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Biomedical subjects

M H Sullivan

Publications and source records attributed to M H Sullivan.

At least 91 records · Page 5Linked to original sources
PubMed1990

Limited transfer of prostaglandin E2 across the fetal membrane before and after labor.

The transfer of prostaglandin E2 (PGE2) across intact fetal membranes (amnion-chorion-decidua) obtained before and after the onset of labor was investigated using a novel system for in vitro fetal membrane culture. Studies using physiological concentrations of PGE2 showed that very little PGE2 will cross the membranes without being metabolised, before or after the onset of labor. It was only when pharmacological concentrations of PGE2 were used that the enzyme activity of the chorion was no longer able to prevent transfer of PGE2 without conversion to inactive metabolites. These results suggest that only small amounts of PGE2 from amnion are normally transferred across the chorio-decidua before or after the onset of labor, but the metabolism of PGE2 subsequent to transfer across the fetal membranes requires further assessment.

Arachidonic Acid↗
PubMed1990

Control of fetal membrane prostaglandin E2 production by bacteria.

The effect of bacteria on the production of prostaglandin E2 (PGE2) and PGE2 metabolites by fetal membranes has been investigated. Live bacteria stimulated a large increase in the levels of PGE2 metabolites, but only a small increase in the levels of PGE2 on the fetal side of the membranes. No significant changes in the levels of PGE2 or its metabolites were found on the maternal side. Bacteria may therefore stimulate PGE2 production by fetal membranes during short-term incubations, but it seems that the metabolic capacity of the chorion was so high that no change in PGE2 levels was detectable on the maternal side of the fetal membranes. This was confirmed by the finding that less than 1% of 3H-PGE2 added to the fetal side of the membrane reached the maternal side without being metabolized.

Culture Techniques↗
PubMed1989

Production of epoxygenase metabolite by human reproductive tissues.

Human amnion, trophoblast and umbilical vein endothelial cells synthesise an arachidonic acid metabolite which is neither a lipoxygenase nor a cyclo-oxygenase product. It is sensitive to stimulants and inhibitors of the cytochrome-P450-dependent epoxygenase system and co-migrates on HPLC with 14,15-epoxyeicosatrienoic acid (14,15-EET), which is an epoxygenase product. The function of 14,15-EET in these reproductive tissues is unknown, but it may be involved in the maintenance of vascular function.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

An investigation of glucose uptake in relation to steroidogenesis in rat testis and tumour Leydig cells.

The mechanisms of the requirement of glucose for steroidogenesis were investigated by monitoring the uptake of the glucose analogue 2-deoxy-D-glucose by rat testis and tumour Leydig cells. The characteristics of glucose transport in both of these cell types were found to resemble those of the facilitated-diffusion systems for glucose found in most other mammalian cells. The Leydig cells took up 2-deoxy-D-glucose but not L-glucose, and the uptake was inhibited by both cytochalasin B and forskolin. In the presence of luteinizing hormone, the rate of 2-deoxy-D-glucose uptake by both cell types was increased by approx. 50%. In addition to D-glucose, it was shown that the Leydig cells could also utilize 3-hydroxybutyrate or glutamine to maintain steroidogenesis.

Animals↗
PubMed1986

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.

Animals↗
PubMed1986

Modulation and role of Ca2+ in LH and LHRH agonist action in rat Leydig cells.

The results of our recent studies on purified rat Leydig cells indicate that there are no major qualitative differences in the stimulating effects of LH and LHRH agonists on steroidogenesis via mechanisms that are dependent on calcium. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. Using the fluorescent indicator quin-2, it was shown that LH and LHRH agonist increase intracellular calcium levels; LH was more potent than LHRH agonist (max increase in concentrations obtained were 500 nM and 60 nM respectively). This difference was probably the result of a direct effect of cyclic AMP (whose production is stimulated by LH but not by LHRH) because cyclic AMP analogues were as potent as LH in increasing calcium levels. These studies indicate a major role for calcium in the control of steroidogenesis in testis Leydig cells.

Animals↗
PubMed1985

Effects of calmodulin and lipoxygenase inhibitors on LH (lutropin)- and LHRH (luliberin)-agonist-stimulated steroidogenesis in rat Leydig cells.

The results of this study, carried out with purified rat Leydig cells, indicate that there are no major differences in the stimulating effects of lutropin (LH) and luliberin (LHRH) agonists on steroidogenesis via mechanisms that are dependent on Ca2+. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. All three calmodulin inhibitors used (calmidazolium, trifluoperazine and chlorpromazine) were shown to block LH- and LHRH-agonist-stimulated steroidogenesis. This probably occurred at the step of cholesterol transport to the mitochondria. Similarly, three lipoxygenase inhibitors (nordihydroguaiaretic acid, BW755c and benoxaprofen), inhibited both LH- and LHRH-agonist-stimulated steroidogenesis. The amounts of the inhibitors required were similar for LH- and LHRH-agonist-stimulated steroidogenesis. Steroidogenesis stimulated by the Ca2+ ionophore A23187 was also inhibited, but higher concentrations of the inhibitors were required. Indomethacin (a cyclo-oxygenase inhibitor) increased LHRH-agonist-stimulated steroidogenesis;this is consistent with the role of the products of arachidonic acid metabolism via the alternative, lipoxygenase, pathway. The potentiation of LH-stimulated testosterone production by LHRH agonist was unaffected by indomethacin or by lipoxygenase inhibitors at concentrations that inhibited LH-stimulated testosterone production by 75-100%. It was not possible to eliminate a role of calmodulin in modulating the potentiation, although higher concentrations of the inhibitors were generally required to negate the potentiation than to inhibit LH- or LHRH-agonist-stimulated testosterone production.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1985

Control and production of leukotriene B4 in rat tumour and testicular Leydig cells.

As part of an investigation into the role of leukotrienes in steroidogenesis, the formation of leukotriene B4 was investigated in purified Leydig cells from rat testes and from a tumour by using a sensitive radioimmunoassay. Detectable levels were found in both Leydig cell types (70 pg/10(6) cells) and these remain unchanged during incubation for 60 min at 32 degrees C. Addition of the Ca2+ ionophore A23187 increased LTB4 production more than 6-fold within 10 min whereas steroidogenesis was not increased until after 20 min. In the presence of luteinizing hormone or luteinizing hormone releasing hormone agonist no increase in LTB4 was detected in the testis Leydig cells whereas luteinizing hormone stimulated testosterone production from 3.2 +/- 0.1 to 148.9 +/- 7.5 ng/10(6) cells during the same time period. Similar results were obtained with the tumour Leydig cells. The LTB4 was found to be rapidly secreted by the cells in all experiments. The basal and A23187-stimulated levels were inhibited by nordihydroguaiaretic acid, a lipoxygenase inhibitor. It is concluded that LTB4 is produced in Leydig cells and can be stimulated by high calcium levels, but that it is probably not required for the control of steroidogenesis.

Animals↗
PubMed1984

Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis.

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1984

The role of calcium in luteinizing hormone-releasing hormone agonist (ICI 118630)-stimulated steroidogenesis in rat Leydig cells.

The luteinizing hormone-releasing hormone (LHRH) agonist ICI 118630 was found to increase testosterone production in purified rat testis Leydig cells in a concentration- and time-dependent manner, but no consistent changes in cyclic AMP levels were detectable. The stimulation of steroidogenesis by LHRH agonist was found to be dependent on the concentration of Ca2+ in the incubation medium; at least 1 mM was required. The calcium ionophore A23187 mimicked the effects of the LHRH agonist on steroidogenesis, and addition of both compounds together did not further increase testosterone production. The calcium ionophore caused a small increase in cyclic AMP which was independent of the concentration of the ionophore and of the calcium concentrations. The evidence obtained in this study indicates that LHRH agonist-stimulated steroidogenesis in rat testis Leydig cells is primarily mediated by calcium and not cyclic AMP.

Animals↗
PubMed1984

LHRH agonist decreases LH- but not forskolin-stimulated cyclic AMP levels in rat Leydig cells in vitro.

The effects of an LHRH agonist on LH- and forskolin-stimulated cyclic AMP and testosterone production have been investigated in purified rat Leydig cells in vitro. In agreement with previous results it was found that preincubation with LHRH agonist inhibited subsequent LH-stimulated cyclic AMP production. At least 2 h preincubation was required and this effect of the LHRH agonist was negated by the protein synthesis inhibitor cycloheximide and by the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Forskolin-stimulated cyclic AMP production was not inhibited by the LHRH agonist. Forskolin increased testosterone production to the same levels attained by LH and preincubation with LHRH agonist increased both forskolin- and LH-stimulated testosterone production. The data obtained suggest that LHRH agonist increases the synthesis of an inactive form of phosphodiesterase (or associated protein) which is activated by LH via a mechanism not involving cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1984

The effect of calcium on the potentiation of LH-stimulated steroidogenesis and inhibition of LH-stimulated cyclic AMP production by LHRH agonist (ICI 118630) in rat Leydig cells.

A luteinizing hormone-releasing hormone (LHRH) agonist (ICI 118630) potentiated the effects of luteinizing hormone (LH) and dibutyryl cyclic AMP on steroidogenesis during 4 h incubations with rat Leydig cells. LH-stimulated cyclic AMP levels were decreased by the addition of the LHRH agonist. The potentiation of the LH-increased steroidogenesis was dependent on Ca2+; maximum effects required at least 2.5 mM Ca2+ in the incubation medium. The calcium ionophore A23187 negated the potentiation in a dose-dependent manner (ED50 = 0.2-0.3 microM), but had no effect on LH-induced steroidogenesis, despite a 90% decrease in cyclic AMP production. The latter decrease was found to be dependent on the Ca2+ concentration. In the presence of the phosphodiesterase inhibitor methylisobutylxanthine (MIX), the ionophore A23187 induced a dose-dependent decrease in both LH and LH plus LHRH agonist-stimulated steroidogenesis and cyclic AMP production. The results obtained indicate that calcium, rather than cyclic AMP, is the mediator of the potentiating effects of LHRH agonist on LH-increased steroidogenesis in rat Leydig cells. The marked inhibition of the synergism in the presence of calcium ionophore A23187 suggests that Leydig cell calcium homeostasis must be intact for LHRH agonist action to occur. LHRH agonist causes a Ca2+-dependent decrease in LH-stimulated cyclic AMP production.

Animals↗
PubMed1983

Stimulation of cholesterol side-chain cleavage by a luteinizing-hormone-releasing hormone (luliberin) agonist (ICI 118630) in rat Leydig cells.

The action of a luliberin (luteinizing-hormone-releasing hormone) agonist (ICI 118630) and lutropin (luteinizing hormone) on the activity of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat Leydig cells has been investigated. This has been carried out by studying the metabolism of exogenous (22R)-22- and 25-hydroxycholesterol to testosterone. It was found that both hydroxycholesterols increased testosterone production to higher levels than achieved by lutropin alone. Addition of luliberin agonist but not lutropin was found to increase further the metabolism of the hydroxycholesterol to testosterone; this occurred in the presence of saturating and subsaturating levels of the hydroxycholesterols. This effect of luliberin agonist was potentiated in the presence of lutropin. The protein synthesis inhibitor, cycloheximide, inhibited the luliberin agonist-induced stimulation of the hydroxycholesterol metabolism. At low calcium levels (1.1 microM), testosterone production was increased by addition of (22R)-22-hydroxycholesterol but the luliberin agonist effect was negated. The calmodulin inhibitor trifluoperazine inhibited (22R)-22-hydroxycholesterol-stimulated steroidogenesis and negated the luliberin agonist effect. These results indicate that luliberin agonist specifically increases the synthesis of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat testis Leydig cells.

Animals↗