RESUMO
Central obesity is a relevant risk factor for major cardiovascular events due to the atherosclerotic involvement of coronary, cerebral and lower limb arterial vessels. A major role in the increased cardiovascular risk is played by platelets, which show an increased activation and a reduced sensitivity to the physiological and pharmacological antiaggregating agents. This review focuses on platelet dysfunction in central obesity. The mechanisms involved are related to: i) the reduced sensitivity to insulin and other substances acting via intracellular cyclic nucleotides, such as nitrates and prostacyclin; ii) the altered intracellular ionic milieu with elevated cytosolic Ca(2+); and iii) the increased oxidative stress, which elicits isoprostane production from arachidonic acid. Therapeutic guidelines recommend a multifactorial prevention of cardiovascular disease including antiplatelet drugs in high risk patients, even though, at present, the protective effect of antiplatelet therapy in obese, insulin resistant subjects has not been evaluated by specific trials. Some reports, however, suggest a decreased sensitivity to the antiaggregating effects of both acetylsalicylic acid (aspirin) and thienopyridines in human obesity. Platelet defects may play a pivotal role in the reduced efficacy of antiplatelet therapy in obese subjects in the setting of cardiovascular prevention and acute coronary syndrome treatment. Thus, a specifically tailored antiaggregating therapy is likely necessary in obese, insulin resistant subjects, especially in the presence of type 2 diabetes mellitus.
Assuntos
Plaquetas/metabolismo , Doenças Cardiovasculares/etiologia , Hemostasia , Obesidade/sangue , Ativação Plaquetária , Animais , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/prevenção & controle , Resistência a Medicamentos , Hemostasia/efeitos dos fármacos , Humanos , Resistência à Insulina , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/fisiopatologia , Estresse Oxidativo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária , Transdução de Sinais , Trombose/sangue , Trombose/etiologia , Falha de TratamentoRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Resistência à Insulina , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/fisiologia , Estresse Oxidativo , Transdução de Sinais/fisiologia , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Modelos Animais de Doenças , Masculino , Proteínas dos Microfilamentos/metabolismo , Músculo Liso Vascular/citologia , Inibidores de Fosfodiesterase/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos ZuckerRESUMO
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.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Resistência à Insulina/fisiologia , Insulina/farmacologia , Insulina/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Cinética , Músculo Liso Vascular/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Zucker , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: The influence of postprandial blood glucose on diabetes complications is intensively debated. We aimed to evaluate the predictive role of both fasting and postprandial blood glucose on cardiovascular events in type 2 diabetes and the influence of gender. METHODS: In a population of 529 (284 men and 245 women) consecutive type 2 diabetic patients attending our diabetes clinic, we evaluated the relationships, corrected for cardiovascular risk factors and type of treatment, between cardiovascular events in a 5-yr follow-up and baseline values of hemoglobin A1c (HbA1c) and blood glucose measured: 1) after an overnight fast, 2) after breakfast, 3) after lunch, and 4) before dinner. Continuous variables were categorized into tertiles. RESULTS: We recorded cardiovascular events in 77 subjects: 54 of 284 men (19%) and 23 of 245 women (9.4%). Univariate analysis indicated that cardiovascular events were associated with increasing age, longer diabetes duration, and higher HbA1c and fibrinogen in men, and higher systolic blood pressure, albumin excretion rate, HbA1c, and all blood glucose values in women. Smoking was more frequent in subjects with events. When all blood glucose values and HbA1c were introduced simultaneously in the models, only blood glucose after lunch predicted cardiovascular events, with hazard ratio of the third tertile vs. the first and the second tertiles greater in women (5.54; confidence interval, 1.45-21.20) than in men (2.12; confidence interval, 1.04-4.32; P < 0.01). CONCLUSIONS: Postprandial, but not fasting, blood glucose is an independent risk factor for cardiovascular events in type 2 diabetes, with a stronger predictive power in women than in men, suggesting that more attention should be paid to postprandial hyperglycemia, particularly in women.
Assuntos
Glicemia/metabolismo , Doenças Cardiovasculares/epidemiologia , Diabetes Mellitus Tipo 2/sangue , Angiopatias Diabéticas/epidemiologia , Jejum/fisiologia , Período Pós-Prandial/fisiologia , Caracteres Sexuais , Idoso , Biomarcadores/sangue , Pressão Sanguínea , Índice de Massa Corporal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos TestesRESUMO
AIM: Statins reduce cardiovascular morbidity and mortality in the general population with an excellent risk-benefit profile. The most frequent adverse events are myopathy and increase in hepatic aminotransferases. In this review, we consider the role of liver in metabolism of statins, their potential hepatic toxicity and the guidelines for their prescription in patients affected by different liver diseases. DATA SYNTHESIS: Statin-induced hepatic toxicity: i) occurs in 1-3% of patients; ii) is characterized by increased aminotransferase levels; iii) is dose-related; iv) is frequently asymptomatic; v) usually reverts after dosage reduction or treatment withdrawal. Finally, after recovery, a rechallenge with the same or other statins may not result in increased aminotranferases. CONCLUSIONS: Caution is needed when prescribing statins to patients with liver disease, and liver toxicity should always be monitored during statin treatment. In particular, i) the potential hepatic toxicity requires frequent control of biochemical parameters related to hepatic cytolysis and cholestasis in all patients on statins; ii) administration of statins is counterindicated in patients with advanced or end-stage parenchymal liver disease due to the relevant impairment of their metabolism; iii) cholestatic disorders with secondary dyslipidemia do not require statin treatment even if relevant alterations of the lipid pattern are detected; iv) patients with acute liver disease of viral or alcoholic etiology should not receive statins until normalization of cytolysis enzymes; v) chronic hepatitis patients may be treated by statins if their cardiovascular risk is elevated and provided that careful follow-up is carried out to rapidly recognize the onset of further liver damage; vi) liver transplantation recipients affected by dyslipidemia induced by immunosuppressive therapy can be treated with statins under careful clinical control; vii) the benefits of statins should likely overcome the risks in the large majority of dyslipidemic patients affected by non-alcoholic hepatosteatosis, a disease frequently diagnosed in insulin-resistant subjects.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Fígado/efeitos dos fármacos , Alanina Transaminase/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Fígado/enzimologia , Hepatopatias/enzimologia , Medição de Risco , SegurançaRESUMO
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.
Assuntos
GMP Cíclico/análogos & derivados , Resistência à Insulina/fisiologia , Insulina/fisiologia , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiologia , Animais , Western Blotting , Células Cultivadas , GMP Cíclico/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , MAP Quinase Quinase Quinases/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Ratos , Ratos Zucker , Fator A de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Iloprosta/farmacologia , Obesidade/sangue , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/fisiologia , Adulto , Índice de Massa Corporal , Feminino , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacosRESUMO
OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.
Assuntos
GMP Cíclico/metabolismo , Glucose/metabolismo , Insulina/fisiologia , Proteínas Musculares , Músculo Liso Vascular/fisiologia , Óxido Nítrico/fisiologia , Transdução de Sinais/fisiologia , Transporte Biológico , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Imunofluorescência , Transportador de Glucose Tipo 4 , Guanilato Ciclase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/fisiologia , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Óxido Nítrico/biossínteseRESUMO
AIMS AND BACKGROUND: Intraoperative localization, during open and laparoscopic surgery, of small, nonpalpable colonic lesions located at peculiar sites or with concurrent inflammatory bowel alterations (diverticulosis, perivisceritis) is often difficult. The aim of our work was to assess the validity of radioguided identification after preoperative labeling. METHODS AND STUDY DESIGN: Patients who were candidates for colon surgery for occult lesions that, because of their size and location, were assumed to be difficult to detect, underwent colonoscopy 1 to 2.5 hours before surgery. A small dose of labeled albumin macroaggregates was injected with a sclerotherapy needle into the subserosa underneath the lesion. Immediately following the injection the lesion was identified with a transcutaneously placed gamma detecting probe. Intraoperative tracer detection was performed either during open surgery or by means of a laparoscopic probe (detection time 3-5 mins). The position of the lesion was marked with a suture or with a clip. Surgery was performed according to the type of lesion to be treated. RESULTS: In our initial clinical experience 15 colon lesions were preoperatively marked in 14 patients and were subsequently detected during surgery (four under laparoscopy) with a gamma detecting probe. This technique allows highly accurate, fast, and inexpensive surgical localization of lesions without irradiation and without complications. CONCLUSION: Our experience shows that preoperative endoscopic marking of nonpalpable colon lesions with 99mTc-labeled albumin macroaggregates followed by intraoperative detection with a gamma probe is a useful clinical method that is highly accurate and without complications.
Assuntos
Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/cirurgia , Neoplasias Primárias Desconhecidas/diagnóstico por imagem , Neoplasias Primárias Desconhecidas/cirurgia , Diagnóstico Diferencial , Humanos , Laparoscopia , Laparotomia , CintilografiaRESUMO
BACKGROUND: To know the relationships between pre- and postprandial blood glucose (BG), i.e. BG profile shape, is a requisite for an appropriate therapy for type 2 diabetic patients. In non diabetic subjects, pre-breakfast, pre-lunch and pre-dinner BG are similar, so that BG postprandial excursions are superimposed on a stable BG preprandial baseline. We aimed to clarify: (a) whether BG preprandial baseline is stable also in type 2 diabetes and (b) whether fasting BG (FBG) influences the slope of BG preprandial baseline and the relationships between pre- and postprandial BG. DESIGN: We evaluated self-measured BG profiles of 237 type 2 diabetic patients on diet alone (M/F, 152/85; age 58.6 +/- 0.7 years; years from diagnosis 4.8 +/- 0.6; BMI 28.0 +/- 0.3 kg m-2): 536 profiles containing preprandial BG (corresponding HbA1c 6.8 +/- 0.06%) and 208 profiles containing both pre- and postprandial BG (corresponding HbA1c 6.8 +/- 0.09%). The profiles, measured by nurses, of 866 type 2 diabetic patients on diet alone were also considered (corresponding HbA1c 6.7 +/- 0.04%). RESULTS: In self-measured profiles containing only preprandial BG: (i) FBG (6.77 +/- 0.07 mmol L(-1)) is higher than pre-lunch BG (6.09 +/- 0.07 mmol L(-1)), P = 0.0001) and pre-dinner BG (5.84 +/- 0.06 mmol L(-1)), P =0.0001); (ii) the delta value between FBG and pre-dinner BG is correlated with FBG (r = 0.57, P = 0.0001), the highest FBG, the steepest the fall of BG preprandial baseline throughout the day. This trend is confirmed in profiles measured by nurses. In profiles containing both pre- and postprandial BG: (i) there is a trend to preprandial BG fall (P = 0.0001) and to postprandial BG increase (P = 0.0001) from morning to evening; (ii) postprandial excursions are influenced and sometimes masked by the slope of BG preprandial baseline, thus, in profiles with FBG < or = 6.7 mmol L(-1), all postprandial values are higher than FBG (P = 0.0001), whereas in profiles with FBG > 7.8 mmol L(-1), postprandial values are not significantly higher than FBG. CONCLUSION: In type 2 diabetes, the shape of BG profiles changes in relation to FBG, because it deeply influences the slope of BG preprandial baseline on which postprandial excursions are superimposed. Thus, before planning treatment policies, not only the extent of fasting and postprandial hyperglycaemia, but also the shape of profiles should be considered, to safely correct hyperglycaemia without inducing hypoglycaemia.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/metabolismo , Jejum/metabolismo , Automonitorização da Glicemia/normas , Ritmo Circadiano/fisiologia , Feminino , Humanos , Hiperglicemia/diagnóstico , Hiperglicemia/metabolismo , Hipoglicemia/diagnóstico , Hipoglicemia/metabolismo , Hipoglicemia/prevenção & controle , Masculino , Pessoa de Meia-Idade , Recursos Humanos de Enfermagem Hospitalar , Período Pós-Prandial , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Acetilcisteína/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Acetilcisteína/sangue , Difosfato de Adenosina/antagonistas & inibidores , Difosfato de Adenosina/farmacologia , Adulto , Calcimicina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colágeno/farmacologia , GMP Cíclico/sangue , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/sangue , Humanos , Ionomicina/farmacologia , Masculino , Oxidiazóis/farmacologia , Inibidores da Agregação Plaquetária/sangue , Purinonas/farmacologia , Quinoxalinas/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
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.
Assuntos
Plaquetas/fisiologia , AMP Cíclico/sangue , GMP Cíclico/sangue , Nitroprussiato/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Adulto , Plaquetas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Cinética , Masculino , Fatores de TempoRESUMO
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.
Assuntos
AMP Cíclico/análise , GMP Cíclico/análise , Expressão Gênica , Insulina/farmacologia , Músculo Liso Vascular/citologia , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Cálcio/farmacologia , Células Cultivadas/efeitos dos fármacos , Citrulina/metabolismo , Humanos , Ionomicina/farmacologia , Músculo Liso Vascular/enzimologia , Óxido Nítrico/biossíntese , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Nitroprussiato/farmacologia , RNA Mensageiro/análise , Transcrição GênicaRESUMO
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.
Assuntos
Acetilcisteína/farmacologia , Arginina/farmacologia , Plaquetas/fisiologia , Sequestradores de Radicais Livres/farmacologia , Guanosina Monofosfato/fisiologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Antioxidantes/farmacologia , Plaquetas/efeitos dos fármacos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Humanos , Óxido Nítrico/fisiologia , Agregação Plaquetária/fisiologia , ômega-N-Metilarginina/farmacologiaRESUMO
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.
Assuntos
Plaquetas/efeitos dos fármacos , Canavanina/farmacologia , Difosfato de Adenosina/fisiologia , Arginina/farmacologia , Plaquetas/fisiologia , Catecolaminas/fisiologia , GMP Cíclico/metabolismo , Interações Medicamentosas , Humanos , Técnicas In Vitro , Óxido Nítrico Sintase/antagonistas & inibidores , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Testes de Função Plaquetária , Serotonina/fisiologiaAssuntos
Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Resistência à Insulina , Insulina/farmacologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismoRESUMO
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.
Assuntos
Plaquetas/fisiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus/sangue , Insulina/farmacologia , Nitroglicerina/farmacologia , Obesidade/sangue , Agregação Plaquetária/efeitos dos fármacos , Magreza/sangue , Difosfato de Adenosina/farmacologia , Adulto , Análise de Variância , Glicemia/análise , Plaquetas/efeitos dos fármacos , Pressão Sanguínea , Índice de Massa Corporal , Colesterol/sangue , HDL-Colesterol , GMP Cíclico/sangue , Diabetes Mellitus/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Hemoglobinas Glicadas/análise , Humanos , Insulina/sangue , Resistência à Insulina , Masculino , Obesidade/fisiopatologia , Agregação Plaquetária/fisiologia , Inibidores da Agregação Plaquetária/farmacologia , Valores de Referência , Triglicerídeos/sangueRESUMO
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
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.