RESUMO
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.
Assuntos
Arginina/análogos & derivados , Plaquetas/metabolismo , GMP Cíclico/sangue , Inibidores Enzimáticos/farmacologia , Insulina/farmacologia , Óxido Nítrico/fisiologia , Adulto , Análise de Variância , Arginina/farmacologia , Plaquetas/efeitos dos fármacos , Feminino , Humanos , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/sangue , Proteínas Recombinantes/farmacologia , ômega-N-MetilargininaRESUMO
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.
Assuntos
Plaquetas/efeitos dos fármacos , AMP Cíclico/sangue , GMP Cíclico/sangue , Insulina/farmacologia , Óxido Nítrico/biossíntese , Adulto , Plaquetas/química , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , MasculinoRESUMO
To investigate the effects of insulin on platelets in obesity and in non-insulin-dependent diabetes mellitus (NIDDM)--classic insulin-resistant states--we determined ADP-induced platelet aggregation and platelet cGMP (guanosine 3',5'-cyclic monophosphate) content in platelet-rich plasma obtained from nine obese subjects and nine age-matched healthy volunteers and from eight NIDDM obese patients and nine age-matched healthy volunteers after a 3-min incubation with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l). Platelet aggregation was evaluated using different ADP doses to measure the ADP concentration determined on the basis of a dose-response curve necessary to elicit a maximal aggregation of 50% (ED50). Insulin induced a dose-dependent decrease of platelet aggregation to ADP (P = 0.0001) in healthy subjects. A significant effect was evident starting from an insulin concentration of 240 pmol/l. On the contrary, in insulin-resistant subjects, insulin reduced platelet sensitivity to ADP only at a concentration of 1,920 pmol/l. When ADP ED50 values obtained in platelet-rich plasma incubated with insulin were expressed in percentage of the ADP ED50 values obtained in platelet-rich plasma without insulin, considered as 100%, we observed that ADP ED50 with 1,920 pmol/l insulin was 153.6 +/- 13.2% in the younger healthy subject group (P = 0.004), 150.0 +/- 3.8% in the older healthy subject group (P = 0.0001), 116.1 +/- 6.1% in obese subjects (P = 0.031), and 120.0 +/- 8.6% in NIDDM patients (P = 0.05). In healthy subjects, insulin induced a dose-dependent increase of platelet cGMP (P = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Plaquetas/fisiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus/sangue , Insulina/farmacologia , Obesidade/sangue , Inibidores da Agregação Plaquetária/farmacologia , Difosfato de Adenosina/farmacologia , Adulto , Plaquetas/efeitos dos fármacos , Pressão Sanguínea , Estudos de Casos e Controles , Colesterol/sangue , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Relação Dose-Resposta a Droga , Feminino , Hemoglobinas Glicadas/análise , Humanos , Técnicas In Vitro , Insulina/sangue , Masculino , Proteínas Recombinantes/farmacologia , Valores de Referência , Triglicerídeos/sangueRESUMO
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.
Assuntos
Insulina/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Adulto , GMP Cíclico/sangue , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Nitroglicerina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/sangue , Inibidores da Agregação Plaquetária/farmacologia , Purinonas/farmacologia , Teofilina/farmacologia , Vasodilatadores/farmacologiaRESUMO
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.
Assuntos
Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Piridonas/farmacologia , Adenosina/antagonistas & inibidores , Difosfato de Adenosina/farmacologia , Adulto , Plaquetas/enzimologia , Plaquetas/metabolismo , Colágeno/farmacologia , AMP Cíclico/sangue , Dipiridamol/farmacologia , Epinefrina/farmacologia , Humanos , Técnicas In Vitro , Masculino , Milrinona , Inibidores de Fosfodiesterase/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Piridonas/antagonistas & inibidores , Teofilina/farmacologiaRESUMO
1. The present study investigated the influence of the organic nitrate glyceryl trinitrate (GTN) on the anti-aggregating effects of adenosine. We determined the effects of adenosine, GTN and their combination on platelet responses in platelet-rich plasma and whole blood, and on intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). 2. Adenosine inhibited the in vitro platelet aggregation in response to different agonists in a dose-dependent way through an elevation of intraplatelet cAMP levels. Effective adenosine concentrations were higher than those detectable under physiological conditions, but very close to levels achieved during myocardial ischaemia or haemorrhagic shock. 3. GTN was able to decrease platelet responses influencing intraplatelet cGMP levels. Furthermore, the drug increased the inhibitory effects of adenosine and enhanced its effects on intraplatelet cAMP levels. 4. The present data provides further evidence that compounds that increase intraplatelet levels of cGMP and cAMP act synergistically on the inhibition of platelet aggregability through the influence of increased cGMP levels on cAMP accumulation. The interplay between GTN and adenosine in the inhibition of platelet function could be effective during nitrate administration in the treatment of acute myocardial ischaemia when blood adenosine levels are significantly increased.