Injections of mild irritants intradermally (carrageenan, zymosan and dextran) and intracaveally (carrageenan) in a tissue cage model of inflammation were used in studies of the pharmacodynamics and pharmacokinetics of tolfenamic acid administered intramuscularly in calves. Inhibition of serum thromboxane (TX)B2 and inflammatory exudate prostaglandin (PG)E2 were used as indicators of the magnitude and time course of blockade of cyclo-oxygenase isoforms COX-1 and COX-2, respectively. Single doses of 2, 4 and 8 mgkg-1 tolfenamic acid partially inhibited irritant-induced rises in skin temperature (non-dose dependently) and skin oedema (dose-dependently). These doses also markedly inhibited serum TXB2 synthesis and the duration of inhibition was dose-related. A dose of 2 mgkg-1 tolfenamic acid also attenuated skin temperature rise over carrageenan-injected tissue cages, and markedly inhibited exudate PGE2 synthesis, even though drug penetration into both exudate and tissue cage transudate was limited. Tolfenamic acid pharmacokinetics were characterized by a relatively short tmax (0.94-2.04 h), a high estimated Vdarea (1.79-3.20 Lkg-1), an estimated t1/2 beta of 8.01-13.50 h and Cl beta of 0.142-0.175 Lkg-1h-1. The actions of tolfenamic acid in inhibiting PGE2 synthesis and in attenuating two of the cardinal signs of inflammation (heat and swelling) suggest that a dosage of 2 mgkg-1 administered intramuscularly should be effective clinically as an anti-inflammatory agent.

Aging is strictly associated with an increased incidence of cardiovascular events (CVEs) in the general population. Mechanisms underlying the risk of CVEs are still unclear. Platelet activation contributes to the onset of cardiovascular complications. The incidence of atrial fibrillation (AF) increases with age, and the natural history of AF is often complicated by CVEs. We prospectively investigated the relationship between age, urinary thromboxane (Tx) B2, which reflects platelet activation, and CVEs in 833 AF patients. Median TxB2 level was 120 [66-200] ng/mg of urinary creatinine. At multivariable linear regression analysis, age (B: 0.097, p=0.005) and previous MI/CHD (B: 0.069, p=0.047) were associated with log-TxB2 levels. When we divided our population into age classes (i.e. < 60, 60-69, 70-79, ≥ 80 years), we found a significant difference in TxB2 levels across classes (p=0.005), with a significant elevation at 74.6 years. During a mean follow-up of 40.9 months, 128 CVEs occurred; the rate of CVEs significantly increased with age classes (Log-rank test, p < 0.001). TxB2 levels were higher in patients with, compared to those without, CVEs in patients aged 70-79 (p < 0.001) and ≥ 80 (p = 0.020) years. In conclusion, TxB2 levels enhance by increasing age, suggesting that platelet activation contributes to CVEs in elderly patients with AF.

Arachidonic acid (AA) and adenosine diphosphate (ADP) are potent stimuli of platelet aggregation. Each agonist may act through separate platelet pathways. In order to evaluate inhibition of ADP and AA on platelet aggregation, we studied the effect of ticlopidine (TC) and aspirin (ASA) alone and in combination on plasma thromboxane levels, platelet deposition, and patency of small-diameter vascular grafts in a canine model. Thirty-four mongrel dogs were classified as thrombosis prone (TP) or thrombosis resistant (TR) on the basis of in vitro platelet aggregation to AA. Four groups were studied: group I, control; group II, TC (100 mg/kg/day); group III, ASA (3 mg/kg/day); and group IV, TC/ASA (same doses). PTFE grafts were implanted bilaterally in the carotid and femoral arteries Ticlopidine inhibited in vitro platelet aggregation to both ADP and AA but had no significant effect on plasma thromboxane (Tx) B2 production. Aspirin inhibited AA-induced platelet aggregation and significantly decreased TxB2 production. Aspirin inhibited AA-induced platelet aggregation and significantly decreased TxB2 levels in both TP and TR animals (p less than 0.01). Although TC and ASA significantly inhibited platelet deposition and improved 1-month patency in both TP and TR animals, maximal patency was achieved in the group in which TC and ASA were combined. We conclude that platelet ADP and AA pathways are important determinants of the thrombogenic potential in vascular graft performance in dogs and that combined inhibition of both pathways achieves maximal vascular graft patency.

To help clarify the mechanism of prostaglandin (PG) E2 and thromboxane (TX) B2 production by macrophages, the effects of three different stimulators (calcium ionophore A 23187, zymosan A and Actinomyces viscosus) on the release of arachidonic acid and the production of PGE2 and TXB2 from guinea pig macrophages were examined. In the present study, we showed that the produced levels of PGE2 and TXB2 were not dependent on the release of arachidonic acid from the phospholipids.

In experimental studies using mongrel dogs, 60 minutes after total thoracic esophagectomy the dog lung transiently released into the systemic circulation up to about 6000 micrograms/ml of thromboxane A2(TXA2) measured by radioimmunoassay as its metabolite thromboxane B2(TXB2). To determine whether lung TX release had effects on pulmonary function, we measured the changes in extravascular lung water (EVLW), lung resistance (RL) before, 10, 30, and 60 minutes after total thoracic esophagectomy in 14 anesthetized dogs. In seven untreated dogs, EVLW and RL increased and CL decreased approximately twofold at 60 minutes after the surgery, which corresponded well with a large transpulmonary plasma concentration gradient of TXB2. In remaining 7 dogs pretreated with intravenous OKY-046 which was TXA2 synthetase inhibitor, increase in EVLW and RL and decrease in CL were minimal and plasma concentration of TXB2 remained low value of a preoperative level. In clinical studies, 20 patients with esophageal carcinoma were evaluated. All of these patients underwent total thoracic esophagectomy with extended lymph node dissection of a similar extent. In 5 control patients, significant increase in EVLW and pulmonary vascular resistance were noted at 60 minutes after surgery. On the other hand, while the patients who had intravenous OKY-046 administration during operation at a dose of 1 microgram/kg/min or 5 micrograms/kg/min showed significant decrease in EVLW and pulmonary vascular resistance at 60 minutes after surgery. Based on these results, it is concluded that TXA2 appears to be one of the most important factors to cause the postoperative pulmonary complication after total thoracic esophagectomy for esophageal cancer.

Sulfinpyrazone (100 microM) and its thioether metabolite G25671 (50 microM) suppressed arachidonic acid-induced platelet aggregation, thromboxane (Tx) B2 formation and ATP release. Platelet activation by the endoperoxide analog U46619 also was prevented by sulfinpyrazone or G25671 (0.3-1 mM). Previous studies have shown that human platelets pre-exposed to arachidonic acid or to U46619 and then washed and resuspended failed to respond to a second challenge by both arachidonic acid and U46619; desensitization by arachidonic acid and U46619 occurred at a site sensitive to endoperoxides/Tx receptor antagonists; and the desensitizing effects of U46619 were direct, whereas those of arachidonic acid were mediated by a cyclooxygenase-dependent metabolite. We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevented desensitization. We also studied the threshold aggregating concentration of arachidonic acid and U46619 in healthy subjects before and after treatment with sulfinpyrazone and we found a good correlation between ex vivo and in vitro findings. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect. Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.

To study the modulatory role of renal eicosanoids on renal hemodynamics and electrolyte excretion, pressor doses of norepinephrine (NE) were infused in 10 control subjects (mean age, 26 y) and 13 patients (mean age, 25 y) with borderline hypertension. The highest NE dose used (150 ng/kg/min) produced comparable increases in mean blood pressure in control subjects (20 +/- 2 mmHg) and in patients (23 +/- 3 mmHg). NE induced a significant increase in renal vascular resistance (p less than 0.01, both groups), with a smaller decrease in glomerular filtration rate resulting in a concomitant increase in filtration fraction (p less than 0.01, both groups). The renal hemodynamic changes tended to be more pronounced in borderline hypertension. NE infusion led to similar decreases in electrolyte clearances in the two groups. Urinary prostaglandin (PG)E2, PGF2 alpha (p less than 0.01), and 6-keto-PGF1 alpha increased with NE infusion. Urinary thromboxane (TX)B2 increased slightly in control subjects and decreased in borderline hypertension (p less than 0.05). The 6-keto-PGF1 alpha/TXB2 ratio, an index of vasodilation, was significantly increased (p less than 0.05) in borderline hypertension. These results demonstrate that in both groups pressor infusion of NE induced significant modifications in renal hemodynamics and in urinary electrolyte and eicosanoid excretion. The vasodilatory component of the renal eicosanoid system appears hyperresponsive in borderline hypertension, which may represent an early antihypertensive defense mechanism.

1. Experiments were designed to determine the effects of low concentrations (5-500 nmol/L) of naftidrofuryl, a 5-hydroxytryptamine (5-HT) antagonist, on renal functions and prostanoid synthesis responses to noradrenaline (NA) and 5-HT. Isolated kidneys of 8 week old male spontaneously hypertensive rats were perfused at a constant flow rate in a single-pass system. 2. In baseline conditions, naftidrofuryl did not modify the renal vascular resistance and the glomerular filtration rate (GFR), although it elicited a significant but not dose-dependent increase in the venous excretion of 6-keto-prostaglandin (PG) F1 alpha and thromboxane (Tx)B2, the stable end-products of PGI2 and TxA2, respectively. 3. NA increased renal vascular resistance and GFR in a dose-dependent manner and enhanced the venous excretion of 6-keto-PGF1 alpha and TxB2. Naftidrofuryl significantly attenuated the effects of NA on renal vascular resistance, abolished those on GFR and enhanced, at the highest concentration (500 nmol/L) only, those on 6-keto-PGF1 alpha excretion. 4. 5-HT increased renal vascular resistance but not GFR. It did not change the sodium excretion and the release of 6-keto-PGF1 alpha and TxB2. Naftidrofuryl blunted the RVR response to 5-HT without change in the prostanoid release. The inhibitory action of naftidrofuryl was not modified by indomethacin which, by itself, prevented the vasoconstrictor response to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandins and thromboxanes (Txs) are produced by polymorphonuclears (PMNs) and macrophages (Mphis) in response to various stimuli. PMNs were separated from other human blood cells and Mphis were separated from rat peritoneal lavage. In this paper we show that human recombinant interleukin-1 (hrIL-1) can stimulate the release of thromboxane B2 (TxB2) by PMNs and Mphis. In addition, we have shown that aggregation of PMNs may occur when calcium ions (7 mM) and hrIL-1 (100 ng/ml) are added to the cell preparation, but not when Ca2+ alone, hrIL-1 alone, or first hrIL-1 then calcium are added to the cell preparation. The treatment of human platelets with hrIL-1 shows that after 15 min incubation TxB2 is released. In addition, we compared the aggregation of platelets caused by ADP with that caused by hrIL-1. Human recombinant IL-1 at a concentration of 100 ng/ml also causes little aggregation of platelets, in this case the aggregation is reversible. In conclusion, hrIL-1 beta stimulates TxB2 release in PMNs, Mphis and platelets and this effect increases with addition of Ca2+ ions. The mixture of hrIL-1 and Ca2+ causes little aggregation of PMNs. In monocyte suspensions, pretreated with human recombinant IL-1 receptor antagonist (IL-1ra) 500 ng/ml for 10 min and then treated with LPS or hrIL-1 beta 10 micrograms/ml, the release of TxB2 was partially inhibited. IL-1ra may play a significant role in the control of IL-1 and LPS induction in the release of TxB2.

The effect of 0.01 microM dipyridamole on prostanoid production was studied in atria from normal, acute diabetic and insulin-treated diabetic rats. Diabetes was induced by i.v. administration of 65 mg/kg of streptozotocin (STZ) and the rats were killed 5 days later. Atria were incubated during 60 min in Krebs solution. The prostanoids 6-keto-prostaglandin (PG) F1alpha (6-keto-PGF1alpha) and thromboxane (TX) B2, stable metabolites of prostacyclin and TXA2, respectively, as well as PGE2 were measured by reversed phase high-performance liquid chromatography-UV. In diabetic atria, 6-keto-PGF1alpha production was reduced by 50% whereas TXB2 release was increased two-fold compared to the controls, with a significant decrease in the 6-keto-PGF1alpha/TXB2 ratio. The preincubation with 0.01 microM dipyridamole for 30 min increased 6-keto-PGF1alpha production in control, diabetic and insulin-treated diabetic atria whereas TXB2 release was not modified. This effects provoked an significant increase in the 6-keto-PGF1alpha/TXB2 ratio. In conclusion, STZ diabetes reduces the 6-keto-PGF1alpha/TXB2 ratio impairing the functional status of the atria. Dipyridamole increased this ratio in atria from diabetic and insulin-treated diabetic rats, thus opposing the effects of STZ diabetes. This fact suggests the possibility of a participation of the drug in pathologies characterized by an imbalance in the production of vasodilator and vasoconstrictor prostanoids.

Microalbuminuria is a predictor of adverse outcome in hypertension.We evaluated in vivo platelet activation, by urinary 11-dehydrothromboxane (TX)B2 and plasma P-selectin, in hypertensives with or without microalbuminuria, and its possible association with oxidative stress, by urinary 8-iso-prostaglandin (PG)F2alpha and endothelial dysfunction. Sixty essential hypertensive patients, with (n=30) or without (n=30) microalbuminuria, and 30 controls were studied. Endothelial function was assessed by nitric oxide products, intercellular adhesion molecule (ICAM)-1, and asymmetric dimethylarginine (ADMA) levels. Urinary 11-dehydro-TXB2 excretion was higher in microalbuminuric (median 805 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (414 and 291 pg/mg, respectively; P<0.0001). Plasma P-selectinwas significantly higher in patients with microalbuminuria (median 136 ng/ml) as compared to those without microalbuminuria or controls (85 and 65 ng/ml; P<0.0001). Urinary 8-iso-PGF2alpha excretion was also enhanced in microalbuminuric (median 279 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (157 and 146 pg/mg, respectively; P<0.0001). A significant impairment in endothelial function was found in microalbuminuric patients, with decreased nitric oxide and increased ICAM-1 and ADMA levels. Multivariate regression analysis showed that urinary 8-iso-PGF2alpha excretion (beta=0.49; P<0.0001) and microalbuminuria (beta=0.36; P<0.001) were independently related to 11-dehydro-TXB2 in hypertensives. Vitamin E supplementation (900 mg daily for 1 month) in 10 hypertensives with microalbuminuria was associated with normalization in median 11-dehydro-TXB2 and 8-iso-PGF2alpha. We conclude that lipid peroxidation is a major determinant of persistent platelet activation in hypertensive patients with microalbuminuria.

1. We studied, in a random sample of 385 nonsmoking men born in 1968-1969 and 31 men born in 1913 or 1923, whether inheritance and environmental factors influenced platelet activity and vessel wall prostacyclin formation, as reflected non-invasively by the urinary excretion of the 2,3-dinor-metabolites of thromboxane A2 (2,3-dinor-thromboxane B2, Tx-M) and prostacyclin (2,3-dinor-6-keto-prostaglandin F1 alpha, PGI-M), respectively. 2. Fathers of young men with high platelet activity did not excrete more Tx-M than fathers of young men with low platelet activity. Men born in 1913 or 1923 displayed higher Tx-M (563 versus 128 pg/mg of creatinine, P less than 0.001) and PGI-M (163 versus 130 pg/mg of creatinine, P less than 0.01) excretion than those born in 1968-1969. Excretion of both Tx-M and PGI-M was correlated to the urinary output of noradrenaline and adrenaline. 3. Well-trained subjects did not differ in their excretion of Tx-M or PGI-M from those who did not exercise regularly. A recent acute infection was also unrelated to the excretion of Tx-M or PGI-M. PGI-M excretion was, however, significantly correlated to Tx-M excretion (r = 0.51, P less than 0.001). 4. This study provides the first non-invasive evidence that advancing age and sympathoadrenal tone are positively correlated to platelet activity in randomly sampled men, and that paternal inheritance, physical fitness and recent infection lack correlation to platelet activity.

Concentrations of thromboxane (Tx) B2 in plasma and its production by platelets were measured in 20 spinal and 10 epidural anesthesia patients scheduled for small operations in the lower extremities. The main metabolite of prostacyclin, 6-keto-PGF1 alpha and prostaglandin (PG) E2 in plasma were also determined. Plasma TxB2 and TxB2 production by platelets increased during both spinal and epidural anesthesia. Plasma TxB2 levels also remained elevated 1 h after anesthesia. The plasma concentrations of 6-keto-PGF1 alpha and PGE2 did not change during spinal or epidural anesthesia. In in vitro studies, only low concentrations of lidocaine (0.5-1.0 micrograms/ml) and bupivacaine (0.5-3.0 micrograms/ml) increased platelet TxB2 production. In platelet rich plasma, neither lidocaine nor bupivacaine in concentrations of 0.5-3.0 micrograms/ml caused constant changes in ADP-induced platelet aggregation, but they inhibited it in toxic concentrations (12 micrograms/ml). The results suggest that the increased TxB2 plasma levels and platelet TxB2 production during regional anesthesia are not caused by local anesthetics itself but by other factors, e.g. tissue trauma. In clinically found concentrations, local anesthetics do not cause any constant changes in platelet aggregation.

The platelet-activating factor (PAF) induced a marked increase of the thromboxane (TX) B2-formation in the incubation medium of isolated myocardium and tissue from other organs. The content of the 6-oxo-prostaglandin (PG)F1 alpha, the inactive metabolite of PGI2, remained uninfluenced or showed a small decrease. PAF, given in a concentration of 2.10(-9) mol/l or a single dose of 100 ng, significantly reduced the contraction force and the coronary flow of isolated guinea-pig hearts. This effect was connected with a high efflux of TXA2. The PAF-antagonist, WEB 2086, nearly abolished the cardiac effects of PAF, and iloprost or a pretreatment with indomethacin markedly reduced the PAF-influence on the heart. The TXA2-antagonist BM 13177 was ineffective. The results indicate a close interaction between the myocardial PAF-effect and the TXA2-formation of the heart tissue, but gave no suggestion for a mediation of the PAF-effect by TXA2. The PAF-antagonistic action of WEB 2086, iloprost and indomethacin could be of some interest in the therapy of cardiovasculatory diseases.

Continuously superfused rat anterior pituitary cells were used to study the effects of prostaglandins (PGs) and a thromboxane (TX) on the secretion of TSH. Indomethacin, a blocker of PG synthetase, inhibited the amount of TSH secreted in response to TRH. This reduction in TRH responsiveness was overcome by administration of PGE2 in combination with the TRH. Arachidonic acid, a prostanoid precursor, increased the amount of TSH released by TRH. Superfusion with TXB2 or imadazole, an inhibitor of TX synthetase, did not change TSH secretion. PGs A2, B2, D2, F1 alpha, F2 alpha, and endoperoxide analogs U-44069 and U-46619 had no effect on hormone release. PGE1 and E2 both increased TRH-stimulated TSH, but neither compound affected basal output; PGI2 was found to stimulate TSH release. Somatostatin inhibited TRH-induced TSH, but failed to block the effects of the PGs. These studies demonstrate that PGs, but no TXs, play a role in TSH secretion. PGE1 and PGE2 appear to modulate TRH responsiveness, while PGI2 directly stimulates hormone output.

Continuously superfused rat anterior pituitary cells were used to study the effects of exogenous prostaglandins (PGs) and thromboxanes (TXs) on the secretion of prolactin (PRL). No change in hormone release was observed upon superfusion with TXB2 (10(-5)M) or the TX synthesis inhibitor, imidazole (1.5 mM). PGs A2, B2, D2, E1, E2, F1 alpha, F2 alpha, and endoperoxide analogs, U-44069 and U-46619, also had no effect on PRL secretion (all at 10(-5)M). In contrast 10(-5)M PGI2 was repeatedly found to stimulate PRL release to a level at least 125% above control, while producing no apparent change in the amount of hormone secreted in response to TRH. Somatostatin (SRIF), at a dose of 10(-6)M, maximally inhibited TRH-induced PRL output, but failed to alter the PRL response to PGI2. These studies indicate that PGI2 may have a direct effect on the anterior pituitary to modify PRL secretion.

Plasma levels of 6-keto-prostaglandin F1 alpha (6kPGF1 alpha) and thromboxane (Tx) B2 have been assessed in sickle cell disease (SCD) with discrepant results. Inasmuch as direct measurement of plasma prostanoids is fraught with the problem of interfering substances, we assessed plasma 6kPGF1 alpha and TxB2 levels in patients with SCD by RIA after extraction of eicosanoids and separation by HPLC. We demonstrate that the 6kPGF1 alpha and TxB2 levels in children with SCD in steady state as well as in vaso-occlusive crisis (VOC) are significantly lower when compared with those from age-matched controls. The VOC plasma 6kPGF1 alpha and TxB2 levels were, however, significantly elevated when compared with those from children in steady state. Changes similar to those noted with unpaired plasma samples were also observed when paired steady state and VOC plasmas from the same patients were assessed. The ratio of TxB2 to 6kPGF1 alpha was, however, significantly elevated in patients with SCD in crisis when compared with eicosanoid ratios obtained during steady state. In an attempt to understand whether the abnormality in 6kPGF1 alpha was due to an impairment in endothelial cell prostacyclin-regenerating ability, we compared the ability of plasma from controls and children with SCD to activate arachidonic acid (AA) release and prostacyclin production by [14C]AA-prelabeled bovine aortic endothelial cells. Our results suggest that the decreased 6kPGF1 alpha levels in plasma from children with SCD was not due to an effect on substrate AA release but rather a modulatory effect of sickle plasma components on endothelial cell cyclooxygenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoxygenase as well as cyclooxygenase pathways of arachidonic acid (AA) metabolism are involved in antigen-induced bronchoconstriction in ovalbumin-sensitized guinea-pig lungs. Chemical lung challenge induced by aerosol containing formaldehyde (5 ppm up to 20 ppm) was effective in increasing thromboxane (TX) B2 release in a dose-dependent manner, without affecting leukotriene (LT) release. This suggests that xenobiotics acting by non-immunologic mechanisms on bronchial mucosa may stimulate the cyclooxygenase pathway of AA metabolism, which, in turn, might cause bronchial and/or lung parenchymal effects of pathophysiological relevance.

The intracellular Ca2+ mobilization in thrombin-stimulated platelets was greater in male rats than in female rats. Thromboxane (TX) B2 production in male platelets was greater than that in female platelets. Aspirin suppressed Ca2+ mobilization in rat platelets, but the inhibitory effect of aspirin was more efficient in males than that in females. Aspirin inhibited TXB2 production, and this inhibitory effect of aspirin was stronger in male platelets than in female platelets. Castration decreased Ca2+ mobilization and TXB2 production and weakened the effect of aspirin on them. It is suggested that the sex difference in the antiplatelet effect of aspirin results from the difference in the inhibition of Ca2+ mobilization via the inhibition of TXA2 production in thrombin-stimulated rat platelets.

In vivo microdialysis of the rabbit hippocampus was used to study the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on dialysate concentrations of thromboxane B2 (Tx B2)- and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha)-immunoreactive materials that are stable metabolites of biologically active thromboxane A2 and prostacyclin. All pharmacological substances were applied in the dialysis medium. The application of 1 mM NMDA for 20 min resulted in five- and eightfold increases in Tx B2 and 6-keto PGF1 alpha concentrations, respectively. An increase in NMDA concentration to 2.5 mM did not potentiate a peak eicosanoid release, but significantly prolonged this effect. Either 10 microM MK-801 or the extrusion of Ca2+ from the dialysis medium inhibited the release by about 50%. Quinacrine, a phospholipase A2 inhibitor (250 microM), decreased the NMDA-evoked eicosanoid release by 30%, whereas 10 microM indomethacin, a cyclo-oxygenase inhibitor, completely suppressed the release. One hundred micromolar furegrelate, an inhibitor of thromboxane synthase, reduced by 75% Tx B2 release with concomitant 100% increase in 6-keto PGF1 alpha formation. Thus, stimulation of NMDA receptors induces calcium-dependent formation of thromboxane A2 and prostacyclin in the hippocampus, which may have pathophysiological implications. The neuronal site of their formation seems probable, although a transcellular mechanism of their synthesis should be also considered.

Variable antiplatelet effects have been described for various antianginal drugs, including beta-blockers, verapamil, and nifedipine. To assess and characterize a possible similar effect of nitrates, platelet-rich plasma (PRP) from 22 healthy donors was incubated with scalar concentrations (1.25, 12.5, 125 micrograms/ml) of isosorbide dinitrate (ISDN) and with the vehicle alone for periods of 5 and 10 min. Platelet aggregation was successively induced by ADP (0.4-1.2 microM), adrenaline (0.8-8 microM), collagen (16.7 micrograms/ml), arachidonic acid (AA; 1 mM) and thrombin (0.5-2 U/ml). At the end of aggregation thromboxane (TX) B2 levels in PRP were assessed by RIA. A dose-dependent decrease in both platelet aggregation and TXB2 levels by all the inducers tested was demonstrated with both incubation times. Maximum inhibition of platelet aggregation was observed with ADP and adrenaline (72.0% and 55.6% respectively with 10-min incubation and the highest ISDN concentration. P less than 0.01 and P less than 0.05 respectively). A reduction in TXB2 levels in PRP was also observed, but reached statistical significance (P less than 0.01) only for AA-induced TXB2 generation. For an in vivo study ISDN was infused for periods of 30 min at 4 mg/h in 11 angina patients, and for periods of 20 min at 30 mg/h in an other eight, under ECG and arterial blood pressure monitoring. ADP- and adrenaline-induced aggregation and TXB2 production, and determination of circulating platelet aggregates (CPA) were performed in basal conditions and at 5, 15, 30 and 90 min from infusion start in the first group, at 5, 15, and 80 min in the second group.(ABSTRACT TRUNCATED AT 250 WORDS)

Isosorbide dinitrate inhibits platelet function in vivo at concentrations about 10 times lower than in vitro (10(-7)-10(-6) vs. 10(-6)-10(-5) M). We investigated two possible reasons for this difference. Isosorbide dinitrate and its in vivo longer-lived metabolites, isosorbide-2- and isosorbide-5-mononitrate were incubated for 5 min with human platelet-rich plasma or washed platelets; irreversible aggregation was induced with threshold doses of ADP, adrenaline, collagen, arachidonic acid and thrombin, and thromboxane (TX) B2 production was measured by radioimmunoassay. Moreover, the concentration of exogenous prostacyclin required to inhibit platelet aggregation by 50% (IC50) after preincubation with isosorbide dinitrate or vehicle was determined. At 10(-7) M, only isosorbide-2-mononitrate inhibited aggregation (-12%, p less than 0.05) and TX production (-36%, p less than 0.01) by ADP. At 10(-6) M isosorbide-2-mononitrate inhibited aggregation by adrenaline more than the dinitrate (-41% vs. -25%, p less than 0.05). In addition, at supra-threshold doses of all the aggregating agents, isosorbide dinitrate decreased IC50 of prostacyclin from 2.7 +/- 1.2 to 0.36 +/- 0.2 nM. Generation of a platelet-active metabolite and synergism with prostacyclin are new properties of isosorbide dinitrate that may account for antiplatelet effects in vivo.

We studied the influence of cardiovascular (CV) risk factors, previous CV events, and cotreatments with preventive medicines, on residual platelet thromboxane (TX)B2 production in 182 patients chronically treated with enteric coated (EC)-aspirin (100 mg/day). The response to aspirin was also verified by assessing arachidonic acid-induced platelet aggregation and urinary 11-dehydro-TXB2 levels. Residual serum TXB2 levels exceeded the upper limit value for an adequate aspirin response in 14% of individuals. This phenomenon was detected at 12 hours after dosing with aspirin. The coadministration of statins (mostly atorvastatin) was an independent predictor of residual serum TXB2 levels, and the percentage of patients with enhanced values was significantly lower in statin users vs. nonusers. We provide evidence in vitro that atorvastatin reduced residual TXB2 generation by increasing the extent of acetylation of platelet COX-1 by aspirin. In conclusion, the coadministration of statins may counter the mechanisms associated with reduced bioavailability of aspirin detected in some individuals with CV disease.

Inflammation plays a central role in the pathogenesis of several brain disorders and neuronal injury, and it develops as a consequence of glial cell activation. Activated microglial cells generate potentially damaging nitric oxide, oxygen free radicals, prostanoids, and pro-inflammatory cytokines. Naturally occurring polyphenols have recently received attention for their potential protective effect on neurodegenerative disorders characterized by microglial activation, due to their anti-inflammatory and antioxidant properties. In the present study, we investigated, using an in vitro model of primary microglia, the ability of 1-phenyl-6,7-dihydroxy-isochroman (encoded L 137), a natural polyphenolic compound, to inhibit microglia activation induced by an inflammatory insult. So, L137 effects (1-100 μM) on production of pro-inflammatory mediators in lipopolysaccharide (LPS)-activated microglial cells were evaluated. The expression of inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) as well as of the nuclear transcription Factor-kappa B (NF-κB) was also performed in cellular lysates by Immunoblot. L137 significantly reduced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 secretion, as well as nitric oxide (NO) and prostanoids [Thromboxane (TX)B2, prostaglandin (PG)E2] production in activated microglial cells. Western blot analyses showed an inhibitory effect of L137 on the iNOS and COX-2 expression, mediated by a modulation of redox-sensitive nuclear transcriptional factor (NF)-κB, known to control a wide array of genes involved in inflammation. In conclusion, this study demonstrate that L137 is able to inhibit the production of pro-inflammatory and neurotoxic mediators by LPS-activated microglial cells thus suggesting L137 as a potential lead compound for drug development for neurodegenerative disorders where microglia-mediated inflammatory responses play an important pathogenic role.