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Biomedical subjects

B A Cooke

Publications and source records attributed to B A Cooke.

At least 37 records · Page 2Linked to original sources
PubMed1992

Calmidazolium is a potent stimulator of steroidogenesis via mechanisms not involving cyclic AMP, calcium or protein synthesis.

This study reports an unexpected effect of calmidazolium on steroidogenesis. In contrast with previous work, which established that calmidazolium inhibits hormone-stimulated testosterone production in rat Leydig cells, the present study demonstrates that this compound is a potent stimulator of steroidogenesis when added by itself; this stimulation (approx. 10-fold in a 2 h incubation), was obtained over a narrow dose range (e.g.1-10 microM) in mouse and rat Leydig cells and in rat adrenocortical cells. The same concentrations of calmidazolium decreased basal cyclic AMP to undetectable levels in rat Leydig cells. Also, cyclic AMP stimulated with luteinizing hormone (LH), cholera toxin and forskolin was inhibited by calmidazolium (ED50 2 microM). In contrast with the actions of LH and cyclic AMP analogues on steroidogenesis, the effect of calmidazolium was not inhibited by removal of extracellular Ca2+, or by the addition of La3+ (a Ca(2+)-entry blocker), or the addition of cycloheximide (an inhibitor of protein translation). However, like dibutyryl cyclic AMP, calmidazolium-stimulated steroidogenesis was inhibited by aminoglutethimide, an inhibitor of cholesterol side-chain cleavage. Another calmodulin inhibitor, trifluoperazine, did not stimulate steroidogenesis. It is concluded that calmidazolium has a similar effect on steroidogenesis to LH, but by-passes the requirements for cyclic AMP, Ca2+, and protein synthesis. Calmidazolium is therefore a potentially important probe for elucidating the mechansims of control of steroidogenesis.

Adrenal Cortex↗
PubMed1992

Control of steroidogenesis in Leydig cells.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to stimulate steroidogenesis not only via cyclic AMP but also other pathways which include arachidonic acid and leukotrienes and regulation of chloride and calcium channels. The same stimulatory pathways may lead to desensitization and down-regulation of the LH receptor and steroidogenesis. The LH receptor exists in a dynamic state, being truncated, or internalized, degraded or recycled. Desensitization is controlled by protein kinase C (PKC) in the rat and by cyclic AMP dependent protein kinase and PKC in the mouse Leydig cells. Using an adapted anti-sense oligonucleotide strategy we have shown that the cytoplasmic C-terminal sequence of the LH receptor is essential for desensitization to occur. In contrast, these sequences of the LH receptor are not required for the stimulation of cyclic AMP and steroid production. We have also shown that the extracellular domain of the LH receptor is secreted from the Leydig cell and may act as a LH-binding protein.

Animals↗
PubMed1992

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C.

The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1991

Evidence for the requirement of proteolysis in LH stimulated cyclic AMP production and steroidogenesis in Leydig cells.

We have investigated the effect of protease activity on cyclic AMP production and steroidogenesis in rat testis, mouse testis and mouse tumour Leydig (MA10) cells. LH-, dibutyryl cyclic AMP-, and forskolin-stimulated steroidogenesis, but not 22R(OH) cholesterol conversion to pregnenolone, was inhibited by protease inhibitors. In mouse Leydig cells, LH but not forskolin or cholera toxin stimulated cyclic AMP production was inhibited by protease inhibitors. These results suggest that steroidogenesis in Leydig cells requires proteolysis before the conversion of cholesterol to pregnenolone. In the mouse but not rat Leydig cells, LH-stimulated cyclic AMP production is also dependent on proteolysis.

Animals↗
PubMed1991

Effect of cell purity, cell concentration, and incubation conditions on rat testis Leydig cell steroidogenesis.

This study examines the effects of cell purity and incubation conditions on testosterone production by rat testis Leydig cells in short-term primary culture. Both basal and luteinizing hormone (LH)-stimulated testosterone production were affected by the purity of the cell preparation, i.e., as the purity of the cell preparation was increased the amount of testosterone produced per Leydig cell was also found to increase. The stimulation ratio of testosterone production, calculated as the secretion of testosterone in the presence of LH (100 ng/ml) divided by the basal secretion of testosterone, increased with the increase in plating density (20,000 to 200,000 cells per well). This pattern of change was independent of the vessel and volume of incubation. In terms of the absolute amount of testosterone produced, increasing the plating density led to a decrease in the amount of steroid produced both basally and in response to LH. Composition of the incubation medium also had an effect on testosterone production; phenol red and sodium bicarbonate exerted negative effects. At all temperatures studied (4 degrees, 24 degrees, 34 degrees, and 37 degrees C), LH increased testosterone production and the degree of stimulation increased with temperature. We conclude that cell purity and incubation conditions markedly affect rat Leydig cell steroidogenesis in vitro. Furthermore, the manner in which the results are presented can affect their interpretation.

Animals↗
PubMed1991

Differences in LH receptor down-regulation between rat and mouse Leydig cells: effects of 3',5'-cyclic AMP and phorbol esters.

The role of cyclic AMP and phorbol esters in luteinizing hormone (LH) receptor down-regulation in Leydig cells has been studied. Dibutyryl cyclic AMP (db-cAMP) (0.01, 0.1 and 1 mM), forskolin (80 microM) and cholera toxin (1.19 nM) caused a 30-50% loss of [125I]hCG binding sites and an inhibition of receptor-[125I]hCG complex internalization in mouse tumour Leydig (MA10, MLTC-1) cells during 2 h. In contrast, db-cAMP had no effect on the level of binding sites or internalization of the hormone receptor complex in rat testis Leydig cells or a rat tumour (R2C) Leydig cell. Phorbol 12-myristate 13-acetate (PMA) at concentrations from 10(-9) to 10(-5) M had no effect on hormone binding or hormone-receptor complex internalization in any of the Leydig cells. In contrast a 2 h preincubation of MLTC-1 cells with 10(-7) M PMA caused a loss of subsequent LH-stimulated cyclic AMP and pregnenolone production. These results indicate that LH receptor down-regulation is mediated by cyclic AMP dependent kinase, but not protein kinase C, in mouse Leydig cells. No down-regulation of rat Leydig cell LH receptor occurs with either kinase.

Animals↗
PubMed1991

A novel method to modulate desensitization and truncation of luteinizing hormone receptors using antisense oligodeoxynucleotides.

We report a novel method to study the mechanisms of luteinizing hormone (LH) receptor desensitization and truncation, using antisense oligodeoxynucleotides that code for regions of the NH2-terminus, the third extracellular loop and the C-terminus of the LH receptor. Mouse tumour (MA10) Leydig cells were incubated for 48 h with the addition of 2.5 microM antisense oligodeoxynucleotides at time 0 and 24 h. It was found that the NH2-terminus oligodeoxynucleotide completely inhibited synthesis of the LH receptor. Pretreatment with the third extracellular loop oligodeoxynucleotide inhibited LH-, dibutyrylcyclic AMP (db-cAMP)- and phorbol 12-myristate 13-acetate (PMA)-induced desensitization and truncation of LH receptors. Truncation but not desensitization, of the LH receptor was prevented in cells pretreated with the C-terminus oligodeoxynucleotide. These results indicate that different sites of the C-terminal intracellular tail of the LH receptor are involved in the regulation of desensitization and truncation of the LH receptor.

Animals↗
PubMed1991

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells.

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A2 (PLA2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chorionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F2 alpha formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosterone (50 and 25 microM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA2.

Adrenal Cortex Hormones↗
PubMed1991

Regulation of the truncation of luteinizing hormone receptors at the plasma membrane is different in rat and mouse Leydig cells.

Regulation of the truncation of LH receptors was investigated in two types of mouse tumor Leydig cells (MA10 and MLTC-1), rat testis Leydig cells (RTL), and a rat tumor Leydig cell (R2C). Receptor numbers were measured by binding [125I]hCG to the cells cultured in monolayers. Addition of 3.3 nM LH for 2 h at 34 C had no detectable effect on binding sites in RTL or R2C cells, but in MA10 and MLTC-1 cells it caused a loss in binding sites. The effect on MA10 and MLTC-1 cells could be mimicked by inhibiting receptor internalization with 5 mM NaN3 and prevented by the addition of protease inhibitors. Incubating RTL and R2C cells with protease inhibitors caused a 2- to 3-fold increase in binding sites and a 2- to 3-fold increase in LH (0.033 and 0.33 nM)-stimulated cAMP production. When RTL and MA10 cells were incubated in the presence of [125I]hCG, a radioactive protein complex with an approximate mol wt of 80,000-90,000 was released into the incubation medium. We conclude that LH receptors are regulated by proteolysis at the plasma membrane in both mouse and rat Leydig cells. Furthermore, truncation of the LH receptor in the mouse Leydig cells is involved in down-regulation, whereas in the rat it is a continuous process.

Animals↗
PubMed1991

Separation and characterization of Leydig cells and macrophages from rat testes.

A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1-F8) (9 to 74 ml/min at 386 g). Each of these fractions was then further purified by density gradient centrifugation on 0-90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1-F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11-20%) were found in fractions F1-F3 (average density 1.045 g/ml), containing 11-37% Leydig cells. Less than 3% of the cells in fraction F4-F8 (average density 1.075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated- and Percoll-purified fractions F4-F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for 125I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35.7 mm/h.g) from fractions F7 and F8 were approximately twofold more responsive to LH (3.3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20.7 mm/h.g).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Induction of lutropin receptors by lutropin and cyclic AMP in cultured mouse tumour (MA10) Leydig cells.

The role of cyclic AMP in the regulation of lutropin (luteinizing hormone, LH) receptors has been investigated in cultured mouse tumour (MA10) Leydig cells. The LH receptors were quantified by measuring the binding of 125I-labelled human chorionic gonadotropin (hCG). LH (0.03 nM) in the presence of 1 mM-dibutyryl-cyclic AMP [(Bu)2cAMP] caused a 3-8-fold increase in subsequent 125I-hCG binding. (Bu)2cAMP (1 mM), cholera toxin (11.9 nM) and forskolin (1 microM) each caused a 2-4-fold increase in binding. In the presence of translation (cycloheximide) and transcription (actinomycin D) inhibitors, there was a loss of detectable binding sites. (Bu)2cAMP increased the rate of recovery of binding sites after trypsin treatment of MA10 cells, with a concomitant 2-fold increase in the level of binding sites. Under conditions where receptor levels were increased by 3-8-fold there was also a significant increase in pregnenolone production. It is concluded that LH and cyclic AMP have positive regulatory effects on LH receptors in MA10 cells by inducing the synthesis of new receptors. These induced receptors are functionally coupled to steroidogenesis.

Animals↗
PubMed1990

Evidence for the involvement of phospholipase A2 in the regulation of luteinizing hormone-stimulated steroidogenesis in rat testis Leydig cells.

In this study the effects of modulating the release of arachidonic acid by phospholipase A2 (PLA2) on luteinizing hormone (LH)-stimulated testosterone production in rat testis Leydig cells have been investigated. Exogenously added PLA2 significantly stimulated both basal and LH-stimulated testosterone production. The effects of three structurally unrelated PLA2 inhibitors (dexamethasone, quinacrine and p-bromophenacyl bromide (pBPB)) were determined. Dexamethasone and quinacrine caused a dose-dependent inhibition of LH-induced testosterone production but had no effect on LH-induced cyclic AMP accumulation. Dibutyryl cyclic AMP-, and forskolin-stimulated testosterone production were also inhibited by all three inhibitors used. 22R-OH-cholesterol-stimulated testosterone production was not inhibited by quinacrine or dexamethasone showing that they were not exerting their inhibitory effect on LH-induced testosterone production by decreasing the activity of the steroidogenic enzymes. However, pBPB exerted an inhibitory effect on LH-induced testosterone and cyclic AMP production. Furthermore pBPB also inhibited 22R-OH-cholesterol-induced testosterone production illustrating that apart from its well-documented effect on PLA2, it also exerts a direct inhibitory effect on the steroidogenic enzymes. The finding that PLA2 inhibitors inhibit testosterone production without affecting cyclic AMP accumulation provides further indirect evidence for second messengers in addition to cyclic AMP being involved in the action of LH in Leydig cells. These results indicate that PLA2 is involved in LH-induced testosterone production and that cyclic AMP may exert its actions via this pathway.

Acetophenones↗
PubMed1990

Evidence for two independent pathways in the stimulation of steroidogenesis by luteinizing hormone involving chloride channels and cyclic AMP.

The possible role of chloride channels in luteinizing hormone (LH) action on steroidogenesis in rat Leydig cells had been investigated. A chloride channel blocker, SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid), inhibited LH-stimulated steroidogenesis at low (less than or equal to 1 ng/ml), but not at high (100 ng/ml) LH concentrations. In addition, dibutyryl cyclic AMP- and forskolin-stimulated steroidogenesis was unaffected by SITS. The removal of extracellular chloride potentiated steroidogenesis stimulated by submaximal but not maximal doses of LH. These results suggest that at low levels of LH, steroidogenesis depends on chloride channels whereas with high levels, cyclic AMP is the mediator of LH action.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1990

The levels and possible involvement of leukotriene B4 and prostaglandin F2 alpha in the control of interstitial fluid volume in the rat testis.

The possible involvement of two arachidonic acid metabolites, prostaglandin F2 alpha (PGF2 alpha) and leukotriene B4 (LTB4), in the stimulatory effect of human chorionic gonadotrophin (hCG) on the volume of interstitial fluid (IF) in the rat testis has been investigated. Administration of hCG caused a time- and dose-dependent increase in the IF levels of PGF2 alpha while LTB4 showed no clear dose-dependence, but did decrease significantly at 2-8 h after injection of 100 IU hCG. Administration of ethane dimethane sulphonate (EDS), which specifically destroys Leydig cells, decreased the volume of IF but the IF levels of LTB4 and PGF2 alpha were unchanged. This indicates that although the absence of Leydig cells results in significant changes in IF volume, LTB4 and PGF2 alpha are probably not involved in these changes. Furthermore, these findings suggest that Leydig cells are not the only contributors to LTB4 and prostaglandins E2 and F2 alpha in testicular IF. In rats injected peripherally with hCG, intratesticular administration of the cycloxygenase inhibitor, indomethacin, decreased PGF2 alpha levels in IF after 2 and 4 h but not at later times. However, no inhibitory effect of indomethacin on the hCG-stimulated increase in IF volume was detected; indeed, at 2 h after injection of indomethacin + hCG there was a significant increase in IF volume in the indomethacin-injected testis. It is concluded that, although prostaglandins and LTB4 are secreted into IF in the rat testis, these arachidonic acid metabolites are apparently not the primary mediators of the increase in IF volume that follows hCG treatment.

Analysis of Variance↗
PubMed1989

Mechanisms of hormone-induced desensitization of adenylate cyclase.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to activate the formation of cyclic AMP via the regulatory GTP binding protein (Gs). This is followed by a desensitization of that same hormonal response which is caused by an uncoupling of the LH receptor from Gs. The coupling between Gs and the adenylate cyclase catalytic unit remains intact. Treatment of Leydig and other cell types with phorbol esters mimics hormone-induced desensitization. However, differences between hormone- and phorbol ester-induced desensitization have been found. In testis Leydig cells phorbol esters, as well as uncoupling the LH receptor from Gs, also inactivates the subunit of the inhibitory GTP binding protein (Gi). These studies suggested that activation of protein kinase may be involved in the hormone-induced desensitization of adenylate cyclase. Paradoxically, it has also been found that two inhibitors of protein kinase C, sphingosine and psychosine also inhibited LH-induced cyclic AMP production. These effects were mainly found during the initial stimulatory period with LH. It is suggested that activation of adenylate cyclase may require a protein kinase C-mediated phosphorylation step which is followed by further phosphorylation resulting in uncoupling of the receptor from Gs. No direct stimulation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), diacylglycerol and/or activation of protein kinase C by LH in Leydig cells has been demonstrated. An alternative mechanism of protein kinase C activation has been proposed for brain cells, i.e. that involving arachidonic acid activation of protein kinase C instead of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗