scholarly journals HISTOCHEMICAL DEMONSTRATION OF PHOSPHOLIPASE B (LYSOLECITHINASE) ACTIVITY IN RAT TISSUES

1966 ◽  
Vol 14 (12) ◽  
pp. 907-914 ◽  
Author(s):  
ATHOS OTTOLENGHI ◽  
JOHN P. PICKETT ◽  
WILLIAM B. GREENE
Keyword(s):  
Fatty Acid ◽  
Incubation Medium ◽  
Copper Ions ◽  
Isotonic Saline ◽  
Histochemical Demonstration ◽  
Interstitial Tissue ◽  
Rat Tissues ◽  
Positive Staining ◽  
Phospholipase B

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 µ frozen sections, fixed in cold calcium-formol and incubated at 37°C in Tris buffered medium at pH 6.6 containing 2.2 x 10–3 M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a blackbrown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60°C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent. diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.

BREAKDOWN OF [3H]OXYTOCIN BY RAT TISSUES IN VITRO

1973 ◽  
Vol 59 (2) ◽  
pp. 305-312
Author(s):  
SANDRA M. EGAN ◽  
A. LIVINGSTON
Keyword(s):  
Skeletal Muscle ◽  
Mammary Gland ◽  
Incubation Medium ◽  
Rat Tissues ◽  
Tissue Uptake ◽  
Tissue Extracts ◽  
Rat Mammary Gland

SUMMARY Tissue uptake of oxytocin by rat mammary gland, uterus, heart and skeletal muscle was demonstrated by incubation of these tissues with 3H-labelled oxytocin. Chromatography of tissue extracts showed that some breakdown of oxytocin had occurred after only 30 s. However, in all cases there was no breakdown of oxytocin in the corresponding incubation medium. This indicated that the breakdown of oxytocin occurred within the tissues.

An additional role for insulin in the control of fatty acid synthesis independent of glucose transport

1970 ◽  
Vol 48 (11) ◽  
pp. 1228-1233 ◽  
Author(s):  
M. L. Halperin
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Glucose Transport ◽  
Fatty Acid Synthesis ◽  
Incubation Medium ◽  
Fold Increase ◽  
Acid Synthesis ◽  
Epididymal Adipose Tissue ◽  
Exogenous Substrate

Glucose conversion into pyruvate and fatty acids was studied in epididymal adipose tissue incubated in vitro from normal, 36-h-fasted and fasted–refed rats.Insulin at optimal concentrations caused a 30-fold increase in the rate of glucose incorporation into fatty acids and an increased lactate/pyruvate output rate. Pyruvate, lactate, and N,N,N′,N′,-tetramethyl-p-phenylenediamine (TMPD) addition to this incubation medium resulted in a further 20–75% increase in the rate of fatty acid synthesis as well as a further increase in the pyruvate concentration of the incubation medium. These results suggested that it was the decreased pyruvate concentration secondary to the elevated NADH/NAD+ of the cytoplasm which limited further glucose conversion to fatty acid.With glucose as substrate, TMPD caused the medium pyruvate concentration to be at least as high or higher than that seen with insulin in both nutritional states. However, fatty acid synthesis rates were eightfold greater with insulin. Insulin in the absence of glucose caused a twofold increase in the fatty acid synthesis from pyruvate at a medium concentration of 250 μM in normal and 25 mM in the 36-h-fasted rat. Therefore, insulin augments the rate of fatty acid synthesis both by increasing the supply of substrate (pyruvate) and also by directly increasing pyruvate incorporation into fatty acid by a mechanism distinct from the known stimulation of glucose transport.In fat pads from fasted–refed rats incubated in the absence of exogenous substrate, the rate of fatty acid synthesis was doubled by insulin. This occurred when the rate of pyruvate output was half that in the control condition. This also suggests that insulin stimulated pyruvate conversion to fatty acid in the absence of the known augmentation of glucose transport by insulin.

EVIDENCE TO SUGGEST THAT RAT GROWTH HORMONE IS MODIFIED WHEN SECRETED BY THE PITUITARY GLAND

1979 ◽  
Vol 80 (1) ◽  
pp. 69-81 ◽  
Author(s):  
M. A. VODIAN ◽  
C. S. NICOLL
Keyword(s):  
Incubation Medium ◽  
Young Male ◽  
Inhibitory Effect ◽  
Male Rats ◽  
Dynamic State ◽  
Rat Tissues ◽  
Gh Secretion ◽  
Rat Growth ◽  
Highly Correlated

The relationships were examined between the concentrations of GH determined by bioassay (rat tibia test) and radioimmunoassay in homogenates of rat adenohypophysial tissue and in the incubation medium in which the tissue had been maintained. Adenohypophyses from young male rats were incubated in Medium 199 with or without a putative synthetic GH-releasing factor (GRF) to achieve a dynamic state of GH secretion, but the GRF did not consistently stimulate the release of GH as measured by either bioassay or radioimmunoassay. Both assays gave closely similar values for the concentration of GH in homogenates of the adenohypophyses of undisturbed rats. When the two assays were used to measure the concentrations of GH in incubated pituitary tissue, the values showed no correlation, although the concentrations of GH in the incubation medium were again highly correlated. However, the bioassay: radioimmunoassay ratio for the secreted hormone [2·14 ± 0·09 (s.e.m.)] was significantly higher than that for the intrapituitary form from the unincubated gland (0·93 ± 0·06). By radioimmunoassay, secreted rat GH was found to be less stable than purified rat GH when the two forms were incubated in vitro with slices of various rat tissues. Similarly, the rate of clearance of secreted rat GH from the plasma of hypophysectomized rats, as determined by radioimmunoassay, was much faster than that of the purified GH. The biological activity of purified bovine GH was blocked by aprotinin, an inhibitor of proteolysis, but the drug had no inhibitory effect on the bioactivity of rat GH secreted into the incubation medium. Overall, these results indicate that secreted rat GH has properties intermediate between those of the intrapituitary form and the form found in the circulation.

Impairment of Lipid Metabolism Due to Deficiency of Pyridoxal-5-phosphate and/or Activated Immune system: its Interpretation

Pteridines ◽  
2000 ◽  
Vol 11 (4) ◽  
pp. 107-120 ◽  
Author(s):  
Vera Rudzite ◽  
Edite Jurika ◽  
Matthias Jäger ◽  
Dietmar Fuchs
Keyword(s):  
Cell Cycle ◽  
Fatty Acid ◽  
Immune System ◽  
Membrane Fluidity ◽  
Incubation Medium ◽  
Phospholipid Biosynthesis ◽  
P Deficiency ◽  
Acid Incorporation ◽  
Fatty Acid Incorporation

Abstract Impairment of lipid metabolism due to excess metabolite accumulation induced by pyridoxal-5-phosphate (P-5-P)-deficiency and/or stimulated immune system has been studied and interpreted. Decreased amounts of phospholipids as well as deviations in phospholipid classes and fatty acid composition of phospholipids have been demonstrated due to kynurenine accumulation in the blood of P-5-P-deficient cardiovascular patients and white rats as well as in cardiovascular patients with activated immune system identified by an increased neopterin concentration in the blood (dilated cardiomyopathy). The addition of P-5-P to the incubation medium for phospholipid biosynthesis in vitro did not change fatty acid incorporation into phospholipids, whereas it normalised fatty acid incorporation into phospholipids in liver homogenates received from P-5-P-deficient rats: The addition of kynurenine, neopterin and noradrenalin (accumulated m isolated heart tissue after addition of kynurenine and neopterin to incubation medium for isolated heart) to incubation medium for phospholipid biosynthesis in vitro induced an increase of saturated and a decrease of polyunsaturated fatty acid incorporation into phospholipids. These changes in fatty acid incorporation into phospholipids were followed by increased cholesterol concentrations in samples and an increased cholesterol/phospholipid ratio. Our results suggest that these changes in lipids are characteristic for decreased membrane fluidity, depressed cell cycle and lowered possibility of phospholipids to keep cholesterol in solution. P-5-P-deficiency is also accompanied with excess accumulation of homocysteine in the blood. The addition of L-homocysteine to the incubation medium for phospholipid biosynthesis in vitro was followed by inverse changes in fatty acid incorporation into phospholipids when compared with kynurenine, neopterin and noradrenalin. L-homocysteine induced a decrease of saturated and an increase of polyunsaturated fatty acid incorporation into phospholipids. The cholesterol concentration decreased in samples and the cholesterol/ phospholipid ratio decreased, too . These findings suggest that changes in lipids induced by L-homocysteine are characteristic for increased membrane fluidity and stimulated cell cycle. In this study, we have observed a similar effect to L-homocysteine effect when L-homocysteine, L-tryptophan and 5,6,7,8-tetrahydrobiopterin were added to the incubation medium for phospholipid biosynthesis in vitro. The comparison of our results with data from the literature allows to suggest that excess metabolite accumulation due to activated formation and inactivated catabolism of it plays a significant role in quantitative and qualitative changes of lipids, especially phospholipids, and therefore participates in the regulation of membrane fluidity, cell cycle of normal and malignant cells as well as in keeping cholesterol in the state of solution.

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

ALDOSTERONE STIMULATION IN VITRO

1965 ◽  
Vol 50 (2) ◽  
pp. 301-309 ◽  
Author(s):  
Jürg Müller
Keyword(s):  
Ammonium Chloride ◽  
Human Urine ◽  
Incubation Medium ◽  
The Other ◽  
Ammonium Ions ◽  
Response Curves ◽  
Urine Extract ◽  
Similar Dose ◽  
Aldosterone Production

ABSTRACT An extract of human urine, which was previously shown to stimulate aldosterone production by rat adrenal sections, was further purified. Evidence was obtained that its aldosterone-stimulating effect was due to the presence of ammonium ions. Addition of ammonium chloride and of urine extract to the incubation medium caused identical increases in aldosterone production in vitro. In addition to ammonium ions, rubidium and caesium ions also stimulated aldosterone production up to 250% that of control values without a significant effect on corticosterone production. Similar dose-response curves were obtained when increasing concentrations of potassium, ammonium, rubidium and caesium ions were tested. Aldosterone production was maximal at concentrations of 7 mval/1 and was significantly lower at higher concentrations. When ammonium chloride and ACTH were simultaneously added to the incubation medium, the production of aldosterone and of corticosterone was lower than with ACTH alone. On the other hand, the stimulating activity on aldosterone and corticosterone production by »TPN« (NADP) and glucose-6-phosphate was enhanced by the simultaneous addition of ammonium chloride.

IN VITRO UPTAKE OF TRITIATED OESTRADIOL BY THE ANTERIOR HYPOTHALAMUS AND HYPOPHYSIS OF THE RAT

1970 ◽  
Vol 64 (4) ◽  
pp. 687-695 ◽  
Author(s):  
Junzo Kato
Keyword(s):  
Incubation Medium ◽  
Posterior Hypothalamus ◽  
Anterior Hypothalamus ◽  
Ovariectomized Rats ◽  
Free Medium ◽  
Cortex Cerebri ◽  
Anterior Hypophysis ◽  
In Vitro Uptake

ABSTRACT The anterior, middle, and posterior hypothalamus, the cortex cerebri, the anterior hypophysis as well as the diaphragm of adult ovariectomized rats were incubated in vitro with tritiated 17β-oestradiol. The uptake of tritiated oestradiol was differentially distributed intracerebrally with higher accumulation in the anterior hypothalamus and the hypophysis. Lowering the temperature of the incubation medium caused a reduction in the uptake of radioactivity by the anterior hypothalamus as compared to that found in other brain tissues. Tritiated oestradiol taken up in vitro by the anterior hypothalamus and the hypophysis tended to be retained after further incubation in a steroid-free medium. The addition of non-radioactive 17β-oestradiol to the medium inhibited the uptake of tritiated oestradiol by these tissues. Moreover, pretreatment with non-radioactive 17β-oestradiol in vivo prevented the preferential accumulation of tritiated oestradiol in vitro in the anterior hypothalamus and the hypophysis. These results indicate that oestradiol is preferentially taken up in vitro by the anterior hypothalamus and the hypophysis of the rat.

ÉTUDE DE L'ACTIVITÉ BIOLOGIQUE IN VITRO DES HORMONES GONADOTROPES

1960 ◽  
Vol XXXIII (III) ◽  
pp. 444-450 ◽  
Author(s):  
Maria de la Luz Suarez Soto ◽  
Jean Legault Démare
Keyword(s):  
Paper Chromatography ◽  
Incubation Medium ◽  
Ultraviolet Absorption ◽  
Rat Ovary

ABSTRACT Serum gonadotrophin (PMS) when added to the incubation medium of rat ovary slices increases the amount of Δ4-3-ketosteroids produced. This enhancement is proportional to the logarithm of dose. The ketosteroids were determined by their ultraviolet absorption; paper chromatography has shown that only androst-4-en-3,17-dione is present.

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