Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.
Production and metabolism of platelet‐activating factor (PAF) in the fetal rat brain under normal and under ischemic stress conditions were examined. Endogenous PAF levels, determined by a bioassay using PAF‐stimulated platelet release of [3H]serotonin, averaged 2.32 ± 2.14 pg/mg in control brains and was reduced to 1.10 ± 1.06 pg/mg after 20 min of maternal‐fetal blood flow occlusion. [3H]PAF administered intracranially into the fetuses in utero was removed in a biphasic, time‐dependent manner: a rapid component with an estimated elimination rate constant of 0.067 min−1 and t1/2 of 10 min and a slower component with an elimination rate of 0.017 min−1 and t1/2 of 41 min. In fetal brains subjected to ischemia a delayed elimination of [3H]PAF was noticed in the slow component (t1/2 = 59 min), indicating a possible difference between the clearance of exogenous and endogenous PAF. The disappearance of [3H]PAF was accompanied by an increase in the radioactivity associated with lyso‐PAF that reached a plateau after 2.5 min, possibly indicating the degradation of the fast component. A steady increase in the alkyl‐acyl‐glycerophosphorylcholine radioactivity commenced after 5 min and continued up to 30 min. The endogenous production of PAF and the rapid degradation due to maternal‐fetal blood flow occlusion indicate an additional target for therapeutic intervention in the pathology of intrauterine ischemia. Addition of the calcium ionophore A23187 stimulated in vitro formation of PAF and lyso‐PAF from [3H]‐choline‐labeled fetal brain phospholipids, suggesting that intracellular calcium may play a major stimulatory role in PAF production. Degradation of polyphosphoinositides by a phospholipase C may constitute a major target for PAF generated either by decapitation or after blood flow occlusion, as evident from the protective effect of the in vivo administered BN52021 PAF antagonist.
Intact cerebral hemispheres from 20-d-old rat fetuses incubated at 37 degrees C in Dulbecco's Modified Eagle Medium (DMEM) synthesize and release a number of arachidonic acid derived metabolites, such as thromboxane B2 (TxB2), 6-keto prostaglandin F1 alpha (6k-PGF1 alpha), and prostaglandin E2 (PGE2) eicosanoids. Synthesis is time-dependent and is stimulated upon addition of the calcium ionophore A23187 (10 microM). Ionophore stimulation is prevented by EDTA/EGTA (5 mM each) ion chelators, dextran-70 (5%), and indomethacin (10 microM), a potent cyclooxygenase inhibitor. Ca2+ (2 mM) enhances ionophore-mediated formation of TxB2, 6K-PGF1 alpha, and PGE2 by 2.5-, 2.9-, and 4.2-fold, respectively; Mg2+ blocks ionophore stimulation. Freezing and thawing enhances release of eicosanoids to a level nearly the same as that obtained in the presence of A23187, indicating a common mode of action.
In a rat model of intrauterine growth retardation and fetal brain ischemia, the maternal-fetal circulation was obstructed for up to 40 min in 20-day gestational age rats by occluding (restriction) the uterine blood vessels supplying the placenta. After restriction, flow was returned (reperfusion) for designated times. A time-dependent depletion of cerebral pyruvate levels (from 0.2 ± 0.02 to 0.06 ± 0.01 pmol/g wet weight) accompanied by an elevation in lactate concentration (from 1.95 ± 0.03 to 7.00 ± 0.56 umol/g wet weight) was observed after 20 min restriction. During 20 min, reperfusion lactate levels continued to increase, then gradually decreased as the reperfusion continued for approximately 2 h. A drastic increase in the lactate/pyruvate ratio (from 10 to 117) suggested that the fetal brain was relying on anaerobic glycolysis to meet its energy demands. In addition, a time-dependent decrease in fetal brain phosphocreatine (PCr) content from 2.54 ± 0.26 to 1.52 ± 0.15 mM was observed after 20 min of maternal-fetal blood flow obstruction. ATP levels gradually decreased after 20 min restriction from 1.62 ± 0.13 to 0.59 ± 0.09 mM. After 30 min reperfusion ATP, PCr and pyruvate returned to their normal values. These metabolic changes observed are concordant with the ability of the ischemic fetal brain to sustain adequate levels of ATP for energy-requiring cellular processes. The capacity of glucose transporters to facilitate transport of glucose into brain tissue was assessed ex vivo, using [3H]2-deoxyglucose (2D-Glu). A statistically significant increase of 2D-Glu uptake, from 48.9 ± 2.3 to 69.5 ± 4.5 pmol/mg (42%) was noticed in fetal brains after 20 min restriction. Kinetic analysis revealed a 2.2-fold increase in the maximal uptake of [3H]-2D-Glu after 20 min blood flow restriction. Facilitation of specific glucose transporters triggered by oxygen depletion and glucose reduction, may contribute to the partial resistance of the fetal brain to ischemia and account for the moderate decrease in ATP energy levels.
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