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Vasoactive substances produced by cultured rat brain endothelial cells

The vasoactive substances synthesized by primary cultures of rat brain endothelial cells were investigated and compared to those from two, immortalized cell lines, RBE4 and GP8. The vasoactivity of endothelium-derived substances was measured on isolated canine coronary artery. Vascular tone was significantly decreased by both primary and GP8, but not by RBE4 cells. Indomethacin pretreatment of primary and GP8 cells turned vasorelaxation into contraction while N(omega)-nitro-L-arginine pretreatment decreased the vasorelaxation induced by primary, but not by GP8 cells. Eicosanoid production was determined after incubation with [14C]arachidonic acid. The predominant vasoactive eicosanoid was prostaglandin E2 in both primary and GP8 cells. RBE4 cells synthetized mainly prostaglandin E2 and thromboxane B2 and significantly less prostaglandin E2 than did either primary or GP8 cells. The capacity of cerebral endothelium to regulate vascular tone by production of dilator and constrictor substances can be preserved under certain circumstances in immortalized cell lines.

Valproate Treatment and Platelet Function : The Role of Arachidonate Metabolites

Summary: Purpose: Valproate (VPA) is an extensively used drug in the therapy of epilepsies. One of the most frequently reported side effects of VPA is hemorrhagic diathesis. Some authors emphasized the decreased platelet count as the basis of VPA‐induced hemorrhagic diathesis, but some reports suggested that a significant proportion of patients with normal platelet count may still have an altered platelet function. The mechanism of the VPA‐induced platelet dysfunction has not yet been elucidated. A determining element of platelet functions is the arachidonate cascade. Present ex vivo experiments were designed to determine whether a relation exists between the incidence of hemostasis caused by VPA and the effect of this drug on the arachidonate cascade of platelets.

The effects of valproate on the arachidonic acid metabolism of rat brain microvessels and of platelets

Long-term administration of the antiepileptic drug valproate can induce hematologic, hepatic and endocrine abnormalities and morphologic alterations in the brain capillaries and glial cells. Valproate elicits bone marrow suppression, reducing the number of red blood cells and platelets, and causes platelet functional abnormalities. Various data suggest that more than one mechanism of valproate-associated toxicity may exist, but the pathomechanism of cell function alterations elicited by valproate has not yet been elucidated. The reported ex vivo experiments were designed to investigate the effects of valproate on the arachidonic acid cascade of rat brain capillaries and platelets. Valproate was administered (300 mg/kg body weight/day) in the drinking water to male Wistar rats for 2 weeks. Isolated platelets and brain microvessels were labelled with [14C]arachidonic acid and the released [14C]eicosanoids were separated by overpressure thin-layer chromatography and determined quantitatively by liquid scintillation counting. Valproate treatment reduced the synthesis of cyclooxygenase and lipoxygenase products in rat platelets. In brain microvessels valproate stimulated the synthesis of lipoxygenase metabolites and attenuated the cyclooxygenase pathway. Modifications of the arachidonate cascade in platelets and brain microvessels may contribute to the cell function alterations caused by valproate.

Platelet arachidonate cascade of migraineurs in the interictal phase

Morphological and functional alterations of platelets in migraineurs may be linked to the development of migraine. We examined the eicosanoid synthesis of platelets of untreated female migraineurs in a headache-free period and compared it to that of age- and blood group-matched healthy female volunteers. In the platelets of headache-free migraineurs significantly less amounts of anti-aggregatory prostaglandin D 2 and prostacyclin, as well as of 12-L-hydroxy–5,8,10-heptadecatrienoic acid (a potent endogenous inducer of endothelial prostacyclin production) were produced, while the synthesis of platelet aggregatory thromboxane did not differ when compared to that of healthy women. These results suggest that the platelet eicosanoids of migraineurs in the headache-free period might promote the development of cellular, vascular and neurological events inducing headache.Morphological and functional alterations of platelets in migraineurs may be linked to the development of migraine. We examined the eicosanoid synthesis of platelets of untreated female migraineurs in a headache-free period and compared it to that of age- and blood group-matched healthy female volunteers. In the platelets of headache-free migraineurs significantly less amounts of anti-aggregatory prostaglandin D2 and prostacyclin, as well as of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (a potent endogenous inducer of endothelial prostacyclin production) were produced, while the synthesis of platelet aggregatory thromboxane did not differ when compared to that of healthy women. These results suggest that the platelet eicosanoids of migraineurs in the headache-free period might promote the development of cellular, vascular and neurological events inducing headache.

Sex differences in prostaglandin metabolism.

Abstract Prostaglandin E2 (PGE2), PGF2alfa and PGD2 were synthetized from [1-14C]-arachidonic acid by rat kidney medulla microsomal fraction. The formation of each prostaglandin was significantly less in female animals than in males. The rate of inactivation of [3H]-PGF2alfa by kidney cortex cytosol was almost linear with the time of incubation during the first 30 min. The production of PGF2alfa metabolite (13,14-dihydro-15-keto PGF2alfa) was higher in male rats than in females.

Effects of Inflammatory Neuropeptides on the Arachidonate Cascade of Platelets

Background: During neurogenic inflammation, neuro–peptides (substance P, SP, calcitonin gene–related peptide, CGRP, and neurokinin A) are released from nerve endings, and these peptides initiate inflammatory reactions in the microcirculation. Platelets are one of the most important elements of the microcirculation. Methods: Our in vitro experiments were carried out to determine the effects of inflammatory neuropeptides (SP, and CGRP) on the arachidonate cascade of isolated rat platelets. Cells were labeled with 1–14C–arachidonic acid, then the eicosanoids were separated with overpressure thin–layer chromatography or high–performance liquid chromatography and were quantitatively determined with a liquid scintillation analyzer. Results: SP (10–9 and 10–8 mol/l) significantly increased the activity of the arachidonate cascade. The lipoxygenase pathway was significantly stimulated by SP (10–11, 10–9 and 10–8 mol/l), while the cyclooxygenase system was inhibited by 10–12 mol/l, and stimulated by 10–9 mol/l SP. The dose–response curve of TxA2 to SP exhibited a similar pattern to that detected for the cyclooxygenase pathway. Among the vasodilator cyclooxygenase metabolites, only the synthesis of PGE2 was significantly elevated by SP (10–9 mol/l ). CGRP either in low (10–12–10–11 mol/l) or in high concentrations (10–6 mol/l) activated the cyclooxygenase pathway, while it had no effect on the lipoxygenase pathway. CGRP (8–37), a specific CGRP1 receptor antagonist, inhibited the effects of CGRP. Conclusions: Our data suggest that the arachidonate metabolites of platelets may play a role in the process of neurogenic inflammation.

The effect of bradykinin and substance P on the arachidonate cascade of platelets

Several peptide receptors are expressed on the surface of platelets, including B1 and NK1, through which Bk and SP might influence platelet functions including their arachidonate cascade. The metabolites of the arachidonate cascade might play a regulatory role in the inter- and intracellular functions of platelets. Platelets were separated from fresh rat blood by differential centrifugation. Platelets (10(8) ml-1 in each sample) were preincubated with Bk or SP. The arachidonate cascade was investigated with [1-14C]arachidonic acid, as tracer substrate. The synthesised [14C]eicosanoids were isolated and quantitatively determined. Bradykinin elicited a biphasic dose-response curve in the formation of the vasoconstrictor and platelet aggregating thromboxane A2 (TxA2). Bk both inhibited (10(-8) mol/l), and elevated (10(-6) mol/l) the synthesis of TxA2 in the thrombocytes. The 12-HETE synthesis was inhibited by Bk (10(-8), 10(-7), 10(-5) mol/l); 12-HETE is an endogenous regulator of prostacyclin synthesis. The formation of 12-HETE in platelets was stimulated by SP (10(-11), 10(-9), 10(-8) mol/l). The synthesis of TxA2 in platelets was either attenuated (10(-12) mol/l), or stimulated (10(-9) mol/l) by SP. According to our observations Bk and SP might play a regulatory role in the activation or deactivation of platelets.

Dietary manipulation with high marine fish oil intake of fatty acid composition and arachidonic acid metabolism in rat cerebral microvessels

Male weanling Wistar rats were maintained on one of two semisynthetic diets, differing only in the type of oil used: (i) 10% by weight marine fish oil (MFO group) containing 20% eicosapentaenoic acid (EPA) and 17% docosahexaenoic acid (DHA), or (ii) 10% by weight sunflower oil (SFO group). The control group was kept on standard diet for 4 weeks. Blood-free microvessels were isolated from brain cortex by a rapid micromethod, and their fatty acid composition was determined by gas chromatography. It was found that the proportion of n-3 fatty acids (including EPA and DHA) increased significantly in the microvessels of the MFO group, accompanied by a decrease of the n-6 fatty acid series. The changes in fatty acid composition of endothelial cells were not significant in the SFO group in comparison to the control. The amounts of lipoxygenase and cyclooxygenase metabolites were determined. Dietary fish oil decreased the percentage of total products of arachidonate by 50%, while the SFO diet had no effect on it. The amount of lipoxygenase products in the MFO group decreased significantly from 16931±3131 dpm to 6399±357 dpm/300 mg wet weight of brain. Significantly less PGF-1α, PGF-2α and 12-hydroxyhepta-decatrienoic acid (HHT) were found in the capillaries of MFO treated animals, in comparison to the SFO group. The ratios of vasoconstrictor and vasodilator metabolites of arachidonate cascade were not modifed by the diets. Our results suggest that fish oil diet reduces the arachidonate cascade in cerebral microvessels. This effect may explain for the efficiency of n-3 fatty acids in vascular diseases.

Platelet eicosanoids and the effect of captopril in blood pressure regulation.

We investigated the eicosanoid synthesis of platelets of Wistar and of Okamoto spontaneously hypertensive rats (SHR), and the effect of captopril in vitro, using [14C]arachidonic acid as a tracer substrate and chromatographic determination. Lipoxygenase activity was elevated, while the formation of cyclooxygenase products was reduced in SHR platelets, compared to those of Wistar rats. This difference might play a role in the pathomechanism of hypertension in SHR. In SHR with lower blood pressure, captopril reduced thromboxane synthesis, while in SHR with higher blood pressure thromboxane synthesis was unchanged, but the synthesis of prostaglandin D2, a potent vasodilator, and of 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a stimulator of endothelial prostacyclin formation, was increased. We may conclude that, in spite of the missing angiotensin converting enzyme in platelets, a direct effect on platelet eicosanoid synthesis could contribute to the blood pressure decreasing effect of captopril.

Further studies on the anti-inflammatory effect of insulin

Experiments performed on rats showed that insulin, when applied i.v. or s.c., inhibited the foot edema induced by carrageenin, thermic effect of 45.7° C, compound 48/80 and 5-HT, but moderately increased the paw swelling evoked by kallikrein, a kinin-forming enzyme. The increased vascular permeability elicited by intradermal injection of histamine, 5-HT, bradykinin, PGE1, carrageenin and compound 48/80 was also suppressed. The anti-inflammatory effect was not significantly altered by propranolol and adrenalectomy on the thermal and carrageenin edema, it was variably inhibited on the skin test, and was completely abolished on the paw swelling induced by 5-HT and compound 48/80. Since insulin had little or no effect on the vascular response when given topically together with the vasoactive agents, its complex effect on the acute inflammation appears to be brought about via indirect mechanisms.