G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb.

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.

Platelet dysfunction in central obesity.

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.

Resistance to the nitric oxide/cyclic guanosine 5'-monophosphate/protein kinase G pathway in vascular smooth muscle cells from the obese Zucker rat, a classical animal model of insulin resistance: role of oxidative stress.

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.

Postprandial blood glucose is a stronger predictor of cardiovascular events than fasting blood glucose in type 2 diabetes mellitus, particularly in women: lessons from the San Luigi Gonzaga Diabetes Study.

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.

Insulin directly reduces platelet sensitivity to aggregating agents. Studies in vitro and in vivo.

The aim of this study was to investigate the influence of insulin on platelet function, both in vitro and in vivo. For the in vitro investigation, we evaluated whether insulin affects platelet function at a physiological hormone concentration by incubating the platelet-rich plasma (PRP) of fasting subjects with human regular insulin at the final concentration of 40 microU/ml for 30 min; we observed a significant reduction of platelet sensitivity to all the aggregating agents employed, i.e., ADP, platelet-activating factor (PAF), epinephrine, collagen, and Na+ arachidonate. To investigate whether the insulin effect on platelets is dose dependent, we incubated the PRP of fasting subjects with different concentrations of human regular insulin (40, 80, 120, and 160 microU/ml) for 5 min, and we observed that the insulin-induced reduction of platelet sensitivity to aggregating agents is a dose-dependent phenomenon. Furthermore, the comparison between the platelet responses after 5 and 30 min of incubation with insulin showed that the insulin effect on platelet aggregation is time dependent. The lack of specificity of its inhibiting activity suggests that insulin does not interfere with the initial binding of each aggregating agent at specific sites but does influence a common step of platelet aggregation. Our study rules out the possibility that insulin reduces platelet-function-modifying intraplatelet cAMP levels or thromboxane A2 production, because this hormone decreases the platelet concentrations of cAMP--a phenomenon that, per se, promotes platelet aggregation--and does not modify collagen or Na+ arachidonate--induced platelet production of thromboxane A2, measured by radioimmunoassay of its stable-metabolite thromboxane B2.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin increases guanosine-3',5'-cyclic monophosphate in human platelets. A mechanism involved in the insulin anti-aggregating effect.

To investigate whether insulin reduces platelet aggregability through a modulation of the guanosine-3',5'-cyclic monophosphate (cGMP) concentrations, we determined by a radioimmunoassay the cGMP values in the platelet-rich plasma (PRP) obtained from 17 healthy volunteers and incubated for 3 min with different concentrations of human recombinant insulin (0, 240, 480, 720, 960, and 1,920 pM). Insulin induced a dose-dependent cGMP increase, from 18.5 +/- 3.3 to 42.0 +/- 6.4 pmol/10(9) platelets (P = 0.0001). This increase was completely blunted when PRP was preincubated for 20 min with the tyrosine kinase inhibitor genistein (10 microM) or with the guanylate cyclase inhibitor methylene blue (10 microM), but the increase remained highly significant (P = 0.003 and 0.009) when PRP was preincubated for 20 min with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 microM) or with the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine (L-NMMA, 30 microM). Finally, the insulin-induced decrease of platelet aggregability to collagen and ADP was completely blunted when PRP was preincubated with 10 microM of the guanylate cyclase inhibitor methylene blue. This study demonstrates that the platelet anti-aggregatory effect exerted by insulin is attributable to the insulin-induced increase of cGMP that is due to a direct receptor-mediated platelet guanylate cyclase activation.

The insulin-induced increase of guanosine-3',5'-cyclic monophosphate in human platelets is mediated by nitric oxide.

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.

Studies on mechanisms involved in hypoglycemia-induced platelet activation.

The aim of our study was to investigate the mechanisms involved in hypoglycemia-induced platelet activation. Sixteen healthy male subjects received a 60-min intravenous infusion of human regular insulin at the rate of 64 mU . m-2 . min-1: throughout 150 min, we serially measured plasma concentrations of glucose, insulin, and counterregulatory hormones; platelet sensitivity to ADP, thrombin and platelet-activating factor; plasma concentrations of platelet markers for specific proteins of in vivo release reaction (beta-thromboglobulin and platelet factor 4). Our study showed that insulin-induced hypoglycemia causes a significant increase in platelet sensitivity to aggregating agents in vitro and a platelet release reaction in vivo. Hypoglycemia-induced platelet activation was not correlated with plasma glucose concentrations at nadir and occurred before the increase of plasma growth hormone and cortisol. To further elucidate the mechanisms of hypoglycemia-induced platelet activation, we incubated in vitro platelet-rich plasma (PRP) of seven fasting healthy subjects with the same concentrations of insulin, epinephrine, glucagon, growth hormone, and cortisol measured in vivo during insulin-induced hypoglycemia. Only epinephrine was able to increase platelet sensitivity to aggregating agents. To investigate the role of alpha-adrenergic receptors in this phenomenon, we also studied four healthy subjects on another occasion, repeating the above-described insulin infusion together with intravenous infusion of phentolamine (-15 to +150 min), 5 mg over 2 min followed by 500 micrograms/min. alpha-Blockade was able to suppress hypoglycemia-induced increase of platelet sensitivity to aggregating agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired insulin-induced platelet antiaggregating effect in obesity and in obese NIDDM patients.

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)

Insulin stimulates nitric oxide synthesis in human platelets and, through nitric oxide, increases platelet concentrations of both guanosine-3', 5'-cyclic monophosphate and adenosine-3', 5'-cyclic monophosphate.

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.

Insulin stimulates glucose transport via nitric oxide/cyclic GMP pathway in human vascular smooth muscle cells.

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.

Postprandial exercise in type I diabetic patients on multiple daily insulin injection regimen.

This study shows the influence on plasma glucose concentrations of 45 min of mild exercise (48 +/- 4% of maximum aerobic capacity) performed 180 min after breakfast and 195 min after a subcutaneous injection of regular insulin by six type I (insulin-dependent) diabetic patients on a three-daily insulin injection regimen (regular insulin before breakfast and lunch, regular + intermediate insulin before supper). It has been observed that such exercise does not induce a large plasma glucose decrease. Actually, plasma glucose concentrations were 99 +/- 18 mg/dl before exercise, reached a nadir of 78 +/- 17 mg/dl at 35 min, and were 81 +/- 15 mg/dl at the end of exercise. During the control study at rest, in the same 45-min time interval, plasma glucose decreased from 146 +/- 31 to 128 +/- 31 mg/dl. In the exercise study, one patient began exercising while hypoglycemic, and another patient developed asymptomatic hypoglycemia during exercise. In the control study at rest, one patient showed hypoglycemic glucose concentrations. Throughout the exercise study, plasma free-insulin concentrations decreased (from 32 +/- 5 to 20 +/- 4 microU/ml) as a result of the pharmacokinetics of subcutaneously injected insulin.

Moderate exercise increases platelet function in type I diabetic patients without severe angiopathy and in good control.

OBJECTIVE: The aim of this study was to investigate whether a 45-min moderate exercise, performed postprandially with a timing that partially prevented the risk of hypoglycemia, was able to modify platelet function in patients affected by insulin-dependent (type I) diabetes mellitus without severe late complications and in a good metabolic control. RESEARCH DESIGN AND METHODS: We submitted 6 male type I diabetic patients (27.2 +/- 3.4 yr; body mass index, 21.4 +/- 0.6 kg/m2; HbA1c, 7.6 +/- 0.9%) on a daily three-insulin injection regimen, without severe late complications of diabetes, to a 45-min moderate exercise (about 50% of maximal oxygen consumption) with a cycle ergometer, beginning 180 min after breakfast and 195 min after a subcutaneous shot of regular insulin. Serial venous blood samples were conducted to measure plasma glucose, free insulin, counterregulatory hormones (glucagon, growth hormone, cortisol, and catecholamines), platelet sensitivity to ADP, platelet activating factor and collagen, and plasma concentrations of the platelet-specific protein beta-thromboglobulin (a marker of the platelet release reaction in vivo). RESULTS: Exercise was accompanied by a decrease of plasma glucose (from 5.9 +/- 1.2 to 4.6 +/- 1 mmol/L, P = 0.067) and free insulin (from 180 +/- 36 to 114 +/- 30 pmol/L, P = 0.003), and by a significant increase of growth hormone (from 5 +/- 1 to 15 +/- 4 micrograms/L, P = 0.045), cortisol (from 240 +/- 30 to 406 +/- 69 nmol/L, P = 0.018), epinephrine (from 1005 +/- 240 to 5143 +/- 1753 pmol/L, P = 0.077), and norepinephrine (from 5.04 +/- 1.08 to 13.48 +/- 2.98 nmol/L, P = 0.009). Platelet sensitivity to the agonists and plasma concentrations of beta-thromboglobulin increased during the exercise period. In particular, ADP ED50 reached during exercise 61 +/- 16% of basal values (P = 0.048), platelet activating factor ED50 reached 73 +/- 11% (P = 0.043), and collagen ED50 reached 68 +/- 9% (P = 0.008). beta-Thromboglobulin rose from 24 +/- 2 to 32 +/- 3 micrograms/L (P = 0.007). CONCLUSIONS: Moderate exercise enhances platelet function in type I diabetic patients without severe angiopathy and in a good metabolic control.

Platelet resistance to nitrates in obesity and obese NIDDM, and normal platelet sensitivity to both insulin and nitrates in lean NIDDM.

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.

Insulin influences the renin-angiotensin-aldosterone system in humans.

This study investigates whether insulin influences the renin-angiotensin-aldosterone system in humans. Six healthy male volunteers were placed on a 30-minute euglycemic insulin clamp at 160 microU/mL; euglycemia was maintained also in the following 60 minutes by means of appropriate dextrose infusion. Throughout the study, plasma renin activity, angiotensin II, aldosterone, and factors involved in the regulation of the renin-angiotensin-aldosterone system were measured: catecholamines, angiotensin-converting enzyme, sodium, and potassium. A significant increase of plasma renin activity and angiotensin II was observed, and a decrease of aldosterone was also detected. These changes can be ascribed to the effects of the rapid insulin-induced plasma potassium decrease on plasma renin activity and aldosterone secretion because they did not occur in a control clamp study with a potassium infusion.

Insulin influences immunoreactive endothelin release by human vascular smooth muscle cells.

This study sought to investigate whether insulin influences immunoreactive endothelin release from cultured human vascular smooth muscle cells. For this purpose, we incubated cultured vascular smooth muscle cells (VSMC) obtained from human microvessels with 0, 80, and 320 microU/mL insulin with or without arginine vasopressin (10 nmol/L) and angiotensin II (10 nmol/L). After 6 hours, the culture supernatant was collected and immunoreactive endothelin was determined by radioimmunoassay. Insulin at a concentration of 320 microU/mL induced a significant increase of immunoreactive endothelin levels in medium (from 15.2 +/- 0.8 to 20.6 +/- 0.8 pg/200 microL, P < .01) and potentiated arginine vasopressin- and angiotensin II-induced immunoreactive endothelin release (P < .0001 and P < .04, respectively). Insulin at a concentration of 80 microU/mL did not induce a significant increase of spontaneous immunoreactive endothelin release, but significantly increased the effects of arginine vasopressin (P < .05). In conclusion, insulin influences immunoreactive endothelin release from human VSMC in culture.

Nonenzymatic glycation of fibronectin impairs adhesive and proliferative properties of human vascular smooth muscle cells.

Nonenzymatic glycation of proteins is involved in the pathogenesis of diabetes vascular complications. Extracellular matrix proteins are a prominent target for nonenzymatic glycation because of their slow turnover rates. The aim of this study was to investigate the influence of human fibronectin (F) nonenzymatic glycation on adhesion and proliferation of cultured human vascular smooth muscle cells (hVSMC). Incubation of human F with 500 mmol/L D-glucose at 37 degrees C induced a time-dependent increase in fluorescence detectable at 440 nm after excitation at 363 nm. Nonenzymatic glycation did not affect binding of F itself to the plates. Adhesion of hVSMC to F increased with the increase of incubation time of the cells on the protein from 30 minutes up to 120 minutes and remained stable thereafter. Adhesion to glycated fibronectin (GF) was reduced in comparison to control F at all the different adhesion times. Adhesion of hVSMC to GF was reduced when F was exposed to glucose for 4, 9, or 28 days (P=.0417 to .0025), but not when F was exposed for 1 day. Adhesion of hVSMC to GF was reduced compared with adhesion to nonglycated F at all coating concentrations from 0.2 to 10 micrograms/mL (P=.05 to .014). Thus, nonenzymatic glycation of F impairs adhesion of hVSMC in vitro. Proliferation of hVSMC on F increased with increasing concentrations of the protein as coating agent (ANOVA:P<.0001 for both nonglycated F and GF). Proliferation with F glycated for 4, 9, and 28 days was reduced at concentrations of 1, 3, and 10 micrograms/mL as compared with proliferation with nonglycated F (P=.0253 to .0001). Proliferation on F glycated for only 1 day was not significantly reduced. When the number of hVSMC plated on control F was reduced by 25% to take into account the reduced adhesion, the number of cells that proliferated on F was still reduced. In conclusion, nonenzymatic glycation of F impairs adhesive and proliferative properties of hVSMC.

Effects of diltiazem on thromboxane B2 production from platelet-rich plasma and whole blood.

The present study evaluated the effects of the calcium-channel blocking agent diltiazem on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of thromboxane A2) from platelet-rich plasma (PRP) and whole blood samples. Our results showed that diltiazem inhibits collagen- and thrombin-induced platelet aggregation and TXB2 production from PRP. Since no significant interference with conversion of arachidonate to thromboxane A2 was demonstrated, inhibition of phospholipase A2 activity may be the prevailing mechanism of the diltiazem effect. The drug demonstrated a dose-related inhibitory activity on TXB2 synthesis from whole blood samples during spontaneous clotting or following stimulation with collagen or thrombin. The present results give further evidences for an antiplatelet activity of diltiazem and support the hypothesis that inhibition of platelet function contributes to the therapeutic efficacy of this drug in the treatment of cardiovascular diseases.

Influence of propranolol on platelet aggregation and thromboxane B2 production from platelet-rich plasma and whole blood.

The beta-adrenoceptor antagonist propranolol is used in the therapy of hypertension and ischemic heart disease. The aim of our study was to evaluate the effects of this drug on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of Thromboxane A2) from platelet rich plasma (PRP), whole blood samples and during spontaneous clotting. The results indicate that propranolol at concentrations near the therapeutic range, significantly inhibit collagen and thrombin-induced platelet aggregation and TxB2 synthesis from PRP. Furthermore the drug demonstrates inhibitory activity on B-TG release and TxB2 production from whole blood samples and on spontaneous clotting. The results suggest that some benefits of propranolol in the treatment of patients with coronary artery disease or cardiovascular conditions associated with platelet hyperaggregability may also be related to interference with platelet activation "in vivo" and with TxA2 generation.

Organic nitrates and compounds that increase intraplatelet cyclic guanosine monophosphate (cGMP) levels enhance the antiaggregating effects of the stable prostacyclin analogue iloprost.

The present study investigated the effect of a combination between the stable prostacyclin (PGI2) analogue iloprost and compounds, glyceryl trinitrate (GTN) and L-arginine-, which enhance the intraplatelet cyclic guanosine monophosphate (cGMP) levels on platelet aggregation, release reaction and cyclic nucleotide content: in particular cyclic adenosine monophosphate (cAMP) and cGMP. Iloprost inhibited in a dose-dependent way the platelet aggregation in response to collagen, adenosine diphosphate (ADP) and adrenaline and it increased the intraplatelet cAMP concentrations. GTN directly decreased the platelet responses and increased the intraplatelet levels of both cGMP and cAMP. GTN (20 x 10(-6) mol/l) and L-arginine (0.2 x 10(-3) mol/l) potentiated the inhibitory effects of iloprost on platelet aggregation and release reaction. Our results suggest: 1. A synergistic effect of the simultaneous increase of both cAMP and cGMP on the biochemical steps involved in the inhibition of the platelet response; 2. An influence of cGMP on cAMP accumulation.