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Biomedical subjects

G S Boyd

Publications and source records attributed to G S Boyd.

At least 19 recordsLinked to original sources
PubMed1988

Biologic stability of tauro-23-[75Se] selena-25-homocholic acid.

The stability of tauro-23-[75Se]selena-25-homocholic acid (SeHCAT) towards deconjugation by the enzyme cholylglycine hydrolase was compared with that of taurocholate: whereas taurocholate underwent 58% deconjugation within 2 hr, SeHCAT suffered only 8% deconjugation plus 5% conversion to an unknown product within 24 hr. Incubation of SeHCAT under anaerobic conditions for 48 hr at 37 degrees C with human fecal organisms resulted in considerable deconjugation, 7 alpha-dehydroxylation, and dehydrogenation. Twenty-four hours after the simultaneous administration of SeHCAT and tauro-[24-14C]cholate to a rabbit the recovery of 75Se in bile was 90% of that of 14C. Forty-eight hours following administration of SeHCAT to a second rabbit residual bile radioactivity revealed 80% deconjugation and dehydroxylation and 60% reconjugation with glycine. Although SeHCAT is more resistant than taurocholate towards modification by fecal bacterial enzymes, within the rabbit it follows the principal metabolic pathways of the natural bile acids.

Amidohydrolases↗
PubMed1985

Characterisation of rat hepatocyte monolayers for investigation of the metabolism of bile salts.

Rat hepatocyte monolayers were maintained for periods up to 24 h during which time their viability was greater than 85%. Using specific radioimmunoassays, the hepatocyte monolayers were shown to synthesise conjugated cholic, chenodeoxycholic and beta-muricholic acids. Feeding the bile salt sequestrant, cholestyramine, to donor animals increased synthesis of the major bile salt conjugates by the cells. Incubation of hepatocyte monolayers with bovine serum albumin decreased total synthesis of the three bile acids measured, but increased the amount of conjugated chenodeoxycholic acid detected. In order to test whether the effect of bovine serum albumin on bile salt synthesis was due to binding of bile salts, hepatocyte monolayers were incubated with antiserum to conjugated chenodeoxycholic acid. This treatment increased conjugated chenodeoxycholic acid production but had no effect on the other bile salt conjugates. It is concluded that the increase in conjugated chenodeoxycholic acid synthesis seen with bovine serum albumin and antiserum to conjugated chenodeoxycholic acid is caused by binding of the bile salt in the medium.

Animals↗
PubMed1985

Metabolism of cholesteryl ester in monolayers of bovine adrenal cortical cells. Effect of an inhibitor of acyl-CoA: cholesterol acyltransferase.

The effect of Sandoz compound 58-035 on cholesterol metabolism in monolayers of bovine adrenal cortical cells was studied. 58-035 did not inhibit cholesterol ester hydrolase, cholesterol side-chain cleavage, cholesterol synthesis from acetate, or cortisol synthesis in cells stimulated with ACTH or in unstimulated cells. It was, however, an effective inhibitor of formation of cholesteryl ester. The rate of formation of cholesteryl ester in the cells was increased by additional cholesterol derived from mevalonic acid or from the hydrolysis of intracellular lipid droplets. 58-035 caused an increase in the secretion of cortisol from cells maintained on a limited supply of cholesterol from bovine lipoproteins added to the medium when the cells were not stimulated with ACTH. This effect was not observed in stimulated cells. The results suggest that the bovine adrenal cortical cell can direct the flux of exogenous cholesterol very precisely according to its metabolic state.

Adrenal Cortex↗
PubMed1985

The effect of a rat plasma high-density lipoprotein subfraction on the synthesis of bile salts by rat hepatocyte monolayers.

The effect of a rat high-density lipoprotein subfraction (HDL2) on the synthesis of bile salts by rat hepatocyte monolayers prepared from rats fed a diet containing cholestyramine, was investigated. The synthesis of bile salts as measured by radioimmunoassay of conjugated cholic, chenodeoxycholic and beta-muricholic acids was significantly increased when hepatocytes were incubated with a physiological concentration (500 micrograms HDL2 protein X ml-1) of HDL2.

Animals↗
PubMed1984

Cholesterol ester turnover in isolated liver cells. Effects of cholesterol feeding.

Isolated hepatocytes from rats that had been kept in a steady state of [3H]cholesterol were incubated in a salt medium with or without serum. The cells released esterified cholesterol into the incubation medium as lipoproteins. This secretion, 18.1 +/- 0.5 nmol/h per g of cells, was increased when the cells were incubated in a medium containing serum (46.3 +/- 4.9 nmol/h per g of cells). This secretion was strikingly enhanced by cholesterol feeding (1% in the diet, 30 days) to 323-620 nmol/h per g of cells, and inhibited by cycloheximide, colchicine or EDTA. After removal of EDTA and addition of calcium, the cholesterol ester secretion was restored. Free cholesterol of previously labelled high-density lipoproteins (HDL) was exchanged (t1/2 = 30 min) with that of liver cells and esterified. The esterification rate (25.8 +/- 2.5 nmol/h per g of cells) was increased by cholesterol feeding (1% in the diet, 8 days) to 63.2 +/- 2.8 nmol/h per g of cells. No cholesteryl ester hydrolysis was detected with the isolated liver cells. Consequently, it is suggested that the turnover of hepatic cholesteryl ester was caused mainly by secretion in lipoproteins.

Animals↗
PubMed1984

The effect of glucagon-induced adenosine 3' ,5' -monophosphate concentrations on bile acid synthesis in isolated rat liver cells.

The effect of increased intracellular adenosine 3' ,5' -monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 microM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.

Animals↗
PubMed1984

The role of lysine-rich proteins in the acute steroidogenic response of rat adrenal cells to ACTH.

The possibility that a lysine-rich protein was involved in the acute stimulation of steroidogenesis by ACTH was investigated using [3H] labelled lysine and isolated adrenal cells. The results demonstrated that cycloheximide inhibited steroidogenesis in a dose-dependent, rapid fashion and inhibited the incorporation of radioactive lysine into protein. However cells incubated in a lysine-free medium showed the same response to ACTH as cells incubated in a lysine-containing medium. It was also demonstrated that ACTH had no effect on the incorporation of tritiated lysine into the protein or small peptide fractions. These observations suggest that a rapidly synthesised, lysine-rich protein is not involved in the acute response to ACTH.

Adrenal Glands↗
PubMed1983

Investigation of the apolipoprotein fraction of isolated rat adrenal and bovine adrenocortical lipid droplets.

The lipid droplet fractions from rat adrenal and bovine adrenocortical tissue were isolated by density ultracentrifugation. The droplet fractions were delipidated and the protein components investigated by SDS-polyacrylamide slab gel electrophoresis. The adrenal lipid droplets from both species displayed a qualitatively similar protein profile, and both contained a major apolipoprotein subunit of Mr 40 000. Incubation of intact, non-delipidated lipid droplets with [gamma-32P]ATP in vitro resulted in the phosphorylation of the Mr 40 000 apolipoprotein subunit in the case of rat lipid droplets, but not in the case of bovine lipid droplets. However, following delipidation of the droplets with diethyl ether/ethanol, the Mr 40 000 apolipoprotein subunit was phosphorylated in both cases upon incubation of the delipidated protein fractions with [gamma-32P]ATP in vitro. Labelling with [gamma-32P]ATP and [3H]diisopropyl phosphorofluoridate indicated that the cholesterol ester hydrolase enzyme protein was not a major constituent of the adrenal lipid droplet protein fraction.

Adrenal Cortex↗
PubMed1983

The effect of dibutyryladenosine 3',5'-monophosphate on the synthesis of bile salts in isolated hepatocytes from rat.

The effect of dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) on the synthesis of conjugated cholic, chenodeoxycholic and beta-muricholic acids has been investigated. Hepatocytes were incubated with 1 mM Bt2cAMP for 3 h at 37 degrees C. In cells from rats with a basal rate of bile salt synthesis (soft-diet-fed rats) production of conjugated cholic acid was increased about two fold, synthesis of conjugated chenodeoxycholic acid was increased 10-20-fold but formation of its metabolite, conjugated beta-muricholic acid, was decreased by 30-50% in the presence of the cyclic nucleotide. The sum of the amounts of the three bile salts produced (total bile salt synthesis) was increased 30-50% by Bt2cAMP. When hepatocytes were prepared from rats in which bile salt synthesis had been stimulated by feeding the bile salt sequestrant, cholestyramine, Bt2cAMP had no effect on conjugated cholic acid synthesis, increased conjugated chenodeoxycholic acid production 3-5-fold and decreased conjugated beta-muricholic acid synthesis by about 50%. Total bile salt synthesis was unchanged. The ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic acid + conjugated beta-muricholic acid produced, an indication of the activity of 7 alpha-hydroxycholest-4-en-3-one 12 alpha-hydroxylase, was raised by Bt2cAMP in hepatocytes from soft-diet-fed but not in those from cholestyramine-fed rats. The effects of the cyclic nucleotide on the synthesis of the three bile salts in hepatocytes from soft-diet-fed rats were found to be saturable at a concentration of about 2 mM. Responses were half-maximal at concentrations of Bt2cAMP varying between 0.5 and 1.5 mM. These results suggest that in hepatocytes from rats with a basal rate of bile salt synthesis Bt2cAMP has effects at three different stages in the pathway, at the level of cholesterol 7 alpha-hydroxylase, 7 alpha-hydroxycholest-4-en-3-one 12 alpha-hydroxylase and chenodeoxycholine acid 6 beta-hydroxylase. In cells from rats in which bile salt synthesis has been stimulated only the effect at the chenodeoxycholic acid 6 beta-hydroxylase level is apparent. Bt2cGMP and Bt2cCMP had no effect on the synthesis of any of the bile salts measure, showing that the effects are specific for Bt2cAMP. The ratio of the amounts of the three bile salts found inside the cells to those found in the medium was decreased by about 90% when Bt2cAMP was present in the hepatocyte incubations. This effect was mimicked by Bt2cGMP and to a lesser extent by Bt2cCMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

In vitro regulation of bovine adrenal cortical acyl-CoA: cholesterol acyltransferase and comparison with the rat liver enzyme.

Acyl-CoA:cholesterol acyltransferase activity in bovine adrenal cortical microsomes was increased by preincubation of microsomes in vitro in the presence of MgCl2. The acyltransferase activity in the microsomes could be inhibited by further incubation in the presence of ATP/MgCl2. These effects appear to complement the known ATP-dependent activation of adrenal cytosolic cholesterol ester hydrolase, which is consistent with the role of the hydrolase in supplying cholesterol for steroidogenesis. These effects are, however, opposite to those recently demonstrated for the rat liver and intestine. Acyl-CoA:cholesterol acyltransferase activity in rat liver can be increased by the addition of cholesterol, as substrate, or by 25-hydroxycholesterol. Such activation was not observed in adrenal microsomal preparations, further suggesting that the mechanisms of regulation of cholesterol esterification differs between these tissues.

Acyl Coenzyme A↗
PubMed1983

The effect of hypocholesterolemic drug treatment on adrenocortical cell function.

The role of exogenous lipoprotein cholesterol versus endogenous cholesteryl esters as substrates in adrenal steroidogenesis was studied in isolated rat adrenal cells. Hypocholesterolemic drugs were used in rats to depress the plasma cholesterol concentration and the adrenal cholesterol concentration. Adrenal cortical cells were prepared in the usual way. The steroidogenic response to ACTH in normal adrenal cells and in cells which have been cholesterol-depleted was studied. Normal adrenal cells responded specifically over a 6 h incubation period to low doses of ACTH (half-maximal response equivalent to 40 microunits ACTH). These normal cells exhibited no altered response over a 3 h period to ACTH in the presence of serum or serum lipoproteins. The hypocholesterolemic drugs, 4-aminopyrazolo-[3,4-d]-pyrimidine, hexestrol and 17 alpha-ethinyl estradiol were used to lower plasma cholesterol, and after 1 day of 4-aminopyrazolo-[3,4-d]-pyrimidine and 5 days of hexestrol or 17 alpha-ethinyl estradiol treatment the plasma total cholesterol concentrations were similar. After 3 days of 4-aminopyrazolo-[3,4-d]-pyrimidine treatment the adrenal total cholesterol content was lower than after 1 day of this treatment, or 5 days of hexestrol treatment or 5 days of 17 alpha-ethinyl extradiol treatment. Lipoproteins had no significant effect on ACTH-stimulated steroidogenesis in cells isolated from rats treated for 1 day with 4-aminopyrazolo-[3,4-d]-pyrimidine, or for 5 days with hexestrol or 17 alpha-ethinyl estradiol. However, lipoproteins did stimulate steroidogenesis in cells from rats treated for 3 days with 4-aminopyrazolo-[3,4-d]-pyrimidine. The results show that normal adrenal cells contain a reserve of intracellular cholesterol so that the supply of endogenous cholesterol for steroidogenesis does not limit the response to ACTH and exogenous lipoproteins have no effect on steroidogenesis. However, if the cells are severely depleted of cholesterol then exogenous lipoproteins must be added for maximal steroidogenesis to occur.

Adrenal Cortex↗
PubMed1983

Cholesterol metabolism in the adrenal cortex.

Adrenal cortical mitochondria contain a mixed function oxidase capable of converting cholesterol to pregnenolone; this enzyme requires NADPH, oxygen and cholesterol. This cholesterol side chain cleavage enzyme system contains a Flavoprotein, an iron sulphur protein and a specific cytochrome P450 termed cytochrome P450scc. ACTH stimulates the adrenal cortex by activating adenyl cyclase producing an elevated intracellular concentration of cAMP. This in turn increases the activity of a cytosolic cAMP dependent protein kinase. Adrenal cortical cytosol contains a cholesterol ester hydrolase which is activated by ATP and a protein kinase. This enzyme may be deactivated by a phosphoprotein phosphatase. The adrenal cortex contains lipid droplets that are rich in esterified cholesterol. Cholesterol ester hydrolase can release free cholesterol from the lipid droplets. The free cholesterol released may be used to supplement the mitochondrial cholesterol as a pregnenolone precursor. Steroid hormone production by the adrenal cortex exhibits a diurnal rhythm and correlates with the activity of the cytosolic cholesterol ester hydrolase. The acute steroidogenic response to ACTH may be in part attributed to the availability of free cholesterol to the mitochondrial cholesterol side chain cleavage enzyme complex. The intracellular movement of free cholesterol from lipid droplets to mitochondrial inner membranes may be impeded by protein synthesis inhibitors such as cycloheximide. The precise mechanism of this block in steroidogenesis remains to be elucidated. Various drugs and oestrogenic hormones suppress the plasma and adrenal cholesterol concentrations. If adrenal cells are deficient in cholesterol, these cells exhibit a diminished response to ACTH. The response to this hormone can be corrected by supplying cholesterol via exogenous plasma lipoproteins. The route that free cholesterol follows within the adrenal cortical cell and the physiological factors influencing free cholesterol movement in such cells are important issues to be explored in future.

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↗
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↗
PubMed1979

The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure.

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Mixtures of analogues and phospholipid were also studied in monolayers. In all the bilayer systems studied cholesterol caused the greatest 'rigidifying' effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.

Cholesterol↗