Selective incorporation of 14C-arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to 14C-prostaglandins

A method is described for the efficient incorporation of radioactive arachidonic acid into the lipids of rabbit hearts and kidneys. Infusion of ¹⁴C-arachidonate through perfused tissues resulted in the quantitative removel of label from the media. Analysis of the lipids from tissues labeled by this procedure revealed that the majority of the ¹⁴C-arachidonate was incorporated into phospholipids. Essentially all of the radioactivity in phosphatidylcholine was found in the 2-position. Subsequent to the ¹⁴C-arachidonate infusion, stimulation of prostaglandin biosynthesis (e.g. by bradykinin) resulted in the release of radioactive prostaglandins. This suggests that the ¹⁴C-arachidonate is incorporated in a manner such that it is available for homone-stimulated prostaglandin biosynthesis. The method described allows both qualitative and quantitative analysis of arachidonate metabolism in intact tissues and offers significant advantages over other presently used methods.     

Leukotriene C4 action and metabolism in the isolated perfused bullfrog heart

The effects of leukotrienes (LTs) have been widely studied in the isolated perfused mammalian heart; however, little is known about the effect or metabolism of LTs in the isolated bullfrog heart. Isolated perfused bullfrog hearts were administered randomized doses of LTC4, LTD4, or LTE4. The cardiac parameters of heart rate, developed tension, and its first derivative (dT/dt) were recorded. LTC4 was the most potent of the leukotrienes tested in eliciting positive inotropic effects. LTD4 and LTE4 were equally effective but about one order of magnitude less potent than LTC4. None of the LTs showed any chronotropic effects in this preparation. A series of [3H]LTC4 metabolism experiments were carried out using whole perfused hearts and minced bullfrog heart tissue. Isolated perfused bullfrog hearts administered [3H]LTC4 converted significant amounts to [3H]LTD4, and to a lesser degree, [3H]LTE4, during the 6-min course of collection. Both minced atrial and ventricular tissue converted [3H]LTC4 to radioactive metabolites that co-migrated with authentic LTD4 and LTE4 standards. In both tissues, the major product was [3H]LTD4, with smaller amounts of [3H]LTE4 produced. The atrium converted significantly more [3H]LTC4 to its metabolites than did the ventricle. The metabolism of [3H]LTC4 to [3H]LTD4 by both tissues was virtually abolished in the presence of serine borate. Cysteine had no effect on [3H]LTE4 production. The data in this study demonstrate that leukotrienes have the opposite inotropic effect on the heart when compared with mammals. Also in contrast to mammals, frogs metabolize LTC4 to a less potent compound and may use the LTC4 to LTD4 conversion as a mechanism of LTC4 inactivation.     

Topography and ontogeny of the neurons expressing vasopressin,oxytocin, and somatostatin genes in the rat brain: An analysis using radioactive and biotinylated oligonucleotides

1. The use of radioactive and biotinylated oligonucleotide probes has been optimized to detect and analyze by in situ hybridization, neurons expressing neuropeptide genes (vasopressin, oxytocin, somatostatin). 2. In situ hybridization was performed on cryostat-cut sections obtained from tissues perfused with 1% formaldehyde. Radioactive probes were labeled by tailing with 35S-dATP and revealed with autoradiography. Biotinylated probes were obtained either by the incorporation of 11-biotin dUTP or by the addition of biotinylated nucleotides to the oligonucleotide during its synthesis. Biotin was revealed with streptavidin alkaline phosphatase and the appropriate substrate. 3. In the adult rat brain, radioactive and biotinylated probes revealed peptidergic neurons. The biotinylated probes provided an optimal cellular and subcellular resolution with a sensitivity similar to that observed with radioactive probes. Staining was selectively restricted to the cytoplasm and to the proximal part of processes. 4. Biotinylated vasopressin probes with 10 biotins added demonstrated magnocellular neurons and parvocellular neurons in the suprachiasmatic nucleus and the bed nucleus stria terminalis. 5. Vasopressin gene expression was studied during ontogeny in the rat fetus and neonate. Vasopressin mRNA was first detectable at gestational day 16 in the supraoptic nucleus in neurons of neuroblastic appearance. An aspect similar to the one present in adult was found at gestational day 19 in magnocellular neurons and at day 3 postnatal in parvocellular neurons. 6. The results confirm that radioactive oligonucleotide probes are efficient tools to investigate neuropeptide gene expression by in situ hybridization and demonstrate that biotinylated oligonucleotides are very efficient and provide a much higher resolution than radioactive probes with a reasonable sensitivity.     

Removal of 5-hydroxytryptamine by rat lungs.

Isolated rat lungs perfused with Krebs solution removed free radioactive 5-Hydroxytryptamine (5 HT or serotonin) from the fluid perfusing them. However, when platelets labelled with 111In-oxine to tag them individually, and with 14C-labelled 5 HT were also perfused via the lungs, there was no significant removal of the labelled 5 HT from the platelets. Paper chromatography showed that 46.7% of the platelet bound 5 HT was metabolished to an unidentified material. Rat lungs remove free 5 HT from plasma but not when it is platelet bound.     

Oxidation and esterification of cis- and trans-isomers of octadecenoic and octadecadienoic acids in isolated rat liver

The metabolism of 9-octadecenoic and 9,12-octadecadienoic acids with different geometrical configurations was compared in isolated perfused rat liver. More ketone bodies were produced when the trans-isomers were infused. In contrast, only the cis-isomer augmented the triacylglycerol secretion almost entirely as very-low-density lipoprotein (VLDL). Although these responses were independent of the difference in the degree of unsaturation in both the cis- and trans-isomers, the trans-monoenic acid compared to the trans-dienic acid was incorporated more readily into perfusate and hepatic lipids. Quantitative information was obtained with radioactive tracer experiments. The hepatic uptakes of 9-[10-14C]octadecenoic acids were comparable in the cis- and trans-isomers. The trans-octadecenoic acid compared to the cis counterpart was oxidized more readily and incorporated more into liver phospholipid but less into perfusate and liver triacylglycerol. These reciprocal responses counterbalanced each other. The lower rates of triacylglycerol synthesis and secretion in the liver perfused with the trans-octadecenoic acid was confirmed using [2- 3H]glycerol as a tracer. The marked difference in the channelling of cis- and trans-fatty acids in the pathways of oxidation and esterification seems to modify the VLDL secretion in perfused rat liver. Present observations indicate a considerable difference in the fate of unsaturated fatty acids with different configurations. trans-Fatty acids are expected to be an efficient energy source in animal tissues and may not be hyperlipidemic.     

Gangliosides released by the perfused rat liver are associated to high density lipoprotein.

We examine here the delivery of gangliosides from the perfused rat liver into the perfusate. One hour after the administration of [3H]GM1 to recirculating perfused livers, almost 80% of the perfusate radioactive gangliosides were recovered associated to the HDL fraction. This fraction was relatively enriched in radioactive GD1a. The pattern of endogenous gangliosides from perfused livers, rat serum and perfusates were very different: GM3 was the main liver ganglioside, GM1 and GD1a were the most abundant in perfusates being GM3 almost absent; GM3, GM1 and GD1a were present in rat serum in similar proportions. Using a non-recirculating perfusion protocol, radioactive gangliosides were found in the HDL fraction since 15 minutes after the administration of [3H]GM1. These results suggest that rat liver supplies the perfusates with some gangliosides and that they are associated to HDL. These facts arise the possibility that the liver is one of the source of serum gangliosides.     

Effects of parenteral palatinose on glucose metabolism in normal and streptozotocin diabetic rats

The present experiment was carried out to investigate the metabolism of palatinose (6-O-alpha-D-glucopyranosyl-D-fructose) in the rat. The bolus injection of palatinose (0.5 g/kg) in the tail vein of normal and streptozotocin (STZ) diabetic rats caused significant increments in glucose and insulin concentrations. However, in severe STZ diabetic rats (greater than 300 mg/dl of fasting plasma glucose) no significant change in glucose and insulin concentrations was observed. In liver perfusion, the gradual decrease in glucose output from the normal and mild STZ diabetic rat livers perfused with 20 mM Krebs-Ringer-Tris buffer pH 7.4 was prevented by the addition of 5.5 mM palatinose in the perfusate and fructose was detected in the effluent during the palatinose infusion. The results indicate that palatinose is metabolized to glucose and fructose in both normal and diabetic rat tissues, and this causes the increase in blood glucose concentration. On the other hand, the direct stimulatory effect of insulin release from pancreatic B-cell was not observed when the palatinose was infused into the isolated perfused rat pancreas. The study suggest that palatinose administered parenterally is metabolized by tissues and expected to be used as a source of fluid and energy supply.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

Sites of lipase activation and prostaglandin synthesis in isolated, perfused rabbit hearts and hydronephrotic kidneys

The tissue lipids of isolated, perfused rabbit hearts and hydronephrotic kidneys were labelled with [¹⁴C]-arachidonic acid by two different techniques: direct infusion of [¹⁴C]-arachidonic acid in a protein free media into the perfused organ (method A), and recirculation of [¹⁴C]-arachidonic acid in a solution containing albumin (method B). Autoradiography of the labelled organs demonstrated that method A resulted in selective labelling of arteries and arterioles in both perfused organs as well as glomeruli in the kidney. Labelling with method B resulted in a non-specific radioisotope incorporation in both the vasculature and myocardial cells in the heart; and of the vasculature and renal tubules in the perfused kidneys. Analysis of the tissue lipids shows similar patterns of incorporation of radioactivity between methods A and B.Peptide hormone stimulation (bradykinin) and non-specific noxious stimulation (with transient ischemia) were employed to elicit lipase activation (i.e., release of [¹⁴C]-arachidonate) and prostaglandin (PG) synthesis. It was found that in both hearts and hydronephrotic kidneys, the radioactive PG release in response to bradykinin and ischemia was much higher with method A (vascular labelling) than with method B (diffuse labelling) despite the appearance of comparable amounts of bioassayable PG release, thus indicating the sites of PG synthesis in these organs is predominantly localized in the vascular tissue. Furthermore, the radioactive arachidonic acid release in response to bradykinin stimulation in the hydronephrotic kidneys was 3 times higher with method A than with method B, suggesting the predominant sites of hormone specific lipase activation in the renal cortex is also in the vasculature. However, the radioactive arachidonic acid release in response to ischemia was much higher with method B than with method A in both hearts and hydronephrotic kidneys, indicating the sites of non-specific lipase activation in these organs are more diffusely distributed, and present also in the myocardial cells and renal tubules.     

Validation of a procedure for exogenous isotopic labeling of lipoprotein triglyceride with radioactive triolein.

A procedure has been developed for the exogenous isotopic labeling of triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) using high specific activity radioactive triglyceride in the presence of aqueous dimethyl sulfoxide. The labeled product lipoproteins showed unchanged chemical and physical properties. When the particles had also been labeled biologically by incorporation of unesterified fatty acids into the triglycerides of lipoproteins secreted by liver or intestine, both endogenous and exogenous labels were removed at the same rates in the isolated perfused heart and liver or in intact or functionally hepatectomized rats. These experiments additonally indicated that the triglyceride fatty acid composition of chylomicrons and very low density lipoproteins was unchanged during triglyceride depletion in the peripheral tissues. Using such labeled lipoproteins it has been shown that uptake of remnant lipoprotein cholesteryl ester and triglyceride by the liver is simultaneous. The labeling procedure described should prove suitable for kinetic studies of the disposition of the various lipoprotein non-polar ('core') lipids.     

Hepatic synthesis of carnitine from protein-bound trimethyl-lysine. Lysosomal digestion of methyl-lysine-labelled asialo-fetuin.

The biosynthesis of carnitine in the rat was studied by following the metabolism of two radioactive derivatives of asialo-fetuin. The first contained 14C-labelled methyl groups covalently bound to the 6-N-amino fraction of its lysine residues as 6-N-monomethyl- and dimethyl-lysine. By treating this protein with iodomethane, a second derivative was produced in which the radioactivity was preferentially incorporated as 6-N-[Me-14C]-trimethyl-lysine. These desialylated glycoproteins, like other asialo-proteins, were immediately cleared from the blood by rat liver. Within hepatocyte lysosomes, the 14C-labelled proteins were rapidly hydrolysed, producing free amino acids containing the various 6-N-[Me-14C]methylated lysine residues. The radioactive amino acids crossed the lysosomal membrane and were further metabolized in the cytosol. Carnitine was the major radioactive metabolite detected in extracts of the rat carcass and liver after intravenous injection of 6-N-[Me-14C]trimethyl-lysine-labelled asialo-fetuin. Within 3h, at least 34.6% of the trimethyl-lysine in the administered protein was converted into carnitine. Similarly, an isolated perfused rat liver converted 30% of the added peptide-bound trimethyl-lysine into carnitine within 90 min. On the other hand, in numerous attempts we failed to detect radioactive carnitine in both rat liver and carcass between 20 min and 22 h after injection of 6-N-[Me-14C]-monomethyl- and -dimethyl-lysine-labelled asialo-fetuin. These data provide evidence for a pathway of carnitine biosynthesis that involves trimethyl-lysine as a peptide-bound precursor as proposed by R.A. Cox & C.L. Hoppel [(1973) Biochem. J. 136, 1083-1090] and V. Tanphaichitr & H.P. Broquist [(1973) J. Biol. Chem. 248, 2176-2181]. The findings also show that rat liver can synthesize carnitine without the aid of other tissues, but cannot convert free partially methylated lysines into trimethyl-lysine.     

Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants

Summary The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H₂O₂), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOCI and H₂O₂ and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOCl. For example, in isolated hearts perfused for 60 min with 100 M HOCI, approximately 18010 of the methionine and 2801o of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOCI, showing no oxidation of proteins. HOCI was much more effective than H₂O₂ in oxidizing proteins, suggesting that HOCI may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC I, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants.     

T4-induced cardiac hypertrophy and the perfused and total microvasculature of the heart

The purpose of the present investigation was to determine the effects of thyroxine (T4), which induces myocardial hypertrophy, on the number per square millimetre and volume per cubic millimetre of both the total and perfused portions of the arteriolar and capillary beds of the heart. Studies were conducted in the subendocardial and subepicardial regions of the left ventricle of anesthetized open-chest rabbits. Fluorescein isothiocyanate-dextran (i.v.) or radioactive microspheres (intra-atrial) were injected to label the perfused microvessels or to determine coronary flow in three groups of rabbits: controls, and rabbits given 0.5 mg/kg T4 for 3 days and for 16 days. Fluorescent photography was used to identify the perfused microvessels. An alkaline phosphatase stain was employed to locate the total microvascular bed. There were 2369 +/- 638 (SD) capillaries/mm2 and 4 +/- 3 arterioles/mm2 in control hearts. These decreased significantly to 1380 +/- 199/mm2 and 1 +/- 1/mm2, respectively, after 16 days of T4. In controls, 60 +/- 5% of the capillaries and 59 +/- 21% of the arterioles were perfused. This increased significantly to 90 +/- 5 and 86 +/- 18%, respectively, by 16 days of T4 treatment. Similar changes, although smaller, were observed after 3 days of T4. Coronary blood flow increased to 1.7 times control after 3 days and 2.9 times after 16 days of T4. No significant subepicardial versus subendocardial differences were observed in any condition or measurement. Thus, the physiological response to the increased work and increase in anatomic minimum diffusion distance is to increase flow and the proportion of the capillary bed perfused to at least maintain physiological diffusion distances.     

Heterogeneity of bile pigments formed by the breakdown of haemoglobin in the isolated male-dog kidney.

Normothermic perfused isolated male-dog kidneys formed radioactive bile pigments by the breakdown of radioactive haemoglobin prepared from [2-14C]glycin. After column chromatographic separation and preparation of dipyrrolic azopigments, 86.3 +/- 2.2% of the bile pigments seemed to be conjugated bilirubin. Thin-layer chromatographic separation of the azopigments of ethyl anthranilate revealed a good correlation between photometric scanning, radiochromatographic scanning and the radioactivity of the azopigments scraped off the thin-layer glass plates and counted in a liquid scintillation counter. Although the same heterogenity of the azopigments was observed as in dog bile, the isolated male-dog kidney formed significantly less alpha2- and significantly more gamma-fractions.     

In vivo lipolysis in adipose tissue from two anatomical locations measured by microdialysis

In this study, the difference in lipolytic response in inguinal subcutaneous and epididymal adipose tissues of male Sprague-Dawley rats was assessed in vivo by microdialysis. Probes were perfused with Ringer solution in which increasing concentrations of isoproterenol (10(-7) - 10(-4) mol/L) were added. Glycerol release, expressed as extracellular glycerol concentration, was used as lipolytic index. The effect of isoproterenol on local blood flow was investigated using the ethanol technique. No differences were found in the interstitial glycerol concentration between both adipose tissues under basal conditions. When isoproterenol was perfused, a dose-response increase in glycerol production was induced in both tissues. Interstitial glycerol concentration from epididymal adipose tissue was higher than that of inguinal subcutaneous depot at all isoproterenol concentrations. No vasodilatory effect of isoproterenol was found. These results suggest that epididymal adipose tissue is more responsive in vivo to beta-adrenergic lipolysis stimulation than is subcutaneous fat pad from the inguinal region.     

Effect of Age on Vitamin E Concentrations in Various Regions of the Brain and a Few Selected Peripheral Tissues of the Rat, and on the Uptake of Radioactive Vitamin E by Various Regions of Rat Brain

Abstract: The concentrations of tocopherols in selected areas of the brains and a few peripheral tissues of 3-, 14-, and 30-month-old male Fischer 344 rats were determined by a high-performance liquid chromatographic method. Throughout the time period studied, α-tocopherol was the only tocopherol detected in the brain. Concentrations of α-tocopherol increased significantly with age in medulla and spinal cord whereas no such change was seen in other brain areas. Among the peripheral tisues, total tocopherol concentrations increased with age in the liver and adipose tissue while no significant changes were observed in the heart. The pattern of uptake of radioactive α-tocopherol from the serum by the various areas of the brain was similar for the 3-and 14-month-old animals even though the brains from the 14-month-old animals took up less of the radioactive compound. Measurable amounts of tocopherol esters were not present in the tissues of the 30-month-old animals.     

EXPERIMENTAL LATHYRISM : An Autoradiographic Study

In normal and lathyritic chick embryos bone collagen was synthesized primarily in the periosteum of the femurs, and was organized as radioactive spicules in these bones. Saline extraction of the lathyritic bones removed the radioactive spicules, although they eventually seemed to become non-extractable. Normal bone seemed to be unaffected by saline extraction. Marked variation in the degree of isotope incorporation was seen in collagenous and non-collagenous tissues. All the tissues of any one embryo, however, showed a similar degree of isotope incorporation. Tritiated β-aminopropionitrile was diffusely distributed throughout bone and was completely removed by saline extraction. This autoradiographic study supports the postulate that a portion of extractable lathyritic collagen is recently synthesized and is organized in fibrous structures in bone.     

Transfer of esterified cholesterol from serum lipoproteins to the liver.

The fate of cholesteryl esters of the serum lipoproteins was studied in intact rats and in isolated perfused rat livers. The lipoproteins of fasting rat serum were labeled in vitro with [3H]cholesteryl oleate. Following intravenous injection, it was found that the majority of the radioactive ester was rapidly taken up by the liver where hydrolysis of the ester bond occurred. At 5 min, 58% of the injected material was recovered in the liver, 85% of which was still in the ester form, while at 30 min only 22% of the liver radioactivity was in cholesteryl esters. There was very little difference in the rate at which radioactivity was taken up from the different lipoprotein classes. Similar phenomena were observed in the perfused liver, but it was found that although the radioactive esters were being taken up, there was no change in the concentrations of free or esterified cholesterol in the perfusing medium, indicating that the lipoprotein cholesteryl ester was gaining access to the liver through an exchange of molecules. After uptake, cell fractionation experiments showed that the plasma membranes had the greatest relative amounts of radioactivity, suggesting that this is the site of exchange. Small amounts of radioactivity were recovered in the bile, demonstrating that serum lipoproteins can serve as precursors of at least some of the bile steroids.     

Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

The generation of an off-the-shelf in vitro engineered living tissue graft will likely require cryopreservation. However, the efficient addition and removal of cryoprotective agents (CPA) to cells throughout the volume of a three-dimensional (3D) tissue graft remains a significant challenge. In this work, we assessed whether a perfusion bioreactor-based method could be used to improve the viability of cryopreserved mesenchymal stromal cell- (MSC) based tissue constructs as compared to using conventional diffusion-based methods. The bioreactor was first used to saturate 3D constructs with CPA under perfused flow. Following cryopreservation, the bioreactor was then also used for the efficient removal of the CPA from the 3D tissues. We demonstrate that addition and removal of CPA under perfused flow significantly increased the viability of MSC within cryopreserved 3D tissue constructs as compared to conventional diffusion-based methods.     

Bile acid metabolism in mammals. VIII. Biliary secretion of cholylarginine by the isolated perfused rat liver.

In studies of cholic acid metabolism using the isolated perfused rat liver system, an unknown conjugate of cholic acid was observed. This conjugate comprised 15-27% of the biliary bile acids in these experiments, was less polar than cholylglycine on thin-layer chromatography using butanol, acetic acid, and water, and had an apparent molecular weight greater than that of cholyltaurine on gas-liquid chromatography. Amino acid analysis of the hydrolyzed conjugate demonstrated the presence of arginine. Perfusion studies with radioactive arginine, and mass spectrometric analysis proved that the conjugate was cholylarginine. Secretion of this conjugate does not represent a deficiency of available glycine and taurine.