Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Heat transfer pathways between fetal lamb and ewe

Heat produced by the fetus exists to the mother by one of two principal routes: by fetal-maternal exchange in the placenta or through the fetal skin to the amniotic fluid and uterine wall. We measured heat conductances along each pathway to estimate the fraction of total heat exiting each route. Thermistors were placed in the fetal aorta, two different sites in the amniotic fluid, and in a maternal artery. Five days after surgery we injected a total of 280 ml of ice-cold saline into the two separate amniotic fluid sites during a 45-s interval and measured the temperature response for the next hour. After one or two such injections the fetus was killed to cut off umbilical blood flow, and the experiment was repeated to measure the heat fluxes in the absence of placental heat exchange. Experimentally obtained temperature curves were compared with the predictions of a mathematical model. Heat conductances of the skin and uterine wall, as well as the fetal heat production, were estimated in the model using least-squares parameter optimization. In 10 fetal lambs, weighing 3.73 +/- 0.40 (SE) kg, total fetal heat production averaged 3.75 +/- 0.33 W X kg-1. The heat conductance of the uterine wall, 6.6 +/- 0.8 W X degrees C-1, was lower than that of the fetal skin, 10.2 +/- 1.0, and of the placenta, 25.7 +/- 2.9 W X degrees C-1, temperature gradient. We estimated that 84.5% of total fetal heat production exists by fetal-maternal exchange in the placenta with the remaining 15.5% exiting through the fetal skin.     

The effect of prostaglandin E2 infusion in the fetal lamb on fetal plasma acth, prolactin and cortisol concentrations

PGE₂ (2 μg/min) has been infused for 1h into the fetal jugular vein of 8 chronically catheterized fetuses on 13 occasions from 112 to 138 days gestation. Infusion of ethanol vehicle alone was conducted in fetuses from 111 – 139 days gestation. PGE₂ administration produced a significant increase in fetal plasma cortisol after 30 min. No significant change was observed in fetal plasma prolactin concentration. Fetal plasma ACTH concentration was significantly elevated above resting concentration after 30 min. of PGE₂ infusion. Metabolic clearance rate of PGE₂ was 860 ml/min or 350 ml/kg/min. Intrauterine pressure was not changed during the infusion at any gestational age.     

The effect of prostaglandin E2 infusion in the fetal lamb on fetal plasma ACTH, prolactin and cortisol concentrations.

PGE2 (2 micrograms/min) has been infused for 1h into the fetal jugular vein of 8 chronically catheterized fetuses on 13 occasions from 112 to 138 days gestation. Infusion of ethanol vehicle alone was conducted in fetuses from 111-139 days gestation. PGE2 administration produced a significant increase in fetal plasma cortisol after 30 min. No significant change was observed in fetal plasma prolactin concentration. Fetal plasma ACTH concentration was significantly elevated above resting concentration after 30 min. of PGE2 infusion. Metabolic clearance rate of PGE2 was 860 ml/min or 350 ml/kg/min. Intrauterine pressure was not changes during the infusion at any gestational age.     

Antibiotics--cloning of biosynthetic pathways

Biosynthetic pathways leading to antibiotics have often been found to be clustered, and new organizational forms of multifunctional enzymes have been discovered. Such polyenzymes accomplish the synthesis of complex metabolites such as peptides or polyketides by a sequence of enzymatic reactions. So, reactions leading to the tripeptide precursor of beta-lactam antibiotics, ACV, or to the cycloundecapeptide cyclosporine have been fused into single polypeptide chain synthetases, respectively. In certain isofunctional sites restricted similarities have been detected.     

Prostaglandins in the circulation of the fetal lamb

Prostaglandin E and PGF have been measured in the plasma of chronically catheterized fetal lambs throughout the last 20–35 days (0.73 onwards) of gestation. The mean concentration of PGE was higher than that of PGF. There was a significant increase in the concentration of PGE but little change in the concentration of PGF in samples of fetal plasma taken within 24 h of parturition. In contrast, at this time in maternal utero-ovarian venous plasma, there was a large increase in PGF, but relatively little change in PGE. There was a significant decrease in the concentration of PGE and PGF in the plasma of lambs within 12 h after birth compared to the levels found in the same animals as fetuses a few hours previously. The physiological importance of these changes is discussed.     

Lipid-transfer proteins in biosynthetic pathways

Compartmentalization is a defining feature of eukaryotic cells that allows the spatial segregation of different functions, such as protein and lipid synthesis, and ensures their fidelity and efficiency. This imposes the need for an intense flux of metabolic intermediates between segregated enzymatic activities, as seen for the sequential transport of neosynthesized proteins through the segments of the secretory pathway during their post-translational modification. For lipid synthesis, the identification of proteins that transfer lipids between membranes has revealed an additional mechanism for this intercompartment exchange. The intense interest elicited by the lipid-transfer proteins over the last few years has led to the definition of their central role in key processes, such as lipid metabolism, membrane trafficking, and signaling.     

Distribution of the 14C-hydrated analog of phenazepam in the organs and tissues of mice

The absorption kinetics of hydrated phenazepam analog into the liver, spleen, brain, kidney, blood, lungs, heart, skeletal and fat tissues is studied at 0.25-24 hour intervals after its intraperitoneal (i/p) administration to mice. Drug concentration in the above mentioned organs was maximal 0.5-1 hour later. The decrease of the drug and its metabolite level in the organs under study is a biexponential process, consisting of "quick" (1-6 hours) and "slow" phases. The rate of absorption of hydrated phenazepam analog into the organs and tissues and its elimination is lower than that of phenanzepam.     

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues

In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-²H₅]-testosterone and [16,16,17-²H₃]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [²H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [²H₅]-Testosterone was converted into [²H₄]-oestradiol-17β, [²H₄]-oestrone and [²H₃]-6-dehydro-oestradiol-17 in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse.

On the basis of GC–MS characteristics (M⁺ m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M⁺). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [²H₄]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed.

In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [²H₃]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.     

Distribution of alpha 2M-globulin and Lpm-protein in the organs and tissues of the American mink

Distribution in the organs and tissues of two proteins of alpha-macroglobulin fraction, that differ in their antigenic structures, has been studied in the American mink. The both proteins (alpha 2M and Lpm) are present in hepatocytes, in cells of the follicular epithelium of the ovary, in the thymic bodies, in the alveolar macrophages of the lungs and in the splenic lymphoid nodes. Joint localization of alpha 2M and Lpm is revealed in the connective tissue of all the organs examined. The exception make the stomach and the uterine, where alpha 2M is revealed but not Lpm. The results obtained demonstrate a similar distribution of the two alpha-macroglobulins in the mink organism. They correspond to the literature data on morphofunctional topography of alpha 2M in the man. Certain individual differences in alpha 2M and Lpm localization can reflect peculiarities inherent in each of these alpha-macroglobulins of the American mink.     

Glucose and lactate oxidation rates in the fetal lamb

Both glucose and lactate are nutrients of the ovine fetus. Each may be used by the fetus as a fuel for oxidation or as a source of carbon for energy storage and net tissue accretion. The present report describes the oxidation rates of glucose and lactate in vivo for the fetal lamb over a relatively short time period. The fraction of fetal glucose or lactate oxidized was defined as the ratio of 14CO2 excretion across the umbilical circulation to the net entry of [14C]glucose or [14C]lactate into fetal tissues. The fraction of glucose oxidized over a 3-hr study averaged 61.2%, accounting for 2.55 mg X min-1 X kg-1 of glucose oxidized and for 28% of the simultaneous net oxygen uptake. The fraction of lactate oxidized averaged 71.5%, accounting for 4.12 mg X min-1 X kg-1 of lactate oxidized. Oxidation fractions and rates for both glucose and lactate increased with their concentrations in fetal blood suggesting sparing of other fuels for oxidation at higher glucose and lactate concentrations.     

Essential fatty acids in the fetal and newborn lamb

The concentrations of linoleic and linolenic acids and their metabolites in the liver, kidney, brain, erythrocytes and plasma of fetal lambs at various stages of gestation, and of newborn and 2-week-old suckled lambs was determined. Throughout gestation the fetal tissues, erythrocytes and plasma all contained low levels of linoleic and linolenic acids together with consistently high levels of their long-chain polyunsaturated metabolites. The triene: tetraene (eicosa-5,8,11-trienoic acid/arachidonic acid) ratio was always 0.4 or less except at birth when it reached 0.6 in liver and 0.9 in plasma. Milk intake significantly increased the linoleic and linolenic acid levels in the lamb by 2 weeks after birth. These results show that the developing fetal lamb should not be regarded as being deficient in essential fatty acids, as suggested by previous investigators. It is proposed that the total metabolites of linoleic and linolenic acids are the most appropriate measure of the essential fatty acid status of the fetal lamb.     

Effects of prostaglandin E1 and indomethacin on fetal and neonatal lamb mesenteric artery responses to norepinephrine.

The effects of prostaglandin E1 (PGE1) and indomethacin on isolated fetal and neonatal lamb mesenteric artery responses to norepinephrine were investigated. PGE1 (1.5 micrometer) significantly reduced vasoconstriction responses to 0.5 to 5 micrometer norepinephrine. Indomethacin (1 micrometer) markedly potentiated the constrictor effects of 0.5 to 10 micrometer norepinephrine. PGE1 prevented the potentiating effect of indomethacin. Neither PGE1 nor indomethacin altered basal muscle tension. These results suggest that endogenous PGs modify adrenergic responses in the isolated mesenteric arteries of preterm and newborn lambs.