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D A Stansfield

Publications and source records attributed to D A Stansfield.

At least 19 recordsLinked to original sources
PubMed1988

Luteal progesterone synthesis and high-density lipoprotein. Evidence implicating a pronase-sensitive HDL-binding site in the mechanism by which HDL supports luteal progesterone synthesis.

We have shown previously that corpus luteum cells isolated from the superovulated ovaries of rats treated with 4-amino-pyrazolo[3,4-d]pyrimidine constitute a suitable experimental system by which to investigate the mechanism in which plasma high-density lipoprotein (HDL) plays a role in luteal cellular progesterone synthesis. In the present study, the rate of luteal cellular progesterone synthesis was shown to be stimulated by 125I-labelled HDL up to about 70% of the rate achieved in the presence of native HDL. The concentration of HDL needed for half-maximal stimulation of progesterone synthesis in the presence of lutropin was not significantly different irrespective of whether radioiodinated HDL or unlabelled HDL was used. Experimental conditions for studying the binding of 125I-labelled HDL to isolated luteal cells have been defined and cellular binding affinity and binding capacity have been measured. Exposure of the luteal cells to pronase virtually abolished their capacity to bind 125I-HDL and made them unable to respond to added HDL by increasing their rate of progesterone synthesis in the presence of lutropin. Control experiments showed this effect of pronase on cellular progesterone synthesis not to be due to destruction of cellular lutropin receptors, nor to general cellular damage. This evidence supports the view that luteal cellular binding of HDL is part of the mechanism by which HDL acts in luteal progesterone synthesis. Cellular binding capacity and affinity for 125I-labelled HDL were the same irrespective of whether or not lutropin was present during incubation. Furthermore, the binding capacity and affinity of cells from the ovaries of rats not treated with 4-amino-pyrazolo[3,4-d]pyrimidine were the same as in luteal cells isolated from rats that had been treated.

Adenine↗
PubMed1986

Stimulation of progesterone synthesis, in corpus luteum cells isolated from 4-amino-3,4-d-pyrazolopyrimidine-treated rats, by cholesterol presented in non-lipoprotein form.

We have previously shown that the administration of 4-amino-3,4-d-pyrazolopyrimidine (4-APP) to rats reduces progesterone synthesis by cells subsequently isolated from their corpora lutea, and that plasma high density lipoprotein (HDL) can restore this progesterone synthesis to normal. In this paper we demonstrate that a dispersion of phospholipid and cholesterol, but not other sterols, can enhance this 4-APP-disabled progesterone synthesis to the same level as can HDL, thus providing the first direct evidence that cholesterol is the component of HDL upon which rat corpus luteum depends for its ability to synthesize progesterone.

Adenine↗
PubMed1981

Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF.

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a ;burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a ;lag' phase with p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.

Adenylyl Cyclases↗
PubMed1980

The preparation and purification of isolated rat corpus-luteum cells and their use in studying the relationship between cholesterol biosynthesis and the lutropin-stimulated formation of progesterone.

Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.

Animals↗
PubMed1980

Cyclic AMP levels in blood cells from a short-cycle manic-depressive subject.

Erythrocyte and leucocyte numbers and cyclic AMP content were monitored weekly in a mentally defective short-cycle manic-depressive woman. The erythrocyte count remained relatively constant but cyclic AMP content when manic was significantly higher than when depressed, and the mood and biochemical data appeared to have the same periodicity, with coincident peaks and troughs. Total leucocytes were related to mood, with peaks in the manic phase. Mononuclear cyclic AMP content varied without relation to manic or depressive phases.

Bipolar Disorder↗
PubMed1979

Emotional disturbance following childbirth: clinical findings and urinary excretion of cyclic AMP (adenosine 3'5'cyclic monophosphate).

Emotional disturbance was assessed in a group of women in the first few days following childbirth and again 2 months and 1 year following childbirth; the clinical features are described. Variables such as social class, age and parity were not related to the level of emotional disturbance, but a history of marital problems, sexual difficulties, poor relationships with immediate family or disrupted family relationships in childhood were so related. Twenty-four hour urinary excretion of cyclic AMP (adenosine 3'5' cyclic monophosphate) was estimated in the same group of women on 2 occasions in the week following childbirth and again 2-3 months later in approximately one third of the original sample. Excretion of cyclic AMP in the few days following delivery was elevated compared with excretion 2-3 months later, and there was a significant rise in urinary excretion of cyclic AMP between the 1st and 2nd urine collections. Those women showing most emotional disturbance on the 3rd day after delivery and women indicating most mood change in the direction of becoming elated had the highest levels of cyclic AMP in the 2nd urine collection.

Adjustment Disorders↗
PubMed1978

Gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum.

1. Sepharose 6B gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum is described. Both zonal and frontal techniques of analysis were used. 2. Under conditions of zonal analysis recoveries of activity were low. It was concluded that dissociation of two or more components of the adenylate cyclase complex was occurring on the column and that the maintenance of the complex was essential for the high-activity state of the catalytic unit. Two peaks of adenylate cyclase activity, of approximate mol. wts. 45,000 and 160,000 were detected. 3. The theory of frontal analysis (or steady-state gel filtration), applied to the study of the interacting components of the adenylate cyclase complex is discussed, and activity profiles are predicted. Activity profiles obtained experimentally be frontal analysis compared well with the theoretically predicted profile and provide evidence that dissociation of a high-activity complex, with concomitant loss of activity, does occur. Recoveries of activity under conditions of frontal analysis were higher than with zonal analysis. 4. The effects of concentration and removal of detergent on the activity of the soluble enzyme are discussed.

Adenylyl Cyclases↗
PubMed1978

Solubilization of bovine corpus-luteum adenylate cyclase in lubrol-PX, triton X-100 or digitonin and the stabilizing effect of sodium fluoride present in the solubilization medium.

1. Adenylate cyclase activity of the washed 600g sediment of bovine corpus-luteum homogenate was solubilized by Lubrol-PX, Triton X-100 and digitonin. Digitonin was the least destructive of NaF-stimulated activity. 2. NaF, present in the solubilization medium together with MgSO4, increased the percentage yields of soluble activity from untreated 600g sediment and 600g sediment which had been preincubated with p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate). The stabilizing influence of NaF was most marked with digitonin. However, the highest specific activities of soluble enzyme were obtained with Lubrol-PX as solubilizing agent, since digitonin solubilized more membrane protein than does Lubrol-PX, and less of the activity of the digitonin-dispersed 600g sediment was recovered in the 105000g supernatant. 3. p[NH]ppG also has a stabilizing effect when present during the solubilization, but less so than NaF. 4. Both NaF and MgSO4 alone have a stabilizing effect during solubilization. The greatest amounts of soluble activity were obtained with both agents present in the solubilization medium, there being a synergistic effect.

Adenylyl Cyclases↗
PubMed1978

Stabilization and solubilization of bovine corpus-luteum adenylate cyclase. The effects of guanosine triphosphate, guanosine 5'-[beta,gamma-imido]triphosphate, sodium fluoride and Tris/hydrochloric acid concentration on enzyme activity.

1. Adenylate cyclase of the washed 600g sediment of bovine corpus-luteum homogenate is stimulated by p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate), the imido analogue of GTP, and to a lesser extent by GTP itself. Activation by p[NH]ppG is not reversed by extensive washing before assay, but can, however, be reversed by NaF. 2. Both p[NH]ppG and NaF stabilize the enzyme during incubation at 37 degrees C. NaF also causes an irreversible activation, but only of part of the potentially NaF-activatable adenylate cyclase; there are possibly two components of the adenylate cyclase system, which can be distinguished by their response to NaF. 3. Solubilization of the adenylate cyclase activity in the 600g sediment, by using the non-ionic detergent Lubrol-PX, gave variable yields. A relationship between the magnitude of NaF stimulation of the 600g-sediment enzyme and the yield of soluble activity derived from the sediment was recognized. The results suggest that the pre-existing state of the enzyme complex in vivo is reflected by the response in vitro to NaF and may determine the success with which activity can be solubilized. 4. The absolute yields of soluble activity could be increased by p[NH]ppG preactivation of the 600g sediment. During the development of the maximally active state by preincubation with p[NH]ppG the enzyme passes through a stage in which Lubrol solubilization is increased, but the maximally active state is itself less amenable to solubilization. p[NH]ppG activation causes the appearance of NaF-inhibited states, which appear to be preferentially solubilized by Lubrol-PX.

Adenylyl Cyclases↗
PubMed1977

Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine 3':5'-cyclic monophosphate.

Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg(2+) and ATP, but at low concentrations of Mg(2+) and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca(2+) and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg(2+) and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues.

Adenosine Triphosphate↗
PubMed1977

Use of a competitive protein-binding assay for adenosine 3':5'-phosphate for the study of bovine corpus luteum adenylate cyclase.

The use of a competitive protein-binding assay for cyclic AMP, utilizing the binding protein purified from bovine adrenal cortex, for the study of adenylate cyclase activity of the washed 600 g sediment of bovine corpus luteum is validated. A specific assay for cyclic AMP could only be achieved by removal of the degradation products of ATP on a precipitate of nascent BaSO4. Simple dilution of the sample before assay was not sufficient to eliminate interference from degradation products of ATP. An observed variability in optimal ATP substrate and tissue concentrations is though to reflect variability in the enzymic profile of the cyclic corpus luteum. Optima with respect to F-, Mg2+ and pH are more clearly defined and are similar to those reported for adenylate cyclase systems for other tissues.

Adenosine↗