Endothelin-1 promotes steroidogenesis and stimulates protooncogene expression in transformed murine Leydig cells.

The effects of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen to various cell types, on proliferation and differentiated functions of the murine Leydig tumor cell line MA-10 were investigated. Kinetic binding experiments at room temperature showed that [125I] ET-1 bound to MA-10 cells and reached equilibrium in 2 h. The data from competitive binding experiments yielded an apparent single class of high affinity binding sites characterized by a Kd and maximum binding capacity of 1 nM and 59 fmol/10(6) cells, respectively. For steroidogenic assays, cells were incubated with ET-1 (1 pM to 1 microM) and with epidermal growth factor (10 ng/ml) for 4 h at 37 C, and the progesterone levels in the medium were measured by RIA. Like epidermal growth factor, ET-1 caused about a 6-fold increase in progesterone production. ET-1 also enhanced the transient expression of the protooncogenes c-jun and c-myc by 3- and 2-fold, respectively. For proliferation studies, ET-1 (1 pM to 1 microM) was added to quiescent MA-10 cells for 24 h, and cell counts were performed; no increase in cell number was observed. The results of this study demonstrate that MA-10 cells possess high affinity binding sites for ET-1 and that ET-1 stimulates progesterone production and protooncogene expression, but not mitosis in this cell line.

Epidermal growth factor modulates intracellular arachidonic acid levels in MA-10 cultured Leydig tumor cells.

Epidermal growth factor (EGF) acts on various cell types, including the mouse Leydig tumor cell line MA-10, where it has been shown to stimulate steroidogenesis, apparently in a cAMP-independent manner. In the process of examining other possible signaling pathways for EGF in these cells, we found rapid changes in the intracellular concentration of arachidonic acid (AA) following addition of EGF. For example, a significant increase in AA was detected 1 min after incubating the cells with EGF, with the maximal effect observed at an EGF concentration of 10 ng/ml. In addition, exogenous AA increased steroidogenesis, and the steroidogenesis enhanced by AA and EGF was reduced by lipoxygenase inhibitors, suggesting a possible role of an AA metabolite(s) in promoting steroidogenesis. Consistent with this hypothesis is our observation that several exogenous lipoxygenase metabolites were capable of enhancing progesterone production. The EGF-stimulated steroidogenesis was also inhibited by two phospholipase A2 inhibitors, again confirming a probable role of AA or a metabolite in this process. Therefore, AA appears to be an important intracellular mediator responsible, at least in part, for some of the acute metabolic effects mediated by EGF in MA-10 cells.

Immunoreactive endothelin-1 concentrations in follicular fluid of women with and without endometriosis undergoing in vitro fertilization-embryo transfer.

OBJECTIVE: To determine the concentrations of immunoreactive (IR) endothelin-1 in human follicular fluid (FF) and whether IR-endothelin-1 levels are different in women with endometriosis-associated infertility. DESIGN: Follicular fluid and plasma samples, obtained from women with and without endometriosis undergoing IVF-ET, were collected at the time of oocyte aspiration and analyzed for IR-endothelin-1 levels. SETTING: Infertility clinic in an academic research environment. RESULTS: Overall, 90% of FF samples and 60% of plasma samples contained IR-endothelin-1 detectable above the threshold of assay sensitivity. Immunoreactive endothelin-1 levels (mean +/- SEM) in FF samples from women with and without endometriosis-associated infertility were 74 +/- 12 and 37 +/- 6 pg/mL, respectively. There was no difference in IR-endothelin-1 levels in FF samples between controlled ovarian hyperstimulation cycles with or without leuprolide acetate. No significant differences were detected in plasma IR-endothelin-1 levels in women with endometriosis-associated infertility when compared with those without. CONCLUSIONS: These results demonstrate the presence of IR-endothelin-1 in human FF obtained at the time of oocyte aspiration for IVF-ET and higher levels of IR-endothelin-1 in FF of women with endometriosis-associated infertility.

Effects of hormones and intracellular mediators on differentiated functions of cultured Leydig tumor cells.

Cellular regulation by hormones that utilize a myriad of intracellular signaling pathways is recognized to be quite complex. To investigate some of these effects in an established cell line, we tested a panel of hormones and modulators for their effects on cyclic AMP (cAMP) and progesterone production, both alone and in combination with human chorionic gonadotropin (hCG), using the MA-10 cultured Leydig tumor cell line. None significantly affected intracellular levels of cAMP, and only epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated progesterone production. While EGF, basic fibroblast growth factor, insulin, insulin-like growth factor-1, and transforming growth factor beta all decreased cAMP production only, TPA decreased hCG-stimulated cAMP and progesterone production. Those factors that stimulated progesterone production also induced a characteristic morphological change ("rounding") of these cells. In addition, EGF, insulin, and TPA, like hCG, elevated mRNA levels of competence oncogenes (c-fos and c-myc), albeit to different extents. These data demonstrate the wide range of hormones to which the cultured Leydig tumor cell will respond, as well as the varying degree of responses observed in the intracellular signaling pathways that we examined.

Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps.

Insulin receptor capping and its correlation with calmodulin-dependent myosin light chain kinase.

Both fluorescence microscopy and fluorometric analysis techniques have been used to characterize insulin receptor capping in IM-9 human lymphoblastoid cells. Morphologically, insulin caps appear similar to lectin or antiimmunoglobulin-induced caps displaying a preferential accumulation of actin, myosin, and actin-binding protein directly underneath the cap structure. Using the fluorescent calcium indicator quin2 we have detected no change in the calcium activity following insulin stimulation. However, in the presence of a number of calmodulin inhibitors, such as W-5, W-7, W-12, and trifluoperazine (TFP), insulin capping is significantly inhibited, which implies that a calmodulin-regulated process is involved. Using double immunofluorescence microscopy, we have found that the calmodulin-dependent myosin light chain kinase (MLCK) is concentrated directly beneath insulin caps. Upon treatment with trifluoperazine (TFP), the redistribution of both MLCK and insulin receptors are inhibited concomitantly. Our data indicate that the calmodulin-dependent myosin light chain kinase may be directly responsible for the activation of actomyosin-mediated contractility during insulin receptor capping.

Effect of cyclic AMP on human blood platelets: induction of pseudopod formation and protein phosphorylation.

A variety of techniques, including immunofluorescence, electron microscopy and biochemical analysis, were used to examine shape changes and cytoskeletal reorganization of human blood platelets during treatment with N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and agent known to elevate the intracellular level of cyclic AMP (cAMP). Cytochemical analysis shows that the unstimulated platelets have a discoid shape with no obvious membrane projections. Platelets treated with dbcAMP produce pseudopod-like structures containing cytoskeletal proteins such as actin and microtubules. Biochemical analysis reveals that a 125,000 dalton phosphoprotein (P-125) is preferentially recruited into cytoskeletal fractions of platelets treated with dbcAMP. This protein, which is one of the substrates for cAMP-dependent kinase(s) and/or is closely associated with the cytoskeleton, may play an important role in regulating the shape changes and cytoskeletal reorganization that occur during the early stages of platelet activation.

Lymphocyte activation and capping of hormone receptors.

In this study both a ligand-dependent treatment [concanavalin A (Con A)] and a ligand-independent treatment [high-voltage pulsed galvanic stimulation (HVPGS)] have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interleukin-2 (IL-2)] receptors within the first 5 min of stimulation. When either insulin or IL-2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intracellular Ca2+ activity; (2) aggregation of insulin or IL-2 receptors into patch/cap structures; (3) tyrosine-kinase-specific phosphorylation of a 32-kd membrane protein; and finally (4) induction of DNA synthesis. Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W-5, W-7, and W-12 drugs, implying a need for Ca2+/calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine-kinase-specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca2+/calmodulin, and myosin light-chain kinase are all closely associated with the insulin and IL-2 receptor cap structures. These findings strongly suggest that an actomyosin-mediated contractile system (regulated by Ca2+, calmodulin, and myosin light-chain kinase in an energy-dependent manner) is required not only for the collection of insulin and IL-2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for the activation of mouse splenic T-lymphocytes.

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase.

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.

A label selection approach to assess the role of individual amino groups in human choriogonadotropin receptor binding.

Human choriogonadotropin (hCG) was trace-labeled with [3H]acetic anhydride and then incubated with transformed murine Leydig cells (MA-10). The bound hormone was recovered, subunits (alpha and beta) were separated and then cleaved, and the peptides were purified by high performance liquid chromatography. Analysis of the labeling patterns of peptides from the initial preparation and the bound hCG fraction enabled us to determine the protection factor of several amino groups, which is a measure of the effects of acetylation on receptor binding. The largest protection factors, indicating lower labeling in the bound fraction, were found on beta and involved the alpha-amino group/Lys2 (analyzed together) and Lys104, which exhibited 6-fold and 5-fold selections against binding, respectively. Thus, acetylation at either of these amino groups does not prevent binding but results in selection against hormone association with receptor. Other amino groups were analyzed (e.g. Lys122 on beta; the alpha-amino group and lysines 44/45 (analyzed as a pair), 51, and 75 on alpha), and the selection factors indicated either no change or only modest changes (2-fold) in selection for or against binding. These results indicate that the alpha-amino group/Lys2 and Lys104 of the hormone-specific beta subunit participate, either directly or indirectly, in receptor binding.