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G S Boyd

Publications and source records attributed to G S Boyd.

At least 37 records · Page 2Linked to original sources
PubMed1982

Properties of a solubilised and reconstituted preparation of acyl-CoA:cholesterol acyltransferase from rat liver.

Rat liver acyl-CoA:cholesterol acyltransferase activity was released from the microsomal fraction by treatment with Triton X-100. After fractionation with polyethylene glycol 6000, the solubilised preparation was reconstituted in liposomes of different lipid composition by an octyl glucoside dilution method. The activity of the reconstituted system was dependent on the amount of cholesterol used in the liposomes and could also be stimulated by transfer of cholesterol into the reconstituted system from other membranes. The results are consistent with the hypothesis that substrate supply and the fluidity of the membrane contribute in the regulation of the rate of cholesterol ester formation.

Acyltransferases↗
PubMed1982

Pregnenolone efflux from mitochondria of bovine adrenal cortex.

Pregnenolone efflux from bovine adrenal cortex mitochondria was measured with the aid of rapid separation of the organelles from the incubation by centrifugation through silicon oil. In the absence of bovine serum albumin or cytosolic supernatant (100 000 x g), an equilibrium distribution of newly synthesised pregnenolone was attained after 2 min at 30 degrees C; 20% of total pregnenolone was located outside the mitochondria. The presence of 13 mg albumin/ml or 13 mg supernatant protein/ml increased the exogenous proportion of pregnenolone to over 90% and 75% respectively without affecting the rate of cholesterol side-chain cleavage. The binding of pregnenolone by both albumin and supernatant was non-specific and non-saturable. It is suggested that in vivo the mitochondrial membranes do not present a significant barrier to pregnenolone efflux from the organelle.

Adrenal Cortex↗
PubMed1981

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver.

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.

Animals↗
PubMed1981

Portal blood concentrations of conjugated cholic and chenodeoxycholic acids. Relationships to bile salt synthesis in liver cells.

1. Rats were maintained in a strictly controlled environment of 12 h illumination and 12 h darkness. At regular intervals during the light/dark cycle the portal blood conjugated cholic acid and conjugated chenodeoxycholic acid concentrations were measured. The bile salt concentrations exhibited similar diurnal rhythms, the highest concentrations occurring in the middle of the dark phase. 2. The concentrations of conjugated cholic and chenodeoxycholic acids in the portal blood of rats fed a diet containing the bile salt sequestrant, cholestyramine, were significantly lower than those found in rats given a control diet. 3. During total biliary drainage the portal blood concentrations of conjugated cholic and chenodeoxycholic acids fell to a minimum 6--8 h after the start of the experiment, whereas bile salt synthesis in hepatocytes isolated from the rats was not increased until the least 13 h after the commencement of total biliary drainage. 4. These results suggest that the concentrations of bile salts in the portal blood do not affect directly the diurnal fluctuation in rates of bile salt synthesis, as the response of synthesis to a change in portal blood bile salt concentrations is too slow. 5. When the rats were given a small supplement of cholesterol in the diet to suppress hepatic cholesterologenesis prior to being subjected to total biliary drainage, the changes in the portal blood concentrations of conjugated cholic and chenodeoxycholic acids and the synthesis of the two bile salts by isolated hepatocytes were similar to those found in rats given the control diet. 6. The rise in bile salt production seen during biliary drainage may not be dependent exclusively on a preceding increase in cholesterol synthesis.

Animals↗
PubMed1981

Cholesterol 7 alpha-hydroxylase of rat liver. Studies on the solubilisation, resolution and reconstitution of the enzyme complex.

1. Cytochrome P-450 was prepared from the liver microsomes of cholestyramine-fed rats by solubilisation with the non-ionic detergent Nonidet P42 followed by chromatography on a DEAE-cellulose column and on hydroxyapatite. NADPH--cytochrome P-450 reductase was prepared by a technique of affinity column chromatography using 2',5'ADP Sepharose. 2. The activity of cholesterol 7 alpha-hydroxylase was measured in a reconstituted system of microsomal mixed-function oxidase containing cytochrome P-450 and NADPH--cytochrome P-450 reductase from rat liver plus cholesterol and NADPH. Endogenous cholesterol was largely depleted from the enzyme preparations by the treatment of the microsomes with cold n-butanol/acetone. 3. The reconstituted system of mixed-function oxidase catalysed a highly effective and specific 7 alpha-hydroxylation of cholesterol. The reconstituted system showed a higher activity of cholesterol 7 alpha-hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P-450, NADPH--cytochrome P-450 reductase and NADPH. 4. The apparent Km for cholesterol in the reconstituted system was 15 microM and the V was 1.4 nmol 7 alpha-hydroxycholesterol formed min-1 (nmol cytochrome P-450)-1. 5. The reconstituted system also catalysed the 7 alpha-hydroxylation of taurodeoxycholic acid, the 7 alpha-hydroxylation of 26-norcholesterol and to a limited degree the 12 alpha-hydroxylation of cholest-4-ene-3-one-7 alpha-ol. The ability of this reconstituted system to effect these two 7 alpha-hydroxylation reactions and the 12 alpha-hydroxylation reaction was significantly less than the ability of the system to 7 alpha-hydroxylate cholesterol.

Animals↗
PubMed1981

The effect of azastene, cyanoketone and trilostane upon respiration and cleavage of the cholesterol side chain in mitochondria from bovine adrenal cortex.

Mitochondria prepared from bovine adrenal cortex and incubated with ADP and phosphate respired at about 45% of the rat observed in the presence of an uncoupler of oxidative phosphorylation; there was, however, little inefficiency in the reactions involved in the phosphorylation of ADP. Three inhibitors of the 3 beta-hydroxysteroid dehydrogenase (azastene, cyanoketone and trilostane) were employed with a view to preventing pregnenolone metabolism and thus aiding the assay of cholesterol side-chain cleavage. Freshly made solutions of these inhibitors did not modify mitochondrial respiratory rates, at concentrations of 10 microM. In contrast, solutions maintained at 0-4 degrees C for one week subsequently inhibited the respiratory rate of uncoupled mitochondria. When fresh solutions of the inhibitors were used in the assays of cholesterol side-chain cleavage, 10 microM azastene did not significantly inhibit pregnenolone metabolism. Cyanoketone and trilostane were both significant inhibitors of pregnenolone metabolism, but 10 microM cyanoketone reduced the initial rate of cholesterol side-chain cleavage by 50% in the presence of 10 mM malate, although this inhibition did not occur in the presence of 10 mM DL-isocitrate. Thus trilostane may be the preferred inhibitor of the 3 beta-hydroxysteroid dehydrogenase during studies of cholesterol side-chain cleavage in vitro.

Adrenal Cortex↗
PubMed1981

Effect of LH factors regulating ovarian cholesterol metabolism and progesterone synthesis in PMSG-primed immature rats.

Administration of an ovulatory dose of LH (10 micrograms, i.v.) to PMSG (4 i.u., s.c.)-primed immature rats increased ovarian pregnenolone levels 5-fold and ovarian progesterone levels 40-fold within 6 h and the levels, although fluctuating, remained elevated for 72 h. Serum progesterone levels mimicked those of the luteal phase in the normal cycle. The sustained increase in steroidogenesis was accompanied by a decrease in both basal and cAMP-stimulatable ovarian cholesterol ester hydrolase activity and a net increase in ovarian cholesterol ester content. Ovarian free cholesterol levels were essentially unchanged during the 72 h study. LH does not, therefore, chronically stimulate steroidogenesis by providing additional substrate for the steroidogenic enzymes through activating cholesterol ester hydrolase to bring about hydrolysis of cholesterol esters. Moreover, ovarian cholesterol esters are unlikely to be the primary source of cholesterol utilized to support luteal steroidogenesis. Studies with isolated mitochondria suggested that the mechanisms by which LH stimulated steroidogenesis were by (a) stimulating mitochondrial pregnenolone production probably by facilitating the intramitochondrial movement of cholesterol of the site of side-chain cleavage, and (b) promoting the metabolism of pregnenolone to progesterone.

Animals↗
PubMed1981

Action of gonadotrophic hormones on cholesterol side-chain cleavage and cholesterol ester hydrolase in the ovary of the immature rat.

Immature rats were injected with PMSG (Day 0) and hCG (Day 3). PMSG caused an increase in cholesterol side-chain cleavage activity and the concentration of its terminal oxidase, cytochrome P-450, and an increase in cholesterol ester hydrolase activity in ovarian homogenates; the activity of both enzymes was further increased after hCG injection. Cholesterol ester hydrolase activity/mg ovarian wet weight was unchanged between Days 0 and 13, but the concentration of cytochrome P-450/mg wet weight increased 3-fold between Days 5 and 6. Cholesterol side-chain cleavage activity, measured in terms of pregnenolone production/mg wet weight, showed its largest increase (10-fold) between Days 6 and 7. Addition of exogenous cholesterol maintained the rate of pregnenolone production in incubated ovarian homogenates on Days 7--9.

Adrenal Glands↗
PubMed1981

Control of steroidogenesis in rat corpus luteum: the rate of access of cholesterol to the active site of the cholesterol side-chain cleavage enzyme.

The pool of cholesterol at the active site of the cholesterol side-chain cleavage enzyme was increased by preincubation with substrate. This process occurred at a linear time-dependent rate over 20 min, whereas uptake of cholesterol into mitochondria was complete by 1 min. Both endogenous mitochondrial cholesterol and added extramitochondrial cholesterol showed similar time-dependent substrate preincubation kinetics. The rate of metabolism of exogenous cholesterol was increased by injection of LH and decreased by treatment with cycloheximide. The rate of entry of cholesterol into the active site in both preparations, however, retained linear time-dependent kinetics. Calcium ions (2 mmol/L) increased the rate of steroidogenesis most in mitochondria preincubated for short periods and least in mitochondria preincubated for longer periods. These studies indicated that the pool of cholesterol at the active site of the enzyme can determine the rate of pregnenolone biosynthesis in the mitochondria of rat corpus luteum. Calcium ions, but not trophic hormones or cycloheximide, appeared to alter the access of cholesterol to the active site of the cholesterol side-chain cleavage enzyme during substrate preincubation.

Animals↗
PubMed1981

Se-75-labeled bile acid analogs, new radiopharmaceuticals for investigating the enterohepatic circulation.

Four selenium-labeled free bile acids and four selenium-labeled conjugated bile acids, labeled with Se-75 at the C-19, C-22, C-23, or C-24 position, have been synthesized and their absorption and excretion compared with that of [24-14C]cholic acid, following both oral and intravenous administration. All but one of the compounds is absorbed and excreted in bile to a significant extent. One compound, SeHCAT, has been selected for particular study. It is quantitatively absorbed from the gut at the same rate as cholic acid, and both are excreted into the bile at the same rate. It remains almost entirely confined to the enterohepatic circulation (the gut, liver, and biliary tree) and excretion is exclusively fecal. Whole-body retention, measured for 41 days, and tissue distributions suggest that the absorbed radiation dose would be small compared with that in many established tests. Such a compound offers the possibility of a simple, novel, and aesthetically acceptable method of investigating small-bowel disease. It therefore merits further investigation.

Administration, Oral↗
PubMed1980

Dietary effects on certain adrenal cortical functions in the rat.

The rate-limiting step in adrenal steroidogenesis is associated with the mitochondrial-cytochrome-P450scc-dependent production of pregnenolone from cholesterol. This sterol side-chain cleavage reaction is influenced by the supply of cholesterol to the mitochondria. Cholesterol is stored as cholesterol esters while the cytosol contains a hormone-sensitive cholesterol ester hydrolase. This enzyme is activated by phosphorylation involving a cyclic AMP-dependent protein kinase and ATP; this enzyme preferentially attacks cholesterol oleate or cholesterol linoleate. The lipid composition of the adrenal cortex is influenced by diet so that animals on a low-fat diet tend to store cholesterol oleate and as the linoleate content of the diet is increased, the cholesterol linoleate content of the adrenal cortex increases. Animals maintained on a high erucate diet tend to store large amounts of cholesterol erucate in the adrenal cortex; such animals have an impaired adrenal cortical function. Animals maintained on a low-fat diet (marginally deficient in essential fatty acids), a linoleate-replete diet or a moderate erucate diet, all exhibited normal responses to ACTH and normal corticosterone production rates.

Adrenal Cortex↗