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B A Cooke

Publications and source records attributed to B A Cooke.

At least 73 records · Page 4Linked to original sources
PubMed1984

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.

Adenylyl Cyclases↗
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↗
PubMed1983

The effect of cell damage on the density and steroidogenic capacity of rat testis Leydig cells, using an NADH exclusion test for determination of viability.

The ability of cells to exclude pyridine nucleotides was found to be a more sensitive index of cell viability than trypan blue. The three nucleated cell bands obtained by centrifugation of crude rat testis cells on Percoll density gradients contained different proportions of viable and damaged cells. Band III, the steroidogenically active Leydig cell band was mainly viable cells, whilst bands I and II contained 70 and 35% damaged cells respectively. Leydig cells damaged by freezing were shown to have a lower density than viable Leydig cells and did not produce testosterone after stimulation with LH. It is concluded that the methods used to prepare and purify rat testis Leydig cells contribute to the apparent Leydig cell "heterogeneity".

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

11. Testicular and epididymal function. Effects of isolation and purification procedures on the viability and properties of testis Leydig cells.

The effect of the isolation and purification procedures used to obtain mouse and rat Leydig cells on their viability and steroidgenic properties has been investigated. It was found that mechanical dispersion of rat testes prior to collagenase dispersion had a deleterious effect on the steroidogenic capacity of the cells. Omission of the mechanical disruption and separation of the collagenase-dispersed cells on Percoll gradients yielded 3 bands of cells. Band III (density 1.070 g/ml) contained highly pure viable Leydig cells of high steroidogenic capacity. Diaphorase histochemistry (but not trypan blue exclusion) revealed that bands I and II contained high amounts of damaged Leydig cells. These results are discussed in relation to the previously reported heterogeneity of Leydig cells. Both mouse and rat Leydig cells (band III) in monolayer culture maintained their responsiveness to LH for several days. The rat (but not mouse) Leydig cells contained LHRH receptors and responded to LHRH in a biphasic manner: initially a stimulation of both basal and LH increased steroidogenesis occurred which after 24 h became inhibitory.

Animals↗
PubMed1982

Resensitization of lutropin-desensitized tumour Leydig-cell adenylate cyclase with human erythrocyte membranes.

Purified rat tumour Leydig cells were pretreated with or without lutropin (1 h at 32 degrees C). The plasma membranes were then isolated and the adenylate cyclase activity measured in the presence of freshly prepared or heat-inactivated (1 h at 60 degrees C) human erythrocyte membranes. In plasma membranes from control cells in the presence of heat-inactivated human erythrocyte membranes both guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) plus lutropin and NaF caused a 45--50-fold increase in cyclic AMP production over 30 min compared with 12--13 fold p[NH[ppG and 2--3-fold with lutropin alone. In plasma membranes isolated from lutropin-pretreated cells the NaF- and the p[NH]ppG-stimulated cyclic AMP production rates were unchanged, but no effect of lutropin could be demonstrated with or without added p[NH]ppG. However, after mixing lutropin-desensitized Leydig tumour-cell plasma membranes with freshly prepared human erythrocyte plasma membranes, the adenylate cyclase activity in the presence of lutropin, p[NH]ppG, lutropin plus p[NH]ppG and NaF were similar to those of control cell plasma membranes treated in the same manner. The possible mechanisms of this reversal of lutropin-induced desensitization by human erythrocytes are discussed.

Adenylyl Cyclase Inhibitors↗
PubMed1982

Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein.

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED(50) (dose that produces a response that is 50% of the maximum response) 60+/-5.7ng/ml and 8+/-1.8ng/ml (mean+/-s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/10(6) cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific (125)I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4 degrees C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5'-[beta,gamma-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50-60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

Adenylyl Cyclases↗
PubMed1982

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells.

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues(10-8-10-6 M) on the functional activity (testosterone and cyclic AMP production and [125I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0-90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells. The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10-7 M) and after an initial lag period (2h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10(6) cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (p less than 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased. These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.

Animals↗
PubMed1982

Catecholamine stimulation of testosterone production via cyclic AMP in mouse Leydig cells in monolayer culture.

The effects of the beta-adrenergic hormone agonist, isoproterenol, on testosterone and cyclic AMP production in mouse Leydig cells in culture have been investigated. It was found that isoproterenol increased testosterone production on days 1, 2 and 3 of culture but not in freshly cultured cells. Cyclic AMP production was however increased on all days of culture. In subsequent studies carried out on day 2 of culture the amounts of testosterone formed during incubation with isoproterenol were 20-90% of those obtained with maximum stimulating levels of luteinizing hormone. The amounts of cyclic AMP formed were extremely low compared with those obtained with luteinizing hormone (22 +/- 5.3 and 2320 +/- 100 pmoles/10(6) cells/2 h respectively). Isoproterenol (10(-8) -10(-7) M) gave a significant increase in testosterone production and reached a maximum with 10(-6) M. Similar dose-response curves for cyclic AMP production were obtained. The stimulation of cyclic AMP and testosterone by isoproterenol was highly dependent on the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. Propranolol blocked, in a dose-dependent manner, both isoproterenol-stimulated cyclic AMP and testosterone production. In the presence of excess luteinizing hormone no additional effects of isoproterenol were detected. Epinephrine also stimulated testosterone production. It is concluded that catecholamines stimulate testosterone production in mouse Leydig cells in monolayer culture and that this effect if mediated by cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1982

The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

Purified Leydig cells were obtained from adult mouse testes by mechanical dispersion followed by Percoll density-gradient centrifugation as described by Schumacher et al. (1978). The cells were then established in monolayer culture by maintaining them in medium and 10% serum at 32 degrees C in 95% O2, 5% CO2. The cells rapidly attached to the culture dishes, gradually flattened and became epitheloid in appearance. Testosterone production by the cells in response to maximum stimulating levels of LH (100 ng/ml) and dibutyryl cyclic AMP (1 mM) was maintained for at least 2 days (approximately 1 microgram/10(6) cells/2h) and then declined to lower levels by days 3-4. Cyclic AMP production in response to LH was higher on day 1 than day 0 and then declined to lower levels by days 3-4. Binding of [125I]hCG was similar on day 0 and day 1 (approximately 20 fmoles/10(6) cells) and then declined to lower levels by days 3-4. The functional activity of the cells cultured in 0, 1 and 10% foetal calf serum was also examined; no significant effect of the serum on LH-stimulated testosterone or cyclic AMP production was found; however, a decrease of up to 50% in the binding of [125I]hCG to the Leydig cells occurred in the presence of serum. These results demonstrate that the function of differentiated adult Leydig cells can be maintained for at least 2 days in culture.

1-Methyl-3-isobutylxanthine↗
PubMed1982

The deleterious effect of mechanical dissociation of rat testes on the functional activity and purification of Leydig cells using Percoll gradients.

The effect of mechanically dispersing rat testes prior to collagenase treatment on the activity of the subsequently isolated Leydig cells has been investigated. The latter were obtained by centrifugation of the testes cells on Percoll density gradients. It was found that the lutropin-stimulated testosterone and cyclic AMP production were 5 and 3 times higher, respectively, in the purified Leydig cells when the mechanical dispersion was omitted, whereas the specific binding of human choriogonadotrophin was unchanged. It is concluded that mechanical treatment of rat testes, unlike mouse testes, decrease the activity of the Leydig cells.

Animals↗
PubMed1982

Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour.

An LH sensitive adenylate cyclase from a tumour Leydig cell has been investigated. The plasma membranes, prepared by a 2 phase (dextran-polyethylene glycol) centrifugation method were found to have the following properties: In the presence of LH plus p(NH)ppG (guanosine 5'-beta, gamma-imido triphosphate) or fluoride ions, maximum adenylate cyclase activity was obtained in the plasma membranes with 4 to 6 mM Mg2+ plus 0.33 to 2 mM ATP. LH alone stimulated adenylate cyclase activity 2-fold when compared with basal activity and the time course of cyclic AMP production was linear up to 45 min. With GTP (10(-5)M) and GTP plus LH, adenylate cyclase activity was increased 3 and 6-fold, respectively, for up to 20 min and thereafter declined. In contrast p(NH)ppG (10(-5)M) and p(NH)ppG plus LH increased adenylate cyclase activity 7 and 14-fold which was maintained for at least 45 min. Fluoride ions increased the enzyme activity linearly over 45 min approx 18-fold. When GTP or p(NH)ppG were added alone there was a lag time of activation of approximately 10 min which was abolished by the addition of LH. GTP but not p(NH)ppG at concentrations greater than 10(-4) inhibited basal and LH stimulated adenylate cyclase when compared with 10(-5)M GTP. The tumour Leydig cell adenylate cyclase is thus essentially similar to other hormone sensitive somatic cells. The present study makes it feasible to prepare plasma membranes by a simple method from large quantities of pure Leydig cells.

Adenosine Triphosphate↗
PubMed1982

Guanine nucleotide mediated desensitization of adenylate cyclase in cell free preparations from a Leydig cell tumour.

Cell free desensitization of a tumour Leydig cell plasma membrane adenylate cyclase has been demonstrated in the presence of guanine nucleotides. In experiments in which the membranes were pre-incubated with various nucleotides and LH, it was shown that this decreased adenylate cyclase activity was dependent on the presence of GTP and occurred both in the presence and absence of ATP. While pre-treatment with LH alone appeared to enhance subsequent adenylate cyclase activity, this hormone was able to potentiate the desensitizing effect of GTP. The desensitizing effect of GTP was not inhibited by sodium fluoride. In contrast, the GTP analogue p(NH)ppG (guanosine 5'beta, gamma-imido triphosphate) caused a persistent activation of the adenylate cyclase. GMP and guanosine also initially inhibited the adenylate cyclase activity, but this was entirely reversed by p(NH)ppG plus LH. GDP in addition to GTP caused desensitization but this was only partially reversed by p(NH)ppG plus LH. It is proposed that in similarity with the ovary (Bockaert et al. 1976; Ezra & Salomon 1982a) desensitization of Leydig tumour cell plasma membrane adenylate cyclase may involve a GTP-mediated phosphorylation step.

Adenylyl Cyclases↗