Angiotensin-(1-7), a protective peptide against vascular aging.

Vascular aging is a complex and multifaceted process that provokes profound molecular, structural, and functional changes in the vasculature. Eventually, these profound aging alterations make arteries more prone to vascular disease, including hypertension, atherosclerosis and other arterial complications that impact the organism beyond the cardiovascular system and accelerate frailty. For these reasons, preventing or delaying the hallmarks of vascular aging is nowadays a major health goal, especially in our aged societies. In this context, angiotensin(Ang)-(1-7), a major player of the protective branch of the renin-angiotensin system, has gained relevance over recent years as growing knowledge on its anti-aging properties is being unveiled. Here, we briefly review the main actions of Ang-(1-7) against vascular aging. These include protection against vascular cell senescence, anti-inflammatory and antioxidant effects together with the induction of cytoprotective systems. Ang-(1-7) further ameliorates endothelial dysfunction, a hallmark of vascular aging and disease, attenuates fibrosis and calcification and promotes protective angiogenesis and repair. Although further research is needed to better understand the anti-aging properties of Ang-(1-7) on the vasculature, this heptapeptide arises as a promising pharmacological tool for preventing vascular aging and frailty.

Gender differences in the effect of aspirin on retinal ischemia, prostanoid synthesis and nitric oxide production in experimental type 1-like diabetes.

BACKGROUND: The protective effect of acetylsalicylic acid (aspirin) against cardiovascular events is known to be weaker in women than in men. The present study was designed to test whether this effect of aspirin differed between sexes in an experimental model of diabetes with retinal ischemia. METHODS: We compared nondiabetic rats and rats after 1, 2 and 3 months of diabetes that were given 2 mg/kg/day p.o. of aspirin from the first day of diabetes. The variables recorded were platelet aggregation, production of thromboxane B(2) (TxB(2)), 6-keto-prostaglandin F(1alpha) and aortic nitric oxide, and the percentage of the retinal surface occupied by horseradish peroxidase (HRP)-permeable vessels. RESULTS: In female rats made diabetic, TxB(2) synthesis was more markedly reduced, and the percentage of HRP-permeable retinal vessels was less markedly reduced, than in their male counterparts. The response to aspirin treatment was weaker in female than in male diabetic rats in terms of inhibition of TxB(2) synthesis, increased nitric oxide production, and prevention of the increase in the percentage of retinal surface covered by HRP-permeable vessels. CONCLUSION: Aspirin was less effective in preventing retinal ischemia in experimental diabetes in female than in male rats.

Effects of aspirin plus alpha-tocopherol on brain slices damage after hypoxia-reoxygenation in rats with type 1-like diabetes mellitus.

Diabetes mellitus is a risk factor for cerebrovascular ischemic disease. Aspirin (acetylsalicylic acid) is the most widely used drug for the secondary prevention of thrombotic phenomena. It has been also recently demonstrated that alpha-tocopherol influenced in vitro the antiplatelet effect of aspirin. The aim of the present study is to evaluate the effects aspirin plus alpha-tocopherol on cerebral oxidative stress, prostaglandin production and the nitric oxide pathway in a model of hypoxia-reoxygenation in rat brain slices. Our results show an imbalance in brain oxidative status (reflected mainly as the increase in lipid peroxides) as a result of diabetes itself rather than a failure of the glutathione-based antioxidant system. Moreover, our results also show a higher concentration of prostaglandins in the brain of diabetic animals and a higher nitric oxide concentration, mainly through a high iNOS activity. After 180 min of post-hypoxia reoxygenation, LDH activity was 40.6% higher in animals with diabetes, in comparison to non-diabetic animals. The increase of the LDH efflux observed in non-treated rats was reduced by 31.2% with aspirin, by 34.7% with alpha-tocopherol and by 69.8% with the association aspirin-alpha-tocopherol. The accumulation of prostaglandin E2 observed in diabetic non-treated rats was reduced statistically after the treatment with aspirin (34.2% inhibition), alpha-tocopherol (19.3% inhibition) or the association aspirin-alpha-tocopherol (54.4% inhibition). Nitric oxide production after 180 min reoxygenation was significantly reduced in aspirin (36.4%), alpha-tocopherol (22.7%) and aspirin-alpha-tocopherol (77.8%) treated rats with respect to diabetic non-treated animals; this was related mainly with a reduction in iNOS activity. The association between aspirin and alpha tocopherol could protects against brain ischemic-reperfusion damage with a better profile than aspirin alone.

Antioxidant and antiplatelet effects of the alpha-tocopherol-aspirin combination in type 1-like diabetic rats.

We analyze the effect of the combination of acetylsalicylic acid (2 mg/kg/day p.o.) and alpha-tocopherol (25 mg/kg/day p.o.) in a type-1-like experimental model of diabetes mellitus on platelet factors, endothelial antithrombotic factors and tissue oxidative stress. In diabetic rats, the combination of drugs had a greater inhibitory effect on platelet aggregation than in untreated control animals with diabetes (88.87%). The combination of drugs had little effect on the inhibition of thromboxane production (-90.81%) in comparison to acetylsalicylic acid alone (-84.66%), potentiated prostacyclin production (+162%) in comparison to alpha-tocopherol alone (+30.55%), and potentiated nitric oxide production (+241%) in comparison to either drug alone (acetylsalicylic acid +125%, alpha-tocopherol +142%). The combination of the two drugs improved the thromboxane/prostacyclin balance (0.145+/-0.009) in comparison to untreated diabetic animals (4.221+/-0.264) and in untreated healthy animals (0.651+/-0.045). It did not potentiate the antioxidant effect of either drug alone, but did increase tissue concentrations of reduced glutathione, especially in vascular tissue (+90.09% in comparison to untreated animals). In conclusion, in the experimental model of diabetes tested here, the combination of acetylsalicylic acid and alpha-tocopherol led to beneficial changes that can help protect tissues from thrombotic and ischemic phenomena.

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles]. / Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes.

INTRODUCTION: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. MATERIAL AND METHODS: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. RESULTS: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. CONCLUSION: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage.

Lipid peroxidation and glutathione system in hyperlipemic rabbits: influence of olive oil administration.

We studied the effect of supplementation (10% w/w) of a hyperlipemic diet (1% cholesterol) with olive oil (OLIV) for 6 weeks in four groups of 10 rabbits each. At the end of this period, we determined lipid peroxidation, glutathione content, and glutathione peroxidase, reductase and transferase activities in liver, brain, heart, aorta and platelets. The atherogenic diet increased tissue lipid peroxidation and decreased the protective antioxidant effect of glutathione. Dietary supplementation with olive oil reduced tissue lipid peroxidation by 71.6% in liver, 20.3% in brain, 84.5% in heart, 63.6% in aorta, 72% in platelets. The ratios total/oxidized glutathione were increased in all tissues (49% in liver, 48% in brain, 45% in heart, 83% in aorta, 70% in platelets). Olive oil increased glutathione peroxidase and transferase activities in all tissues. We conclude that in rabbits made hyperlipemic with a diet rich in saturated fatty acids, olive oil decreased tissue oxidative stress.

Protective effect of triflusal and its main metabolite HTB in an in vitro model of anoxia-reoxygenation in rat brain slices: comparison with acetylsalicylic and salicylic acids.

Triflusal is a fluorinated derivative of acetylsalicylic acid (ASA) with demonstrated antithrombotic activity. Recently, evidence for a neuroprotective effect has been obtained. The aim of this study was to compare the neuroprotective effects of the main metabolite of triflusal (2-hydroxy-4-trifluoromethylbenzoic acid, HTB) and the ASA metabolite salicylic acid (SA) in an in vitro model of anoxia-reoxygenation in rat brain slices. Rat brain slices (n=10 per group) were subjected to a period of anoxia followed by 180 min reoxygenation. We measured oxidative stress parameters (lipid peroxidation, glutathione system), prostaglandins (PGE(2)), nitric oxide pathway activity (NO) (nitrites+nitrates, constitutive and inducible NO synthase activity) and LDH efflux, a biochemical marker of cell death. Various concentrations (10, 100 and 1,000 microM) of triflusal, HTB, ASA or SA were tested. Triflusal at 10, 100 and 1,000 microM decreased LDH efflux in rat brain slices after anoxia/reoxygenation by 24%, 35% and 49% respectively. This effect was proportionately greater than that of ASA (0%, 13% and 32%). The results with HTB were similar to those with triflusal, whereas SA showed a greater protective effect than ASA (13%, 33% and 35%). The antioxidant effects of HTB and SA on the biochemical mechanisms of cell damage studied here were also greater than the effects of triflusal and ASA, a finding attributable mainly to the decrease in lipid peroxidation and to the ability of HTB to also increase glutathione levels. The triflusal metabolite reduced inducible NO synthase activity by 18%, 21% and 30%, whereas SA inhibited this activity by 9%, 17% and 23%. Triflusal and HTB led to greater increases in NO synthase than ASA or AS. In conclusion, the metabolite HTB plays an important role in the neuroprotective effect of triflusal, at least in the experimental model of anoxia-reoxygenation tested here.

Effect of dietary supplementation with evening primrose oil on vascular thrombogenesis in hyperlipemic rabbits.

The dietary intake of saturated fatty acids affects arteriosclerosis. We studied the effect of supplementation (15% wt/wt) of a hyperlipemic diet (1.3% cholesterol) with evening primrose oil (Oenothera biennis) in four groups of 10 rabbits each. After 6 weeks the aortic endothelium was analyzed morphologically with scanning electron microscopy, and the arterial wall was studied with morphometric techniques and cell nucleus counts. Endothelial functioning was analyzed by measuring prostacyclin synthesis, and thrombogenicity of the subendothelium was studied by perfusion in a Baumgartner annular chamber. Evening primrose oil reduced hypercholesterolemia (from 29 +/- 3 to 12 +/- 2 nmol/l), increased HDL-cholesterol (from 0.5 +/- 0.06 to 0.8 +/- 0.09 nmol/l) and doubled prostacyclin synthesis (from 2.7 +/- 2 to 6.2 +/- 0.7 ng/mg aorta) in rabbits on the hyperlipemic diet, reduced subendothelial surface occupied by platelets (from 6.9 +/- 0.4 to 4.8 +/- 0.3%), and reduced human platelet adhesion on the subendothelium (from 53.3 +/- 6% to 38 +/- 8%, respect to total occupation). Morphological analyses showed that evening primrose oil diminished endothelial lesions caused by the atherogenic diet, reducing area of the arterial wall (from 6.9 +/- 0.2 to 4.7 +/- 0.2 microm2 x 10(6)) and the degree of neointimal proliferation (from 0.6 +/- 0.02 to 0.4 +/- 0.09 microm2 x 10(6)). We conclude that in our experimental model, this dietary supplement enhanced the antithrombotic capacity of the endothelium, reduced subendothelial thrombogenicity, and diminished the extent of vascular wall lesions caused by the hyperlipemic diet.

Antiplatelet effect of the anaesthetic drug propofol: influence of red blood cells and leucocytes.

1. The present study was designed to investigate the mechanism of the antiplatelet action of the anaesthetic propofol in vitro. 2. Human whole blood was incubated with different concentrations of propofol and its solvent Intralipid(R). We determined, platelet aggregometry in whole blood, platelet-enriched plasma (PRP), PRP plus red blood cells (RBC), and PRP plus leucocytes (LC); platelet production of thromboxane B2 (TxB2), ATP release by platelet dense granules, adenosine uptake by RBC, intraplatelet levels of cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP), and LC production of nitric oxide (NO). 3. Propofol-induced inhibition of platelet aggregation was greater in whole blood (IC50 80 - 136 microM) than in PRP (IC50>600 microM), except when aggregation was induced by arachidonic acid, in which case the antiaggregatory effect of the anaesthetic was similar in both media (IC50 72 - 85 microM). Inhibition of platelet aggregation correlated significantly with inhibition of TxB2 synthesis (r2=0.83). Propofol also inhibited granular ATP release; this effect was greatest in whole blood. 4. The presence of RBC or LC increased the antiaggregatory effect of propofol, mainly when collagen was used as aggregating agent. Intralipid inhibited the uptake of adenosine by RBC, however this effect probably does not contribute significantly to its antiaggregatory effect. 5. The anaesthetic potentiated the NO-cyclic GMP pathway, mainly by increasing the synthesis of NO by LC. Intralipid had no effect on the NO-cyclic GMP pathway in the LC-platelet interaction. 6. Propofol inhibited platelet aggregation in human whole blood, possibly through the sum of the effects of Intralipid on the platelet-RBC interaction and the increased synthesis of NO by LC in the platelet-LC interaction.

Effects of the selective inhibition of platelet thromboxane synthesis on the platelet-subendothelium interaction.

1. Drugs that inhibit TxA(2) synthesis are used to reduce platelet aggregation. The aim of this study was to compare the effects of a cyclo-oxygenase (COX) inhibitor (acetylsalicylic acid, ASA), a thromboxane synthetase (TxS) inhibitor (dazoxiben) and a dual TxS inhibitor and TxA(2) receptor blocker (DT-TX 30) on platelet aggregation and the platelet-subendothelium interaction in flow conditions. 2. The techniques used in this in vitro study were platelet aggregometry in whole blood, and measurement of platelet thromboxane B(2) and prostaglandin E(2) production and leucocyte production of 6-keto-PGF(1alpha). The platelet-subendothelium interaction was evaluated in rabbit aorta subendothelium preparations exposed to flowing blood at a shear stress of 800 s(-1). Morphometric methods were used to calculate the percentage of subendothelium occupied by platelets. 3. The 50% inhibitory concentration (IC(50)) of DT-TX 30 in whole blood was in the range of 10(-7) micro M (induced with collagen or arachidonic acid) to 10(-5) micro M (induced with thrombin) or 10(-4) (induced with ADP). IC(50) values under all experimental conditions were lower with DT-TX 30 than with ASA. For thromboxane B(2) the IC(50) were: ASA 0.84+/-0.05 micro M, dazoxiben 765+/-54 micro M, DT-TX 30 8.54+/-0.60 micro M. Prostaglandin E(2) was inhibited only by ASA (IC(50) 1.21+/-0.08 micro M). Leucocyte 6-keto-PGF(1alpha) was inhibited by ASA (IC(50) 6.58+/-0.76 micro M) and increased by dazoxiben and DT-TX 30. The greatest reduction in percentage subendothelial surface occupied by platelets after blood perfusion was seen after treatment with DT-TX 30 in the range of concentrations that inhibited collagen-induced platelet aggregation (control group: 31.20+/-3.8%, DT-TX 30 at 0.1 micro M: 10.71+/-0.55%, at 1.0 micro M: 6.53+/-0.44%, at 5.0 micro M; 1.48+/-0.07%). All three drugs reduced thrombus formation, although ASA (unlike dazoxiben or DT-TX 30) increased the percentage surface occupied by adhesions. 4. In conclusion, the effect of specific blockage of TxS together with blockage of membrane receptors for TxA(2) can surpass the effect of ASA in inhibiting the platelet-subendothelium interaction in flow conditions.

Differential effects of the pyrimido-pyrimidine derivatives, dipyridamole and mopidamol, on platelet and vascular cyclooxygenase activity.

The chronic administration of 10 mg/kg/day of dipyridamole to rats produced 33.7% inhibition of platelet aggregation induced with ADP and a 93% increase in 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in vascular samples, versus saline-treated rats. Mopidamol, 8.3 mg/kg/day, caused 50.6% inhibition of ADP-induced platelet aggregation, 37.6% inhibition of aggregation induced with arachidonic acid, a 47.6% decrease in serum levels of thromboxane B2 and a 23.7% increase in the vascular production of 6-keto-PGF1 alpha, versus saline-treated rats. Dipyridamole showed a higher in vitro anti-aggregating effect in whole blood (IC50 6.6 microM) than in platelet-rich plasma (PRP) (IC50 210 microM), when ADP was used as inducer, and had no effect in the presence of arachidonic acid. Mopidamol exerted a similar effect in whole blood (IC50 3.7-20 microM, depending on the inducer) and PRP (IC50 11-17.3 microM), and showed a dose-dependent inhibition of platelet aggregation and thromboxane B2 synthesis induced with arachidonic acid (IC50 16.8-22.3 microM). Mopidamol also inhibited enzymatically induced lipid peroxidation) (IC50 89 +/- 5.9 mumol/L) and had no effect on free radical-induced lipid peroxidation. The dose-dependent increase in 6-keto-PGF1 alpha in vascular samples after incubation with dipyridamole showed a negative linear correlation with inhibition of lipid peroxidation (r2 = 0.77). It is concluded that the phosphodiesterase inhibitors, dipyridamole and mopidamol, interfere in a different manner with platelet function. It seems that mopidamol may also exert a selective effect on platelet thromboxane synthesis.

Muscarinic receptor subtypes in human and rat colon smooth muscle.

Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8.01 +/- 1.0 microM for human samples and 6.07 +/- 1.1 microM for rat samples. These values are close enough to suggest that the first subtype of muscarinic receptor may be considered cardiac (M2) and the second subtype glandular (M3). The relative densities of the receptor subtypes are significantly different for both species. Human colon samples show the major densities of subtype M2, 22.62 +/- 1.11 fmol/mg protein, this represents 75.66 +/- 3.73% of the total receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of S-adenosyl-L-methionine on platelet thromboxane and vascular prostacyclin.

We therefore designed the present study to evaluate the effect of S-adenosyl-L-methionine (SAMe) on the synthesis of platelet thromboxane and vascular prostacyclin. The experimental materials were human blood and aortic rings from untreated Wistar rats; and platelets and aortic rings from Wistar rats treated for 7 days with SAMe at 5 or 10 mg/kg/day s.c. The administration of 10 mg/Kg/day of SAMe to rats significantly increased vascular production of 6-keto-PGF1alpha. In vitro vascular production of 6-keto-PGF1alpha increased in a concentration-dependent manner when SAMe was incubated in the range of 10(-7) to 10(-4) M. The greatest increase was 167 +/- 15%, obtained in samples incubated with 5 x 10(-5) M SAMe. In aortic rings, lipid peroxidase production was inhibited in a concentration-dependent manner in the SAMe range of 10(-7) to 10(-5) M. Maximum inhibition (75.3 +/- 6.2%) was obtained with SAMe at 1.5 x 10(-5) M. Vascular 6-keto-PGF1alpha production showed a significant inverse linear correlation with vascular lipid peroxide production (Y = -0.04x + 18.1, r = 0.7309, P < 0.0001).

Effect of propofol on oxidative stress in an in vitro model of anoxia-reoxygenation in the rat brain.

Propofol, an intravenous anaesthetic, is similar in chemical structure to the active nucleus of antioxidant substances such as alpha-tocopherol (vitamin E). The present study was designed to test whether propofol had antioxidant effects in an in vitro model of anoxia-reoxygenation in slices of rat brain. We used seven experimental groups: (1) control oxygenated tissue; (2) tissue subjected to anoxia for 20 min and reoxygenation for 3 h; and tissues processed as described and incubated with (3) Intralipid (commercial solvent for propofol), or propofol at a concentration of (4) 10 micromol/l, (5) 50 micromol/l, (6) 150 micromol/l or (7) 300 micromol/l. The production of lipid peroxides was quantified as thiobarbituric acid reactive substances (TBARS); tissular glutathione production and the activities of glutathione peroxidase (GSHpx), glutathione reductase (GSSGrd) and glutathione transferase (GSHtf) were also measured. Reoxygenation led to tissular oxidative stress, which was curtailed by propofol. The anaesthetic led to a 47% reduction in TBARS, a 165% increase in the reperfusion-inhibiting glutathione content, a 47% decrease in GSHpx activity, and an 87% increase in GSHtf activity. Intralipid had no effect on any of the parameters studied here. We conclude that propofol has a clear antioxidant effect in rat brain tissue subjected to anoxia-reoxygenation.

The pyrimido-pyrimidine derivative RA-642 protects from brain injury in a combined model of permanent focal ischemia and global ischemia reperfusion.

The effects of pyrimido-pyrimidine derivatives (dipyridamole, RA-642 and mopydamole) on lipid peroxidation (inhibition of the production of malondialdehyde, MDA) in different regions of the rat brain were studied. Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions. The antiperoxidative effect of RA-642 (in the microM range) was 10 times more potent than that of dipyridamole. Mopydamole did not exert any inhibitory effect on MDA production. In a model of ischemia reperfusion with bilateral occlusion of the common carotid arteries for 1 h and restoration of circulation for a period of 2 h, dipyridamole inhibited FeAs-induced MDA production but did not protect from postischemic brain tissue damage (measured by mitochondrial reduction of tetraphenyl tetrazolium). RA-642 inhibited FeAs-induced MDA production and showed 50-67% protection from tissue damage as compared with untreated animals, while mopydamole did not inhibit MDA production and showed 30-48% protection. No correlation was found between inhibition of lipid peroxidation and protection from brain tissue damage.

Effect of S-adenosyl-L-methionine on rat brain oxidative stress damage in a combined model of permanent focal ischemia and global ischemia-reperfusion.

We analyzed the effects of S-adenosyl-L-methionine (SAM) on tissue oxidative status in a combined model of permanent focal ischemia and global reperfusion in the rat brain. The production of thiobarbituric acid reactive substances (TBARS) was measured under basal conditions and after induction with ferrous salt as an indicator of brain lipid peroxidation. Total, oxidized and reduced glutathione were measured as indicators of the antioxidant defense capacity of brain tissue. Mitochondrial reduction of tetraphenyl tetrazolium (TPT) was quantified morphometrically. Results obtained in vitro showed that incubation with SAM reduced lipid peroxidation, with a maximum inhibition of 65.12+/-5.99% after incubation with 1 mmol/l; glutathione production was not significantly modified. In the brain ischemia-reperfusion model, TBARS production increased and glutathione content decreased, and mitochondrial reduction of TPT decreased significantly after ischemia-reperfusion in areas dependent on carotid circulation. The administration of 50 mg/kg SAM per day for 3 days led to the inhibition of brain lipid peroxidation and increased total glutathione production. These changes were accompanied by an increase in mitochondrial capacity to reduce TPT. We conclude that SAM reduces oxidative damage in the rat brain in an experimental model of ischemia-reperfusion.

Effects of triflusal on oxidative stress, prostaglandin production and nitric oxide pathway in a model of anoxia-reoxygenation in rat brain slices.

Acetylsalicylic acid (ASA) is the most widely used drug in the prevention of ischemic vascular accidents, mainly because of its antithrombotic effect. Recently, evidence of a neuroprotective effect has appeared. The aim of this study was to evaluate the neuroprotective effect of triflusal, a fluorinated derivative of ASA, in a model of anoxia-reoxygenation in rat brain slices. Rats (n=10 per group) were treated for 7 days with 1, 10 or 50 mg/kg/day p.o. of triflusal or ASA or solvent (control group), then brain slices were obtained and subjected to a period of anoxia followed by 180 min of reoxygenation. We measured oxidative stress parameters (lipid peroxidation, glutathione system), prostaglandins (PGE(2)), nitric oxide pathway activity (NO) (nitrites+nitrates, constitutive and inducible NO synthase activity) and cell death (lactate dehydrogenase (LDH) efflux). Triflusal decreased cell death in rat brain slices subjected to reoxygenation after anoxia by 21%, 42% and 47% with 1, 10 and 50 mg/kg/day, respectively. This effect was proportionately greater than the effect of ASA (0%, 25% and 24%). The antioxidant effects of triflusal on the biochemical mechanisms of cell damage studied here were also greater than the effects of ASA: lipid peroxidation was reduced by 29%, 35% and 36% with triflusal, and 0%, 19% and 29% with ASA. Inducible NO synthase activity was reduced by 25%, 27% and 30% with triflusal, and 0%, 25% and 24% with ASA. Triflusal can be considered an alternative to ASA as a neuroprotective agent, at least in the experimental model of anoxia-reoxygenation used in the present study.

Effect of dipyridamole and aspirin on the platelet-neutrophil interaction via the nitric oxide pathway.

This study was designed to determine the influence of the combination of aspirin and dipyridamole on the interaction in vitro between neutrophils and platelets through the nitric oxide (NO) pathway. Collagen-induced platelet aggregation (impedance method) was determined in platelet-rich plasma and in platelet-rich plasma+neutrophils, and cGMP (enzyme immunoanassay) and NO levels (electrochemical method, with a ISO-200 electrode) were also measured. The 50% inhibitory concentration (IC(50)) of aspirin was 139+/-11 microM in platelet-rich plasma, 367+/-21 microM in platelet-rich plasma+L-N(G)-nitro-arginine-methyl-ester (L-NAME), and 42+/-3 microM in platelet-rich plasma+L-arginine. The IC(50) for dipyridamole in platelet-rich plasma was not affected by L-NAME or L-arginine; the combination of aspirin with 20 microM dipyridamole (which has no effect per se) led to an IC(50) of 51+/-2 microM in platelet-rich plasma, 101+/-7 microM in platelet-rich plasma+L-NAME, and 13+/-2 microM in platelet-rich plasma+L-arginine. The cGMP levels showed the greatest increases in the aspirin plus dipyridamole group. Dipyridamole and aspirin increased the leukocyte production of NO: 50% increases were obtained at concentrations of 285+/-31 microM aspirin, 110+/-9 microM dipyridamole, and 16+/-2 microM aspirin+dipyridamole. Dipyridamole alone at a concentration of 20 microM had no significant effect on NO levels. We conclude that the combination of aspirin and dipyridamole significantly increases the antiplatelet effect of leukocytes, through an increase of NO, and that this effect is further evidence of the therapeutic benefits of this combination of drugs.

Characterization of muscarinic receptors in human submandibular salivary glands.

We studied the binding of the muscarinic antagonist [3H]N-methyl-scopolamine [( 3H]NMS) in order to characterize muscarinic receptors located in human submandibular salivary glands obtained from intrasurgical biopsy. [3H]NMS bound with a Kd value of 1.56 nM to a single class of muscarinic receptors (Bmax 37.3 fmol/mg protein) since pirenzepine exhibited a homogeneous binding profile.

Pharmacological characterization and distribution of muscarinic receptors in human placental syncytiotrophoblast brush-border and basal plasma membranes.

Based on the existence of choline acetyltransferase and acetylcholine in human placenta, we have investigated the presence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta. Radioligand binding assay, using [3H]N-methyl-scopolamine as tracer, showed the existence of acetylcholine muscarinic receptors in brush-border (Kd 0.28 +/- 0.04 nM; Bmax 9.4 +/- 1.6 fmol/mg protein) and basal plasma membranes (Kd 0.24 +/- 0.05 nM; Bmax 34.3 +/- 6.3 fmol/mg protein). In order to perform a pharmacological characterization of these receptors, competition binding experiments were carried out using the muscarinic receptor antagonists pirenzepine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-py rido(14) benzodiazepine (AF-DX 116), himbacine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), dicyclomine and hexahydro-sila-difenidol (HHSD). The results obtained showed that the muscarinic receptors in brush-border and basal plasma membranes belong to different subtypes. In brush-border membranes, the receptor found match in terms of affinity for the antagonists with the muscarinic M1 receptor subtype (Ki pirenzepine, 13.6 +/- 8.2 nM; Ki AF-DX 116, 1680 +/- 271 nM; Ki himbacine, 212 +/- 6.5 nM; Ki 4-DAMP. 1.5 +/- 0.4 nM; Ki dicyclomine, 5.1 +/- 0.8 nM; Ki HHSD, 34.3 +/- 7.3 nM), whereas the receptor in basal plasma membrane seems to be of the muscarinic M2 receptor subtype (Ki pirenzepine, 202 +/- 48 nM; Ki AF-DX 116, 124 +/- 60 nM; Ki himbacine, 20.6 +/- 4.8 nM; Ki 4-DAMP, 4.5 +/- 1.2 nM; Ki dicyclomine, 54.6 +/- 22 nM; Ki HHSD, 89.2 +/- 15.8 nM). The results obtained show the existence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta with a different distribution pattern in terms of number of receptors and distribution of different subtypes. The functional significance of these findings is as yet unknown, but these receptors probably mediate different functions as they belong to different subtypes and are coupled to different second messengers.