (L-NMMA); b) the beta-adrenergic agonists isoproterenol and salbutamol; c) the physiological catecholamines noradrenaline and adrenaline; d) noradrenaline+the beta-adrenergic antagonist propranolol or the alpha 2-adrenergic antagonist yohimbine; e) noradrenaline+methylene blue of L-NMMA. We demonstrated that: 1) the inhibition of the insulin-induced cGMP synthesis blunts the insulin-induced increase of cAMP; 2) insulin induces a significant increase of cAMP also in the presence of isoproterenol, salbutamol, noradrenaline and adrenaline: the combined effects of insulin and catecholamines were additive in some, but not in all the experiments; 3) insulin enhances the cAMP concentrations induced by noradrenaline also in the presence of alpha 2- or beta-adrenergic antagonists; 4) in the presence of methylene blue or L-NMMA insulin does not modify the noradrenaline effects on cAMP.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Epinefrina/farmacología , Insulina/farmacología , Músculo Liso Vascular/metabolismo , Norepinefrina/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Albuterol/farmacología , Análisis de Varianza , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Células Cultivadas , Humanos , Isoproterenol/farmacología , Cinética , Azul de Metileno/farmacología , Músculo Liso Vascular/efectos de los fármacos , Propranolol/farmacología , Receptores Adrenérgicos beta/fisiología , Proteínas Recombinantes/farmacología , Yohimbina/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
1. In this study, we investigated the influence of the inotropic agent and coronary vasodilator milrinone on platelet aggregation and intracellular levels of 3',5' cyclic adenosine monophosphate (cAMP) in human platelet-rich plasma (PRP) and whole blood (WB). Furthermore, we evaluated the influence of milrinone on the effects of adenosine, which reduces the platelet aggregation through an elevation of intraplatelet cAMP levels. 2. Milrinone decreased the platelet aggregation in response to agonists in both PRP and WB. A dose-dependent increase of intraplatelet cAMP levels was demonstrated: this result is in accordance with an effect on platelet phosphodiesterases. 3. Milrinone at low concentration and adenosine exerted additive effects on platelet aggregation and intraplatelet cAMP levels. 4. An interplay between milrinone and adenosine was shown in WB. Furthermore, dipyridamole, which prevents the uptake of endogenous adenosine, markedly enhanced the milrinone antiaggregating effect, whereas the adenosine receptor blocker, theophylline, decreased it. 5. The present data provide evidence that milrinone modulates the platelet function through an influence on intraplatelet levels of cAMP and it is able to interplay with substances stimulating adenylyl cyclase. 6. The interplay between milrinone and adenosine in the inhibition of the human platelet function could be effective during milrinone administration in the treatment of heart failure, when blood adenosine levels are significantly increased. These milrinone effects could be advantageous from a therapeutic point of view, since patients with heart failure are at risk of thrombosis and ischemic heart disease.
Asunto(s)
Adenosina/farmacología , Plaquetas/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Piridonas/farmacología , Adenosina/antagonistas & inhibidores , Adenosina Difosfato/farmacología , Adulto , Plaquetas/enzimología , Plaquetas/metabolismo , Colágeno/farmacología , AMP Cíclico/sangre , Dipiridamol/farmacología , Epinefrina/farmacología , Humanos , Técnicas In Vitro , Masculino , Milrinona , Inhibidores de Fosfodiesterasa/farmacología , Agregación Plaquetaria/efectos de los fármacos , Piridonas/antagonistas & inhibidores , Teofilina/farmacologíaRESUMEN
To investigate whether the insulin-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP) in human platelets is mediated by nitric oxide or is influenced by the nitric oxide precursor L-arginine, we measured cGMP in platelet-rich plasma obtained from healthy volunteers incubated for 3 min with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l) both with and without 1) a 20-min incubation with the nitric oxide-synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (50, 70, 100, and 1,000 micromol/l; n = 5 for each dose) and 2) a 20-min incubation with the nitric oxide precursor L-arginine (300 micromol/l; n = 6). In a first set of experiments, insulin induced a dose-dependent cGMP increase, from 9.8 +/- 0.8 to 45.6 +/- 5.5 pmol/10(9) platelets (P = 0.0001); in the presence of 1 mmol/l L-NMMA, this increase was blunted, cGMP being 8.9 +/- 1.4 and 11.1 +/- 2.2 pmol/10(9) platelets at 0 and 1,920 pmol/l insulin, respectively (NS). In the experiments with 70 and 100 micromol/l L-NMMA, the insulin effect on cGMP was inhibited, whereas 50 micromol/l L-NMMA did not blunt this insulin effect. In another set of experiments carried out to investigate the effects of L-arginine, insulin induced a dose-dependent cGMP increase, from 23.6 +/- 6.9 to 59.0 +/- 12.0 pmol/10(9) platelets (P = 0.0001); with L-arginine, basal cGMP values increased to 35.5 +/- 6.6 pmol/10(9) platelets (P = 0.05), and insulin maintained its ability to enhance dose-dependently cGMP values, which rose to 76.8 +/- 19.4 pmol/10(9) platelets (P = 0.003). This study carried out in human platelets demonstrates that the cGMP increase induced by insulin, which accounts for the antiaggregating effect of the hormone, is mediated by nitric oxide.
Asunto(s)
Arginina/análogos & derivados , Plaquetas/metabolismo , GMP Cíclico/sangre , Inhibidores Enzimáticos/farmacología , Insulina/farmacología , Óxido Nítrico/fisiología , Adulto , Análisis de Varianza , Arginina/farmacología , Plaquetas/efectos de los fármacos , Femenino , Humanos , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/sangre , Proteínas Recombinantes/farmacología , omega-N-MetilargininaRESUMEN
In this study, we investigated the effects of a 3-min insulin incubation both at physiological and at supraphysiological concentrations on platelet aggregation and intraplatelet cyclic guanosine monophosphate (cGMP) levels both in the absence and in the presence of phosphodiesterase inhibition. We observed that insulin at concentration in the range 0.25-2 nmol/L decreases platelet response to adenosine 5-diphosphate (ADP), being Effective Dose 50 (ED50) for ADP with 2 nmol/L insulin 164 +/- 15% of the basal value, p = 0.005; furthermore, insulin increases intraplatelet content of cGMP (from basal 7.3 +/-0.6 pmol/10(9) plts to 14.6 +/- 1.2 pmol/10(9) plts with 2 nmol/L insulin, p=0.0001) and does not affect the platelet cGMP increase induced by nitrates. On the contrary, at very elevated concentrations (25-200 nmol/L) insulin increases platelet aggregation to ADP (ADP ED50 with 200 nmol/L insulin being 81 +/- 4% of the basal value, p = 0.01), decreases intraplatelet content of cGMP (from basal 7.2 +/- 0.1 pmol/10(9) plts to 5.7 +/- 0.2 pmol/10(9) plts with 200 nmol/L insulin, p = 0.01) and attenuates the platelet cGMP increase induced by nitrates. When cGMP catabolism is inhibited by theophylline or the selective cGMP phosphodiesterase inhibitor zaprinast, insulin shows anti-aggregating effects also at highly supraphysiological concentration (25-200 nmol/L). These results indicate that insulin, depending on the concentrations employed, shows opposite effects on platelet function, and they provide information about the mechanisms involved: actually, insulin is able to increase both cGMP synthesis, through guanylate cyclase activation, and cGMP catabolism, through phosphodiesterase activation. At physiological or slightly supraphysiological concentrations the first phenomenon is prevailing, so that cGMP intraplatelet values increase and insulin shows antiaggregating properties, whereas, at supraphysiological concentrations, insulin reduces cGMP levels through a prevailing phosphodiesterase activation, as supported by the fact that, when cGMP catabolism is prevented, insulin shows anti-aggregating properties also at the highest concentrations used.