Biomedical subjects

M Ascoli

Publications and source records attributed to M Ascoli.

At least 73 records · Page 4Linked to original sources

PubMed1990

Studies with insulin and insulin-like growth factor-I show that the increased labeling of phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells.

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.

Animals↗

PubMed1989

The inositol phosphate/diacylglycerol pathway in MA-10 Leydig tumor cells. Activation by arginine vasopressin and lack of effect of epidermal growth factor and human choriogonadotropin.

It is now well established that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells but does not affect cell multiplication. The studies presented herein are the first in a series of experiments designed to characterize the intracellular signaling systems activated by mEGF and their possible roles in mediating the diverse biological actions of this growth factor in MA-10 cells. We show that (i) MA-10 cells express a hormone-sensitive inositol phosphate/diacylglycerol pathway that can be stimulated with arginine vasopressin (AVP), (ii) mEGF does not activate this pathway, and (iii) activation of this pathway with arginine vasopressin does not mimic the biological actions of mEGF. Other data presented show that lutropin/choriogonadotropin, the principal endocrine regulators of Leydig cell function, also do not stimulate the inositol phosphate/diacylglycerol pathway in MA-10 cells.

Animals↗

PubMed1989

Regulation of the differentiated functions of Leydig tumor cells by epidermal growth factor.

The three effects of mEGF on MA-10 Leydig tumor cells that have been discussed here are summarized in TABLE 7. The earliest effect of mEGF on MA-10 cells can be detected within 5 min of addition of mEGF and it lasts for about 60 min. During this time mEGF transiently attenuates hCG-stimulated adenylate cyclase activity. Although the magnitude of this effect is small, it can be correlated with a transient attenuation of the hCG-provoked increase in steroid synthesis. At longer times (i.e., 1-8 h) mEGF activates steroid synthesis by a "cAMP-independent pathway" and it potentiates (in a synergistic fashion) the activation of steroidogenesis by hCG, other compounds that activate adenylate cyclase activity, and cAMP analogues. At even longer times (i.e., 8-48 h) mEGF down-regulates the LH/CG receptors and by doing so, limits the steroidogenic response of the cells to hCG. From a biochemical point of view, our data provide an excellent example of those actions of growth factors that are unrelated to the control of cell multiplication, and of the complexity involved even when dealing with a single cell type and a single growth factor. Admittedly we know very little about the molecular basis of the phenomena described herein. Current work in our laboratory, however, is aimed at filling this gap. Among all the questions that we can address, we believe that it is particularly important to characterize the intracellular signaling system(s) activated by mEGF and to determine if a single signaling system is responsible for the diverse biological actions of mEGF in MA-10 cells. From a physiological point of view, our data may also prove important to the understanding of the regulation of testicular functions. There is increasing evidence for the production of EGF (or related peptides such as transforming growth factor alpha) in several tissues, including the testes and ovaries. These findings, together with the results summarized here suggest that EGF (or related peptides) act within the testes in a paracrine, or autocrine fashion and that they may have important modulatory effects on the activation of Leydig cell steroidogenesis by gonadotropins.

Adenylyl Cyclases↗

PubMed1989

On the structure of the luteinizing hormone/chorionic gonadotropin receptor.

In this review we have tried to argue that the evidence indicating that the LH/CG receptor is composed of a single polypeptide is stronger than the evidence indicating that the LH/CG receptor is a more complex structure composed of several subunits. Clearly, however, this issue has not been resolved and probably will not be resolved by performing additional experiments similar to those summarized here. It is our opinion that this issue will be resolved only by 1) reconstitution experiments in which the ability of the purified LH/CG receptor to bind hCG and activate adenylyl cyclase activity is tested; and/or 2) isolation and expression of a full length complementary DNA (cDNA) for the LH/CG receptor and the demonstration of hCG binding and adenylyl cyclase activation by the expressed receptor. Similar experiments will also clarify the proposed structures for the FSH and TSH receptors. As the second decade of work on the LH/CG receptor draws to an end it appears that these experiments are now possible, and hopefully a resolution of the existing controversy will be forthcoming in the near future.

Animals↗

PubMed1988

Inhibition of gonadotropin-responsive adenylate cyclase in MA-10 Leydig tumor cells by epidermal growth factor.

We have previously shown that mouse epidermal growth factor (mEGF) attenuates the increase in intracellular cAMP provoked by human choriogonadotropin (hCG) in MA-10 Leydig tumor cells (Ascoli, M., Euffa, J., and Segaloff, D. L. (1987) J. Biol. Chem. 262, 9196-9203). The studies presented herein were designed to investigate the mechanism(s) responsible for this phenomenon. We show that mEGF attenuates the increase in cAMP accumulation provoked by hCG primarily, if not entirely, by inhibiting adenylate cyclase activity. This phenomenon has some specificity for the agonist used, but it is not cell-specific. Thus, mEGF inhibited hCG-activated adenylate cyclase in MA-10 cells and in rat luteal cells but had no effect on the forskolin-activated enzyme in MA-10 cells or the isoproterenol-activated enzyme in rat luteal cells.

1-Methyl-3-isobutylxanthine↗

PubMed1988

Ca+2 is an inhibitor of adenylate cyclase in MA-10 Leydig tumor cells.

Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have examined the effects of Ca+2 on the activation of cAMP accumulation and steroid biosynthesis by hCG. Our results show that addition of Ca+2 ionophores (A23187 or ionomycin) leads to inhibition of the activation of cAMP accumulation by hCG. The magnitude of this effect is dependent on the concentrations of ionophore and hCG used, becoming more pronounced as the concentration of hCG increases. A detailed examination of the effects of A23187 and removal of extracellular Ca+2 on the rates of cAMP synthesis and degradation in intact cells revealed that A23187 inhibits the rate of cAMP accumulation activated by hCG, but does not affect the rate of degradation of cAMP. On the other hand, removal of extracellular Ca+2 had no effect on the rate of cAMP accumulation activated by hCG or on the rate of degradation of cAMP. Removal of extracellular Ca+2, however, completely prevented the inhibitory effect of A23187 on the rate of cAMP accumulation. Additional experiments show that the effects of A23187 or removal of extracellular Ca+2 on hCG-activated steroidogenesis closely parallel those described for cAMP accumulation. We conclude that Ca+2 is an inhibitor of the hCG-activated adenylate cyclase in Leydig tumor cells, and that this inhibition imposes a limitation on the ability of hCG to activate steroid biosynthesis.

Adenylyl Cyclase Inhibitors↗

PubMed1988

Characterization of an antiserum to the rat luteal luteinizing hormone/chorionic gonadotropin receptor.

The LH/CG receptor was purified from detergent extracts of rat luteal tissue using hCG affinity and wheat germ agglutinin chromatography. Analysis of the purified material by silver staining of sodium dodecyl sulfate-polyacrylamide gels (with or without reducing agents) revealed a prominent broad band corresponding to a 93K protein and several minor contaminants. That the 93K band represents the LH/CG receptor is supported by the following. 1) This band is absent in material purified from rat luteal tissue in which the LH/CG receptor had been down-regulated. 2) [125I]iodohCG binding to Western blots of both the initial detergent extract and the purified material resulted in binding to a 93K protein. The 93K protein representing the LH/CG receptor was excised after sodium dodecyl sulfate-gel electrophoresis of the purified material and was electroeluted. Half of the electroeluted receptor was incubated with reducing agents. After mixing with nonreduced receptor, this mixture was used to immunize one rabbit. Immune, but not preimmune, serum (or immunoglobulin G purified thereof) recognized a 93K protein on Western blots of partially purified (approximately 10% pure) rat luteal receptor. No other bands were seen. Using this approach it was determined that the antiserum recognized reduced, nonreduced, denatured, and native forms of the receptor. A polyclonal antibody to the LH/CG receptor will be useful for studies on the structure and regulation of this receptor.

Animals↗

PubMed1987

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP.

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.

1-Methyl-3-isobutylxanthine↗

PubMed1987

Inhibition of choriogonadotropin-activated steroidogenesis in cultured Leydig tumor cells by the Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate.

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.

1-Methyl-3-isobutylxanthine↗

PubMed1987

Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells. A 92-kDa Glycoprotein.

The structure of the lutropin/choriogonadotropin (LH/CG) receptor has been studied by immunoprecipitating the receptor from biosynthetically labeled cultured Leydig tumor cells (designated MA-10). This was performed by binding human choriogonadotropin (hCG) to the labeled cells, solubilizing the hormone-receptor complex, partially purifying the complex by lectin chromatography, and immunoprecipitating the complex with an antibody that recognizes receptor-bound hCG. The conditions used for the release of the radiolabeled receptor from the immunoprecipitate and the subsequent analysis of this material on sodium dodecyl sulfate gels allowed us to determine directly the structure of the free (not hormone-occupied) LH/CG receptor. From experiments using cells labeled with [35S]methionine and [35S]cysteine, we show that the LH/CG receptor is composed of a single polypeptide chain that migrates as a 92-kDa protein on sodium dodecyl sulfate gels whether analyzed in the absence or presence of reducing agents. Other studies presented demonstrate that the LH/CG receptor is a glycoprotein.

Cell Line↗

PubMed1987

On the fates of receptor-bound ovine luteinizing hormone and human chorionic gonadotropin in cultured Leydig tumor cells. Demonstration of similar rates of internalization.

Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the fate of the receptor-bound ovine LH (oLH) and human CG (hCG) in cells incubated in the presence or absence of extracellular Na+. We have previously shown that Na+ does not affect the number of LH/CG receptors or the binding affinity of hCG, but it decreases the binding affinity of oLH. Thus, it was possible to compare the fate of these two hormones under conditions where their binding affinities differ by a factor of 8 (i.e. in the presence of Na+) or by a factor of less than 2 (i.e. in the absence of Na+). Moreover, since only the affinity of oLH is affected by Na+, we were able to distinguish between those effects mediated by a change in binding affinity from those effects that are more general in nature by comparing the behavior of hCG in cells incubated in the presence or absence of Na+. The results presented herein show that the rates of internalization of oLH and hCG are very similar regardless of the presence or absence of Na+; and the absence of Na+ leads to a 2- to 3-fold decrease in the rate of degradation of the internalized oLH and hCG. We have found, however, that the binding affinities of oLH and hCG have significant effects on the pathway of receptor-mediated endocytosis under conditions where there is no free hormone present in the medium. The results presented show that in the absence of free hormone in the medium, the rate of hormone internalization can be approximated from the rate of disappearance of the surface-bound hormone only if the binding affinity of the hormone is high enough so that there is little or no dissociation of the hormone from the receptor during the course of the experiment (i.e. hCG in the presence or absence of Na+, but oLH only in the absence of Na+). If the binding affinity of the hormone is low (i.e. oLH in the presence of Na+), then the rate of disappearance of the surface-bound hormone represents the sum of the rates of internalization and dissociation of the hormone and thus cannot be used to approximate the rate of hormone internalization.

Ammonium Chloride↗

PubMed1986

Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor.

The structure of the lutropin/choriogonadotropin (LH/CG) receptor of a clonal strain of cultured Leydig tumor cells (designated MA-10) and primary cultures of porcine granulosa cells was studied by cross-linking 125I-labeled derivatives of human CG and ovine LH with bifunctional succinimidyl esters. We show that in both cell types, both subunits of the receptor-bound hormone become cross-linked to a single cellular component of Mr = 106,000, when analyzed in the absence of reducing agents, and of Mr = 83,000 when analyzed in the presence of reducing agents. We also present a detailed investigation on the effects of several collagenase preparations on the structure and some functions of the LH/CG receptor. Our results show that the LH/CG receptor is exquisitively sensitive to degradation by these preparations of collagenase; degradation products can be detected only in the presence of reducing agents; the enzyme(s) responsible for degradation is not collagenase itself, but rather a contaminating enzyme(s), presumably a protease(s); and receptor degradation has little effect on the ability of the cells to bind hormone or to respond with increased steroid biosynthesis. Since normal gonadal cells are usually isolated following dispersion of the tissue with collagenase, our results suggest that these cells are likely to bear a degraded (albeit functional) form of the LH/CG receptor, and thus should not be used in studies dealing with the structure of this receptor.

Animals↗

PubMed1985

Induction of renin activity by gonadotropic hormones in cultured Leydig tumor cells.

The hormonal regulation of renin activity in cloned and cultured Leydig tumor cells (designated MA-10) was examined. The treatment of Leydig cell cultures with bovine LH (bLH), hCG, or with (Bu)2cAMP elicited a dose- and time-dependent induction of renin activity and a concomitant increase in steroid biosynthesis. The optimum concentration of hCG was 25 ng/ml, which caused an average 25-fold increase in renin activity compared to the control value. bLH action was optimum at 75-100 ng/ml and induced an approximately 35-fold increase in renin activity. The maximum inducible level of renin activity was attained after 8-9 h of hormone treatments. The addition of progesterone (the major steroid product of the MA-10 cells) did not induce a significant increase in renin activity. Treatment of MA-10 cells with epidermal growth factor also failed to produce any increase in renin activity. The optimum concentration of (Bu)2cAMP was 800 microM for the induction of renin activity and caused an approximately 40-fold increase compared to the control value. Renin activity induced by bLH, hCG, or (Bu)2cAMP was completely inhibited by mouse anti-renin antibody, indicating the specific nature of renin. Upon withdrawal of (Bu)2cAMP from the culture medium, renin activity gradually declined to the control level, and with retreatment of these cultures with (Bu)2cAMP, a newly induced state of enzyme activity was resumed. Indirectly, the role of new protein and RNA synthesis was examined during hormonal regulation of renin induction using protein and RNA synthesis inhibitors such as cycloheximide, puromycin, actinomycin D, or rifampicin. Both protein and RNA synthesis inhibitors blocked the induction of renin activity in the presence of all three inducing agents, bLH, hCG, or (Bu)2cAMP. The results provide evidence that the induction of renin activity is modulated by bLH, hCG, or (Bu)2cAMP and represent the de novo synthesis of enzyme molecules.

Angiotensin I↗

PubMed1984

Na+ modulates the affinity of the lutropin/choriogonadotropin receptor.

The studies presented herein were designed to assess the effects of Na+ on the affinity of the lutropin/choriogonadotropin (LH/CG) receptor. We performed competition binding experiments in buffers containing NaCl or an isosmolar concentration of an appropriate Na+ substitute. The results presented show that Na+ decreases the affinity of LH, but not that of CG, in intact cells and membranes. In detergent-solubilized receptors, however, the affinity of LH is higher than that of CG and is not influenced by Na+. In addition, we show that Na+ does not affect the number of LH/CG receptors.

Animals↗

PubMed1984

Lysosomal accumulation of the hormone-receptor complex during receptor-mediated endocytosis of human choriogonadotropin.

The experiments presented herein were designed to determine the fate of the human choriogonadotropin (hCG) receptor during endocytosis of the receptor-bound hCG. Using several biochemical approaches, it is shown that the receptor is internalized together with the hormone into endocytic vesicles and transferred to lysosomes without ligand dissociation. Once delivered to the lysosomes, the hCG-receptor complex dissociates, and the free hormone is degraded. This pathway appears to prevent receptor recycling and probably promotes receptor degradation.

Cell Fractionation↗

PubMed1983

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis.

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.

Binding Sites↗

PubMed1983

On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells.

The MA-10 cells are a clonal strain of mouse Leydig tumor cells that have receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). Exposure of the cells to hCG results in a reduction in the number of surface hCG receptors, and little or no change in the number of surface mEGF receptors. On the other hand, exposure of the cells to mEGF results in a reduction in the number of both surface mEGF receptors and surface hCG receptors. In order to study these phenomena, we assumed that the number of surface receptors is determined by the rate at which receptors appear at the surface and by the rate of receptor internalization. When these rates were measured, we found that hCG and mEGF reduce their respective surface receptors by increasing the rate of receptor internalization, and that mEGF reduces the surface hCG receptors by decreasing the rate of appearance of the receptor.

Animals↗

PubMed1983

Inhibition of steroidogenesis in cultured Leydig tumor cells by 22-amino-23,24-bisnor-5-cholen-3 beta-O1 and (20R) 20-phenyl-5-pregnene-3 beta,20-diol.

Using the cholesterol side-chain cleavage enzyme purified from bovine adrenals, we have previously shown that 22-amino-23,24-bisnor-5-cholen-3-beta-ol (22-ABC) and (20R) 20-phenyl-5-pregnene-3 beta, 20 diol (20-PPD) are potent competitive inhibitors of this enzyme. The studies presented herein were designed to characterize the effects of these new inhibitors on steroid production by intact cells. Using cultured Leydig tumor cells, we compared the effects of 22-ABC, 20-PPD, and aminoglutethimide (a well-known inhibitor of the cholesterol side-chain cleavage enzyme) on hormone-stimulated steroidogenesis. Our results show that these compounds inhibit steroid production in a dose-dependent manner and that 20-PPD and 22-ABC are about 100 and 4 times more active, respectively, than aminoglutethimide. In these cells, the inhibitory effects of the new compounds seem to be localized exclusively at the conversion of cholesterol to pregnenolone.

Aminoglutethimide↗