Blood glucose pre-prandial baseline decreases from morning to evening in type 2 diabetes: role of fasting blood glucose and influence on post-prandial excursions.

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.

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 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.

Preliminary results of a phase I/II study of paclitaxel, cisplatin, and cyclophosphamide in advanced ovarian carcinoma.

The clinical activity and toxicity of the triple combination of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), cyclophosphamide, and cisplatin was assessed in both previously treated and untreated women with advanced ovarian carcinoma. Paclitaxel 175 mg/m2 was administered over 3 hours following standard premedication (prednisolone, dexchlorpheniramine, and cimetidine). Cisplatin 80 mg/m2 and cyclophosphamide 600 mg/m2 were given 6 to 12 hours after paclitaxel. Treatment was given at 3-week intervals for six cycles. Twenty-seven patients entered the study; 23 were evaluable for toxicity and 17 for response. Paclitaxel appeared to add additional efficacy to the standard cisplatin/cyclophosphamide regimen. Both the overall and complete remission rates were very high (88% and 70%, respectively), and histologically confirmed complete remissions exceeded 60%. Longer follow-up is needed to determine the duration of these responses. The primary toxicities included leukoneutropenia, peripheral neuropathy, asthenia, and alopecia. Only two of 23 patients withdrew because of toxicity, however, and only two treatment cycles were complicated by neutropenic fever requiring intravenous antibiotics. No life-threatening toxicities were encountered, although the peripheral neuropathy was poorly and slowly reversible and may have a significant impact on the patients' quality of life.

Influence of protamine on adhesion, chemotaxis and proliferation of human vascular smooth muscle cells.

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.

Phase I/II study of paclitaxel, cisplatin, and cyclophosphamide in advanced ovarian carcinoma: preliminary results.

In this phase I/II study, we assessed the impact of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in the treatment of advanced ovarian carcinoma combined with the standard regimen cisplatin/cyclophosphamide given as follows: paclitaxel 175 mg/m2 (over 3 hours perfusion with standard premedication), cisplatin 80 mg/m2 (6 to 12 hours after paclitaxel), and cyclophosphamide 400 mg/m2. From February 1994 to January 1996, 27 patients (median age, 55 years; age range, 35 to 74 years) were entered into the study. Eight patients had distant metastases and 19 had early locoregional disease (stage III, 18 patients; stage IC, one patient). Twenty-two patients had undergone prior surgery (simple biopsy, six patients; optimally debulked, nine patients; suboptimally debulked, seven patients). Twenty-one patients had received no prior chemotherapy and six were previously treated with at least one platinum-based regimen. A maximum of six courses of paclitaxel/cisplatin/cyclophosphamide were given every 21 days. Twenty-three patients were evaluable for toxicity: neutropenia (World Health Organization grade 3/4), 91% of patients; thrombopenia (World Health Organization grade 3/4), 13% of patients; two episodes of neutropenia with fever; and neurotoxicity grade 3, 17% of patients. Alopecia grade 3 was reported in all patients. No hypersensitivity reactions and no cardiac toxicity was observed. Among 17 patients evaluable for response (patients with stage IV disease or stage III suboptimally debulked), 12 (70%) clinical complete responses (CRs) and three (18%) partial responses were observed. Among the 12 patients with CRs, 10 underwent second-look laparotomy and seven of them (70%) achieved a pathologic CR. In the group of 11 chemotherapy-naive patients evaluable for response, eight (72%) achieved a CR and three (28%) achieved a partial response. This combination seems to be safe, with very acceptable toxicity, and also seems to be highly active in the treatment of patients with advanced ovarian carcinoma.

Studies on the influence of insulin on cyclic adenosine monophosphate in human vascular smooth muscle cells: dependence on cyclic guanosine monophosphate and modulation of catecholamine effects.

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.

Interplay between milrinone and adenosine in the inhibition of human platelet response.

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.

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.

Insulin exerts opposite effects on platelet function at physiological and supraphysiological concentrations.

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.

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.

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)

Glyceryl trinitrate enhances the adenosine-induced inhibition of platelet responses: a mechanism potentially involved in the in vivo anti-aggregating effects of organic nitrates.

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.

A phase II study of tamoxifen combined with cisplatin-interleukin 2 and alpha-interferon in metastatic melanoma.

Tamoxifen (TAM) has been reported to enhance cisplatin (CDDP) cytotoxicity in experimental and clinical melanoma studies. Based on our previous experience with sequential cisplatin-interleukin-2 (IL2)-interferon (IFN), we performed a phase II study of TAM combined with our original CDDP-IL2-IFN regimen in 22 pretreated metastatic melanoma patients. With a 41% response rate (95% CI, 21-61) we confirmed the interesting antitumor activity of CDDP-IL2-IFN combination; however, TAM enhanced neither the response rate nor the duration of response, but appeared to induce significantly more myelotoxicity, as compared to our previous results with CDDP-IL2-IFN alone. Whereas mechanisms by which TAM may modulate CDDP cytotoxicity in melanoma tumors remain unknown, the exact place of TAM, if any, and its safety in chemotherapeutic or chemoimmunotherapeutic combinations require further investigations.

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone.

It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240-960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7 +/- 0.1 to 2.6 +/- 0.4 pmol/10(6) cells, p = 0.0001; cGMP: from 1.3 +/- 0.2 to 3.4 +/- 0.7 pmol/10(6) cells, p = 0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p = 0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor NG-monomethyl-L-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p = 0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p = 0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.

Studies on in vitro effect of picotamide on human platelet aggregation in platelet-rich plasma and whole blood.

Picotamide is a new antiaggregating agent influencing the platelet prostaglandin pathway through an inhibition of thromboxane A2 (TXA2) synthesis and a competitive antagonism of platelet TXA2 receptors. In the present study, we investigated the in vitro effect of this drug on human platelet aggregation induced by different agents (adenosine 5'-diphosphate [ADP], collagen, Na arachidonate) both in platelet-rich plasma (PRP; Born's method) and whole blood (WB; impedance method). For each aggregating agent, ED50 value (agonist concentration necessary to induce a maximal aggregation of 50%) was determined in control samples and following addition of different picotamide concentrations on the basis of dose-response curves. Picotamide decreased the response to each aggregating agent in both WB and PRP samples. In WB, 25 microM picotamide was able to induce a highly significant enhancement of ED50 values for ADP (from 6.6 +/- 1 microM to 12.7 +/- 1.7 microM, p < 0.01), Na arachidonate (from 740 +/- 240 microM to 1,080 +/- 280 microM, p < 0.01) and collagen (from 2.4 +/- 0.3 micrograms/ml to 3.8 +/- 0.15 micrograms/ml, p < 0.01). In PRP, the same picotamide concentration significantly enhanced ED50 for each aggregating agent (from 2.0 +/- 0.1 microM to 3.1 +/- 0.3 microM for ADP, p < 0.01; from 960 +/- 80 microM to 1,850 +/- 260 microM for Na arachidonate, p < 0.001; from 3.0 +/- 0.3 microgram/ml to 5.0 +/- 0.8 micrograms/ml for collagen, p < 0.01). Present results show that picotamide effect on platelet response is present also in WB. Data might support the use of picotamide as antiaggregating agent in vascular diseases.

Effects of forskolin and organic nitrate on aggregation and intracellular cyclic nucleotide content in human platelets.

1. The present study investigated the effect of a combination between forskolin, a naturally occurring diterpene which directly activates adenylyl cyclase, and glyceryl trinitrate (GTN), which enhances intraplatelet cyclic guanosine monophosphate levels, on human platelet aggregation and intracellular content of cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). 2. Forskolin inhibited, in a dose-dependent way, platelet aggregation in response to collagen and adrenaline in platelet-rich plasma. In whole blood samples, forskolin inhibited collagen-stimulated aggregation. In presence of forskolin the intraplatelet cAMP levels were significantly increased. 3. GTN directly decreased the platelet response to collagen in whole blood samples (IC50 = 122 mumol/l) and it increased the intraplatelet levels of both cGMP and cAMP. 4. GTN at 20 and 40 mumol potentiated the inhibitory effects of forskolin on platelet aggregation in both platelet-rich plasma and whole blood. 5. Our results suggest a synergistic effect of the simultaneous increase of both cAMP and cGMP on the biochemical steps involved in the inhibition of the platelet response.

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.

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.

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.