Lutropin is processed much more rapidly than human choriogonadotropin by porcine Leydig cells in primary culture

Lutropin (LH) and human choriogonadotropin (hCG) share the same receptor and stimulate testosterone production in porcine Leydig cells in primary culture. Cells were pulsed with [125I]LH or [125I]hCG. During the chase, more than 80% of cell-bound LH consisted in internalized material which was degraded and excreted (half-time: 25 min) NH4Cl largely inhibited this degradation. On the contrary, hCG remained essentially bound to the cell surface and was not degraded by the cells with or without NH4Cl up to 160 min.     

Internalization and degradation of receptor bound C-reactive protein by U-937 cells: induction of H2O2 production and tumoricidal activity

The presence of a membrane receptor for C-reactive protein (CRP-R) on the human monocytic cell line U-937 was the basis for determining the metabolic fate of the receptor-bound ligand and the functional response of the cells to CRP. Internalized [125I]CRP was measured by removing cell surface-bound [125I]CRP with pronase. Warming cells to 37 degrees C resulted in the internalization of approx. 50% of the receptor-bound [125I]CRP or receptor-bound [125I]CRP-PC-KLH complexes. U-937 cells degraded about 25% of the internalized [125I]CRP into TCA-soluble radiolabeled products. The lysosomotrophic agents (chloroquine, NH4Cl) greatly decreased the extent of CRP degradation without altering binding or internalization. In addition, a pH less than 4.0 resulted in dissociation of receptor-bound [125I]CRP. Treatment of U-937 cell with monensin, a carboxylic ionophore which prevents receptor recycling, resulted in accumulation of internalized [125I]CRP. Therefore, it appears that the CRP-R complex is internalized into an endosomal compartment where the CRP is uncoupled from its receptor and subsequently degraded. CRP initiated the differentiation of the U-937 cells so that they acquired the ability to produce H2O2 and also display in vitro tumoricidal activity. The results support the concept that internalization and degradation of CRP leads to the activation of monocytes during inflammation.     

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.     

Modulation of mannose receptor activity by proteolysis.

The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.     

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.     

Dual internalization pathway for lutropin and choriogonadotropin in porcine Leydig cells in primary culture

The role of the high affinity receptor in the internalization of porcine lutropin (pLH) and human choriogonadotropin (hCG) by porcine Leydig cells in primary culture during short-term stimulation by the two hormones was investigated. The fate of the hormones was followed either by electron microscopy (with colloidal gold-labeled hormones) or by measurement of the cellular distribution of [125I]pLH and [125I]hCG. With both techniques, the internalization of pLH was found to be one order of magnitude greater than hCG, though the recycling rate of the high affinity receptors was the same with both hormones. However, when the cell surface was progressively depleted of its high affinity receptors by preincubation with increasing doses of hCG or pLH, the internalization of [125I]pLH remained high and largely independent of the number of high affinity receptors still available on the cell surface, while that of [125I]hCG was found to be proportional to this number. The endocytosis of [125I]pLH could only be inhibited by the simultaneous presence of micromolar concentrations of unlabeled pLH, hCG or alpha or beta subunits of ovine LH (oLH). The intact alpha-hCG subunit and the deglycosylated alpha-oLH subunit were less potent, while beta-hCG and deglycosylated beta-oLH had no significant effect. These results could be explained by the existence of a "carrier" or "scavenger" receptor for LH, but with a low affinity (congruent to 3.10(6) M-1) and present in excess on the cell surface as compared to the high affinity receptor. The possible physiological significance of this receptor is discussed.     

Porcine follicular fluid containing water-soluble LH/hCG receptor

Porcine follicular fluid has been shown to have a specific water-soluble receptor for human chorionic gonadotropin (hCG). The binding of [125I] hCG to follicular fluid is inhibited by unlabelled hCG, LH but not FSH, ACTH and GH. The binding of hormone to the receptor in follicular fluid is a saturable phenomenon and Scatchard analysis suggested that the receptor has high affinity to hCG with no changes as the follicle enlarges. In contrast, follicular fluid from large follicles (6-12 mm) has higher binding capacity (2.04 +/- 0.12 fmol/mg protein) than follicular fluid isolated from medium (3-5 mm) and small (1-2 mm) follicles (0.60 +/- 0.05 and 0.44 +/- 0.04 fmol/mg protein, respectively). With the aid of affinity chromatography on hCG-CNBr-Sepharose 6-B a homogeneous fraction with Mr about 65,000 as estimated by SDS-PAGE was isolated. Treatment of follicular fluid with several protein-modifying reagents changed interactions of [125I] hCG with both soluble receptor and that bound to granulosa cell membrane in the similar manner. The [125I] hCG binding capacity of follicular fluid represents about 9.5% of the total binding capacity of granulosa cells. Finally, soluble LH/hCG receptor is probably secreted actively by follicular cells into follicular fluid. Dead granulosa cells do not release receptor into follicular fluid or incubation medium.     

The processing of receptor-bound [125I-Tyr11]somatostatin by RINm5F insulinoma cells

The peptide somatostatin (SRIF) is secreted by delta cells of the endocrine pancreas and inhibits the secretion of insulin from pancreatic beta cells. We have previously shown that [125I-Tyr11]SRIF binds to specific, high affinity receptors on RINm5F insulinoma cells and that these receptors mediate the action of SRIF to inhibit insulin release. In the present study we investigated the processing of receptor-bound [125I-Tyr11]SRIF in this clonal cell line. Surface-bound and internalized peptides were distinguished by the ability of an acid/salt solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to dissociate only exposed ligand-receptor complexes. Surprisingly, greater than 80% of saturably bound [125I-Tyr11]SRIF was removed by this acid wash independent of the time or temperature of the binding incubation. In contrast, the processing of receptor-bound [125I]EGF (epidermal growth factor) in RINm5F cells was markedly temperature-dependent. Although over 90% of saturably bound [125I]EGF was dissociated by acid after a 4 degrees C binding incubation, less than 10% was removed by acid treatment after 37 degrees C binding. The radioactivity released upon dissociation of receptor-bound [125I-Tyr11]SRIF was analyzed by high performance liquid chromatography and shown to consist of a mixture of intact peptide (40%) and [125I]tyrosine (60%). However, neither the rate of [125I-Tyr11]SRIF dissociation nor its degradation were affected by NH4Cl, methylamine, or leupeptin at concentrations which inhibited the lysosomal degradation of [125I] EGF. Of 11 other protease inhibitors tested, only the metalloendoprotease inhibitor, phosphoramidon, substantially reduced the degradation of receptor-bound [125I-Tyr11]SRIF. These data indicate that, unlike [125I] EGF, receptor-bound [125I-Tyr11]SRIF is not rapidly internalized by RINm5F cells and is degraded by a nonlysosomal process which may involve a metalloendoprotease.     

Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumour cells (MLTC-1)

The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumour cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with [125I]hCG indicated that the bound hormone was lost much more rapidly from the tumour cells than from the luteal cells (t1/2 = 4.5 and greater than 12 h, respectively). The tumour cells were also found to internalise and degrade the hormone more effectively than the luteal cells, as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloroacetic acid (TCA)-soluble label in the medium. In MLTC-1 cells, over 80% of the radioactivity released was TCA-soluble at all times examined, whereas in the luteal cells most (65-75%) was TCA-precipitable. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-[125I]hCG complexes (Mr 96,000 and 74,000) (Kellokumpu & Rajaniemi, Endocrinology 116 (1985) 707) into the medium, although their amount was negligible in MLTC-1 cells. Possibly, due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the Mr 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalised receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumour cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.     

Degradation of erythrocyte-microinjected and scrape-loaded homologous cytosolic proteins by 3T3-L1 cells.

Homologous cytosol was introduced into 3T3-L1 cells by two different methods. Erythrocytes loaded with radiolabelled cytosolic proteins extracted from 3T3-L1 cells were fused with the aid of Sendai virus to 3T3-L1 cells, which were then seeded to confluent and non-confluent cultures. Cytosolic proteins were also introduced into cells by the technique of scrape-loading. In confluent cells, injected cytosolic proteins were recovered largely (54-93%) in a sedimentable (6 X 10(6) gav.-min) fraction from recipient cells irrespective of the method of introduction or of radiolabelling of the injected proteins [( 125I]iodination, reductive methylation with NaB3H4 and backbone labelling with L-[4,5-3H]leucine). The degradation of microinjected cytosolic proteins was in all cases inhibited by the lysosomotropic agent NH4Cl to a greater extent (32-75%) than that observed for endogenous cytosolic (less than or equal to 19%) proteins (labelled with L-[4,5-3H]leucine). In growing cells both endogenous total cell proteins and microinjected proteins were degraded at a slower rate than in confluent cell monolayers. The inhibition by NH4Cl of the degradation of both the endogenous and microinjected proteins is decreased compared with the inhibition observed in confluent monolayers. The results are discussed in terms of the cytoplasmic capacity to segregate microinjected homologous proteins before protein degradation can take place.     

Multiple inhibitory effects of a luteinizing hormone-releasing hormone agonist on hCG-dependent steroidogenesis and on FSH-dependent responses in ovarian cells in vivo and in vitro

[125I] labelled [D-Leu6, des-Gly-NH10(2)] LH-RH ethylamide (LH-RHa), when injected into immature female rats, bound specifically not only to the pituitary but also to the ovaries. LH-RHa inhibited hCG-stimulated progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-induced ovarian hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by LH-RHa. Progesterone production by rat luteal cells in vitro was inhibited by LH-RHa. LH-RHa did not change the affinity or population of LH/hCG receptor in porcine granulosa cells in short term incubation. However, LH-RHa inhibited induction of LH/hCG receptor stimulated by FSH and insulin in long term culture of porcine granulosa cells. LH-RHa delayed hCG-stimulated cyclic AMP accumulation in porcine granulosa cells. These findings suggest that LH-RHa inhibits hCG-stimulated cyclic AMP accumulation and subsequent progesterone production as well as FSH-stimulated LH/hCG receptor induction by acting directly on ovarian cells.     

Synergistic effect of insulin and follicle-stimulating hormone on biochemical and morphological differentiation of porcine granulosa cells in vitro

Insulin and follicle-stimulating hormone (FSH) have been shown to facilitate granulosa cell differentiation in vitro. To gain insight into this process, we evaluated the effects of these hormones, alone and in combination, upon the biochemical parameters of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction and progesterone secretion concomitantly with morphometric analysis of granulosa cell ultrastructure and LH/hCG receptor distribution by quantitative autoradiography under light microscopy. Granulosa cells isolated from small antral follicles (controls) cultured in the absence of exogenous hormones exhibited few microvilli and gap junctions; the mitochondria, endoplasmic reticulum, and Golgi complex were all poorly developed. Progesterone secretion was negligible and the cells bound little [125I]iodo-hCG. Insulin treatment increased gap junction formation, and the extent of smooth and rough endoplasmic reticulum and Golgi complex development (all p less than 0.05) but did not affect mitochondrial ultrastructure or volume. Insulin treatment modestly but significantly increased [125I]iodo-hCG binding and progesterone secretion relative to controls (p less than 0.001). FSH treatment had a similar effect to insulin on cell ultrastructure and additionally enhanced development of the mitochondria and smooth endoplasmic reticulum as well as formation of the microvilli (p less than 0.05). FSH significantly increased [125I]iodo-hCG binding and progesterone secretion relative to insulin-treated samples (p less than 0.001). Combined treatment with insulin and FSH markedly increased gap junction and microvilli formation and enhanced the development of the smooth endoplasmic reticulum and the Golgi complex relative to treatment with either hormone alone (p less than 0.05). Additionally, the combined treatment produced larger mitochondria with tubular christae. Consistent with the morphological development, the combined treatment of insulin and FSH significantly increased progesterone secretion and [125I]iodo-hCG binding (p less than 0.001). Autoradiographic analysis showed that aggregated cells in general exhibited higher LH/hCG receptor density than nonaggregated cells, and a significantly higher overall receptor density compared to nonaggregated cells or to cells treated either with insulin or FSH alone. Our results indicate that insulin and FSH facilitate morphological differentiation of the granulosa cell in a synergistic manner, stimulating gap junctions and microvilli formation and enhancing development of the mitochondria, endoplasmic reticulum, and Golgi complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

A transplantable rat Leydig cell tumor--5. Cellular localization of beta-adrenergic and prostaglandin receptors and their effects on testosterone production

The cellular localization of beta-adrenergic and prostaglandin (PG) receptors and their effects on adenylate cyclase activity (AC) and testosterone production in vitro were investigated in a transplantable rat Leydig cell tumor (H-540). Separation of the tumor cells in Percoll gradients revealed that the specific binding of [3H]PGE1 and [125I]Cyanopindolol was found in the same fraction as that of [125I]LH. This fraction--judged by light microscopy of smears--consisted of tumor Leydig cells. In addition, [125I]cyanopindolol was found specifically bound in the red blood cell fraction. In the Leydig tumor cells, approx 25% of the beta-adrenergic receptors was identified as beta 1-receptors, whereas approx 75% of the receptors were of the beta 2-subtype. The AC in Percoll purified Leydig tumor cells was stimulated by hCG (6-fold), PGE1 (2-fold), PGE2 (1.5-fold), PGI1 (2-fold) and isoproterenol (2-fold). The AC in the red blood cell fraction was stimulated by isoproterenol whereas the PGs and hCG had little or no effect. hCG, isoproterenol and PGE1 were able to stimulate testosterone production in vitro. At 44 h incubation, PGE1 was the most potent stimulator of testosterone production. In conclusion, tumor Leydig cells possess hCG, PGE1, PGI2 and beta-adrenergic receptors coupled to the AC. PGE1 and beta-adrenergic agonists stimulate testosterone production after prolonged incubation in vitro.     

Retention of glucose on oligosaccharide chains linked to the LH/hCG receptor prevents cell surface expression

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [(125)I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.     

The hemopexin receptor on the cell surface of human polymorphonuclear leukocytes

Promyelocytic leukemia HL-60 cells can be induced to differentiate to granulocytes, under the conditions of cultures in the presence of dimethyl sulfoxide (DMSO). Examination of the binding of 125I-labeled hemopexin to DMSO-induced HL-60 cells showed that the density of hemopexin receptors on the induced-cells was 1.35 times that on the uninduced cells. We proposed that a specific receptor for hemopexin was present on the plasma membranes of polymorphonuclear leukocytes (PMNs). The binding of human [125I]hemopexin to human PMNs at 4 degrees C was saturable with time and with increasing concentrations of [125I]hemopexin. Scatchard analysis of the binding revealed the presence of approximately 5.7 x 10(4) binding sites per cell with an apparent dissociation constant (Kd) of 2.3 x 10(-9) M. [125I]Hemopexin was rapidly bound then dissociated from the cells after the release of heme, when the cells were incubated with radioactive hemopexin at 37 degrees C. Incubation of the cells with the [59Fe]heme-hemopexin complex resulted in an accumulation of [59Fe]heme in the cells, with a temperature of 37 degrees C but not that of 4 degrees C. Ouabain or NaF inhibited not only the binding of [125I]hemopexin to PMNs but also the uptake of [59Fe]heme from [59Fe]heme hemopexin by the cells. Neither NH4 Cl nor chloroquine inhibited the uptake. Detergent extracts of 125I-labeled PMNs were incubated with a hemopexin-coupled Sepharose CL-6B. A polypeptide reacting with hemopexin-Sepharose was estimated to have a molecular weight of 80,000, as determined by polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. We propose that PMNs take up heme from hemopexin, as mediated by the 80,000 dalton receptor for hemopexin.     

Direct regulating effects of transforming growth factor beta on the Leydig cell steroidogenesis in primary culture

The effect of transforming growth factor beta on testicular steroidogenesis was studied by using a model of immature porcine Leydig cells cultured in a chemically defined medium. Leydig cells were cultured in the presence of human or porcine purified TGF beta and the following parameters were measured: cell proliferation, LH/hCG binding, and hCG-stimulated steroid hormone productions (DHEA, DHEAS and testosterone). Whereas TGF beta from the two sources had no effect on Leydig cell multiplication, it markedly inhibited LH/hCG-stimulated DHEA and DHEAS in a time- and dose-dependent manner. The maximal inhibitory effect of this peptide on LH/hCG binding (65% decrease), hCG-stimulated DHEA (77% decrease) and DHEAS (92% decrease) productions was observed with 2 ng/ml for 48 h of treatment. In contrast, TGF beta exerted a biphasic effect on hCG-stimulated testosterone production: stimulating (110% increase) until 2 ng/ml and inhibiting (35% decrease) for higher concentrations. [125I]TGF beta was cross-linked to Leydig cells using disuccinimidyl suberate; cells affinity labelled with [125I]TGF beta exhibit a major labelled band of approx 280 kDa, which has the properties expected from a TGF beta receptor. These data demonstrate that TGF beta is a direct potent regulator of Leydig cell steroidogenic function and its effects are probably mediated via a specific receptor.     

Actions of LH/hCG on accumulation and catabolism of progestins in cultured rat granulosa cells

Immature hypophysectomized rats were treated with estradiol-17 beta and follicle-stimulating hormone. Granulosa cells were isolated and incubated for 24 h with or without varying doses of ovine luteinizing hormone (NIMADD-oLH-24) or human chorionic gonadotropin (NIADDK CR 125) and accumulations of progesterone and 20 alpha-hydroxy-4-pregnen-3-one were determined. The cells were reincubated for 3 h with [4-14C]progesterone (0.5 nmol/mL) and the radiolabelled metabolites were separated and quantified. Both LH (0.04-1.0 ug/mL) and hCG (0.04-1.0 ug/mL) enhanced the accumulation of endogenous progesterone (by up to 300 and 150%, respectively) and 20 alpha-hydroxy-4-pregnen-3-one (by up to 90 and 85%, respectively) producing dose-dependent increases of the ratio of progesterone to 20 alpha-hydroxy-4-pregnen-3-one (by up to 125 and 70%, respectively). Studies of the metabolism of [1-14C] progesterone have demonstrated that both LH and hCG led to a dose-dependent decrease of the utilization of radiolabelled progesterone (down to 64 and 70%, respectively, of the control value). This effect was associated with an LH- and hCG-dependent inhibition of 20 alpha-hydroxysteroid dehydrogenase activity (down to 60 and 70%, respectively, of the control value) but had no significant effect on 5 alpha-reductase. The present results indicate that LH and hCG stimulate accumulation of progesterone at least in part by decreasing the 20 alpha-hydroxysteroid dehydrogenase activity.     

Role of gangliosides in gonadotropin and cholera enterotoxin stimulated steroidogenesis in isolated rat ovarian cells

Ovarian cells isolated from 26 day old rats responded to hCG (10 ng/ml) and cholera enterotoxin (100 ng/ml) $\text{in vitro}$ with a forty-five to fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration of gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20–30 minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under identical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [¹²⁵I]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [¹²⁵I]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.     

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.     

Effect of thyroxine-binding globulin (TBG) on thyroxine (T4) uptake by human peripheral mononuclear cells: evidence of TBG-dependent uptake of T4

The effect of sialylated TBG and desialylated TBG on thyroxine (T4) uptake by human peripheral mononuclear cells was investigated in vitro. [125I]-T4 uptake was observed when the cells were incubated with free [125I]-T4. The uptake was inhibited in a concentration dependent manner when TBG was added. During the incubation, [125I]-T4 binding to TBG was observed. [125I]-T4 incorporation into cells was also observed when the cells were incubated with [125I]-T4-sialylated TBG or with [125I]-T4-desialylated TBG complex. The uptake was related to the temperature and length of time of the incubation. The amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-sialylated TBG was greater than that into the cells incubated with [125I]-T4-desialylated TBG during the early 0-20 min. incubation, whereas the amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-desialylated TBG became greater than that into the cells incubated with [125I]-T4-sialylated TBG after 20 min. of incubation. Pretreatment of the cells with methylamine blocked [125I]-T4 uptake in both cases, i.e. incubated with [125I]-T4-sialylated TBG and incubated with [125I]-T4-desialylated TBG. The results suggest that TBG plays a role not only as a carrier protein for T4 in circulation but also as a protein which can transport T4 from the extracellular into the intracellular space, so that the mechanism of T4 transport mediated by desialylated TBG is different from that mediated by sialylated TBG, and that the T4 transport system in both cases, mediated by sialylated TBG and by desialylated TBG, may be related to the internalization of T4-TBG-TBG receptor complex or of T4-T4 receptor complex if TBG receptors are present in the outer surface of the cell membrane.