Control of multiple transducing systems by LH which results in the modulation of adenylate cyclase, protein kinase C, lipoxygenases and cyclooxygenases.
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Adenylyl Cyclases↗
Biomedical subjects
B A Cooke
Publications and source records attributed to B A Cooke.
The inhibitory GTP-binding protein (Gi) occurs in rat Leydig cells and is differentially modified by lutropin and 12-O-tetradecanoylphorbol 13-acetate.
Luteinizing-hormone (LH)-stimulated cyclic AMP production in rat testis Leydig cells was desensitized by both LH and 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA, but not LH, enhanced the subsequent response to cholera toxin. Treatment of the cells with pertussis toxin potentiated cyclic AMP production in both control and LH-desensitized cells, but did not potentiate further the responses obtained by TPA pretreatment. The results implicate the presence of an inhibitory GTP-binding protein (Gi), which may be inhibited by TPA. The presence of a Gi-like protein within the plasma membrane of Leydig cells was demonstrated by pertussis-toxin-catalysed [32P]ADP-ribosylation of a Mr-40000-41000 protein.
Adenosine Diphosphate↗
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↗
Immunoreactive human chorionic gonadotrophin from the cyst fluid and CSF of patients with craniopharyngioma.
A specific radioimmunoassay for the beta-chain of human chorionic gonadotrophin irHCG beta has demonstrated HCG-like material to be present in craniopharyngioma cyst fluid in nine consecutive patients with craniopharyngioma. There was no detectable LH/HCG bioactivity as assessed using testosterone production from isolated Leydig cells from rat testis in seven samples tested. One patient was also found to have measurable irHCG beta in the cerebrospinal fluid (CSF), which fell to undetectable levels following surgery; in this patient, there was clinical evidence that the cyst fluid had leaked into the CSF pre-operatively. Immunocytochemical staining for HCG beta and intact HCG was positive in five of the tumours. irHCG beta was not measurable in the serum of any of the patients.
Biological Assay↗
Similarities and differences in phorbol ester- and luteinizing-hormone-induced desensitization of rat tumour Leydig-cell adenylate cyclase.
The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was shown to mimic luteinizing hormone (LH; lutropin) in causing desensitization of LH-mediated cyclic AMP production in tumour Leydig cells. However, there were differences between LH- and TPA-induced desensitization: (1) TPA induced a more rapid effect than LH; (2) adenosine did not inhibit TPA-induced desensitization, whereas it completely inhibited the LH-induced desensitization; (3) adenylate cyclase activity in plasma membranes from TPA-desensitized cells was not decreased, whereas similar preparations from LH-desensitized cells lost their response to LH and to LH plus guanosine 5'-[beta gamma-imido]triphosphate; TPA-, but not LH-, treated cells had a decreased capacity to respond to cholera toxin and forskolin. These results indicate that LH and phorbol esters induce desensitization of adenylate cyclase in rat tumour Leydig cells by different mechanisms.
Adenosine↗
Effects of lutropin (LH) receptor internalization and recycling on the apparent numbers of LH receptors in rat testis Leydig cells at different temperatures.
The effect of different temperatures on the Scatchard analysis of binding studies with 125I-hCG and intact rat testis Leydig cells has been investigated. The results show that at temperatures greater than 4 degrees C an overestimation of the number of receptors/cell is likely to occur. A one-site analysis of the data gives values of 13,573, 61,924 and 3802 LH receptors/cell at 34 degrees C, 21 degrees C and 4 degrees C respectively. At 34 degrees C and 21 degrees C (but not at 4 degrees C) a two-site analysis of the data is possible, giving similar high affinity binding components at both temperatures (KD 1 X 10(-10) M) but with dissimilar low affinity binding components (34 degrees C, 2.17 X 10(-9) M and 21 degrees C, 6.3 X 10(-8) M). The calculated total number of LH receptors using a two-site analysis at 21 degrees C is equal to 114,766 receptors/cell, and at 34 degrees C it is 37,987 receptors/cell. It is proposed that the differences in the level of binding at different temperatures and the associated changes in the value of KD, reflect the temperature sensitivity of the endocytic pathway of the 125I-hCG/LH receptor complex within the rat testis Leydig cell. From previous studies it is known that at 34 degrees C and 21 degrees C, a large part of the cell-associated hormone is not receptor bound but rather it represents 'processed' hormone before it is released from the cell. At 4 degrees C the cell-associated 125I-hCG remains bound only to cell surface LH receptors and thus gives a more accurate measure of total receptor numbers/cell.
Animals↗
The dual regulation of cyclic AMP and calcium by luteinizing hormone and the mechanisms of desensitization and recycling of the luteinizing hormone receptor.
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Adenylyl Cyclases↗
The dynamics of LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells at physiological levels of LH.
The LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells in vitro has been investigated using sub-physiological to pharmacological concentrations of LH. Leydig cells pretreated with LH for 1 h in the presence of a phosphodiesterase inhibitor showed a dose-dependent decrease in the subsequent response to a high dose of LH; this was significant with concentrations of greater than 350 fmol/l. The maximum amount of desensitization was 60%. The time-course of LH-induced desensitization of the adenylate cyclase system was investigated; with 3 nmol LH/l cyclic AMP was increased in a linear manner up to 45 min, after which time there was either no further production or (with the higher concentrations) a decreased rate of production. In order to determine whether the 'non-desensitized' adenylate cyclase activity was dependent upon LH, the LH-treated cells were acid-washed to remove residual LH; cyclic AMP production still continued, albeit at a lower rate, thus indicating that this adenylate cyclase activity did not require the further presence of LH. The effect of various concentrations of LH on the level of surface-associated LH receptors was determined in the presence of monensin to prevent receptor recycling. A dose- and time-dependent decrease was found; this was significant after 2 h with 3.5 pmol LH/l and reached a maximum of 63% of the control with 3.5 nmol/l. A dose- and time-dependent reversal of desensitization occurred if the cells pretreated with LH were washed and reincubated; with 35 pmol LH/l and after 2 h the cells were fully responsive to a challenge with fresh LH. With higher concentrations of LH in the pretreatment, partial or no recovery was found. These studies demonstrate that physiological and sub-physiological concentrations of LH induce a rapid desensitization of Leydig cell adenylate cyclase. Internalization of occupied and unoccupied LH receptors also occurred. It is proposed that these two events are linked and may, paradoxically, and because of the low numbers of LH receptors, be necessary to maintain the normal response of Leydig cells to LH in vivo.
Adenylyl Cyclases↗
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↗
Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.
A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.
Animals↗
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↗
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↗
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↗
Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.
The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.
Adenosine↗
The mechanisms of LHRH agonist action in gonadal tissues.
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Animals↗
The functional activity of adult mouse Leydig cells in monolayer culture: effect of lipoproteins, pregnenolone and cholera-toxin.
Further studies were carried out on purified mouse Leydig cells to determine why they lose their hormone responsiveness after several days in monolayer culture. The effects of cholera-toxin on cyclic AMP and testosterone production were examined. It was found that cyclic AMP production could still be maximally stimulated by cholera-toxin after 4 days in culture when response to luteinizing hormone (LH) has declined. Testosterone production was, however, not maintained. Because this decline in testosterone production may have been due to the lack of a suitable substrate after several days in culture, cells were cultured initially in the presence of exogenous pregnenolone and low-density lipoproteins (LDL). Both substances were found to enhance basal and LH-stimulated testosterone production and to extend responsiveness of the cells until at least day 4, but by day 7 response was lost. Cells were then cultured in the presence of rat and human LDL and HDL and in both cases LDL was found to enhance consistently testosterone production, but HDL was much less effective. Scanning and transmission electron micrographs showed that changes in cell shape occurred during culture, but indicated that the cells were not depleted of lipid droplets by the end of culture or after LH stimulation. It is concluded that the eventual decline in testosterone synthesis is not due to lack of substrate, although the addition of exogenous substrate does extend the period of responsiveness. Nor is it due to a decrease in adenylate cyclase activity. At least part of the lesion is caused by a decrease in the enzymes required for the conversion of pregnenolone to testosterone.
Animals↗
Evidence for the involvement of lipoxygenase products in steroidogenesis.
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Animals↗
Endocytosis of beta-adrenergic ligands by rat liver. Comparison of beta-adrenergic receptor and adenylate cyclase distribution in endosome and plasma-membrane fractions.
The internalization of beta-adrenergic receptors was investigated in rat livers perfused with an agonist ([3H]isoprenaline) or an antagonist ([125I]iodocyanopindolol). Analytical centrifugation of liver homogenates indicated that the ligands were transferred rapidly to endosomal and lysosomal positions in sucrose gradients. Endosome fractions contained beta-adrenergic binding sites, but adenylate cyclase activity was low and poorly activated by isoprenaline. The results indicate that the receptor-regulatory-protein-adenylate cyclase complex was disassembled during uptake of beta-adrenergic ligands, with the adenylate cyclase being retained at the plasma membrane.
Adenylyl Cyclases↗