Synergistic actions of estradiol and the insulin-like growth factor somatomedin-C on swine ovarian (granulosa) cells

Estradiol amplified synergistically the dose- and time-dependent stimulatory actions of human somatomedin-C on progesterone biosynthesis by cultured swine granulosa cells. This facilitative interaction was not attributable to inhibition of the catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one, but, rather, reflected time-dependent stimulation of pregnenolone synthesis measured in the presence of exogenous soluble sterol substrate for cholesterol side-chain cleavage. Moreover, treatment with somatomedin-C was accompanied by increased synthesis of two immunoprecipitable cholesterol side-chain cleavage constituents, viz. cytochrome P-450scc and adrenodoxin. The synergism between estradiol and somatomedin-C was associated with significantly greater specific binding of somatomedin-C in estrogen-treated than control cultures, with no change in apparent receptor affinity. In vitro synergism occurred at somatomedin-C concentrations estimated by sequence-specific immunoassay to be attainable in ovarian follicular fluid in vivo and was specific in that it was not mimicked by the insulin-like peptide relaxin or by epidermal growth factor or fibroblast growth factor. However, high concentrations of insulin-like growth factor II (multiplication-stimulating activity) and insulin were able to interact with estradiol in a facilitative fashion to enhance progesterone production. In addition, the estrogenic component of the synergism was specific, since it was antagonized by the selective antiestrogen LY156758 and was mimicked sparingly by the nonaromatizable androgen 5 alpha-dihydrotestosterone. We conclude that estradiol and somatomedin-C interact synergistically in a time- and dose-dependent manner to enhance the biosynthesis of pregnenolone and progesterone by swine granulosa cells. Since estradiol and somatomedins are present in significant concentrations in the antral fluids of maturing Graafian follicles, we suggest that coordinated trophic effects of estradiol and insulin-like growth factor(s) may effectively prepare granulosa cells for the high rates of progesterone biosynthesis ultimately required after ovulation.     

Mechanisms subserving insulin's differentiating actions on progestin biosynthesis by ovarian cells: studies with cultured swine granulosa cells

To characterize the nature of insulin action on ovarian cells, an in vitro system of swine granulosa cells was developed in which 3- to 50-fold stimulation of progesterone production was observed in response to insulin under serum-free or sparsely (1%) serum-supplemented conditions. These studies demonstrate that optimal cell density is critical for the full expression of insulin action, and that the dose-dependent responses of granulosa cells to insulin are also significantly influenced by the maturational status of the parent follicle. The striking (greater than 30-fold) responses of medium-sized and healthy preovulatory follicles to insulin were not attributable to the presence or absence of atresia or to a selective inhibition of progesterone's catabolism to 20 alpha-dihydroprogesterone, since 20 alpha-dihydroprogesterone production was also markedly increased in response to insulin. The mechanisms subserving insulin action were explored further by testing the capacity of insulin to 1) increase progesterone accumulation in response to exogenously provided pregnenolone, and 2) stimulate pregnenolone biosynthesis in the presence of trilostane, an inhibitor of pregnenolone metabolism. The effects of insulin were to increase the biosynthesis of progesterone from available pregnenolone and increase the production of pregnenolone from endogenous sterol substrate. The physiological relevance of these differentiative actions of insulin is suggested by insulin's ability to significantly enhance the stimulatory effects of FSH, LH, epinephrine, prostaglandin E2, and cAMP effectors, cholera toxin and 8-bromo-cAMP. In summary, the present studies delineate culture conditions in which swine granulosa cells exhibit a high degree of responsiveness to the stimulatory actions of insulin on progestin biosynthesis. The effects of insulin are critically influenced by cell density, stage of follicle maturation, and the presence or absence of classical ovarian effector hormones with which insulin can interact synergistically. Moreover, our studies indicate that insulin exerts significant actions at several levels of progestin biosynthesis, including the production of pregnenolone, progesterone, and 20 alpha-dihydroprogesterone by granulosa cells. In view of the high concentrations of insulin-like growth factors and somatomedins attained in porcine Graafian follicles, we suggest that trophic actions of insulin or insulin-like peptides and the synergism of insulin with classical ovarian effector hormones are likely to be of physiological importance to the differentiation of granulosa cells in the developing ovarian follicle.     

Angiotensin II induces calcium release in a subpopulation of single ovarian (granulosa) cells.

The effects of angiotensin II on cytosolic free Ca2+ ion concentrations ([Ca2+]i) were studied in single porcine granulosa cells using the calcium-sensitive fluorescent dye fura-2 and high temporal resolution fluorescent videomicroscopy. Angiotensin II initiated specific, rapid, transient and topographically organized increases in [Ca2+]i in a subpopulation of single swine granulosa cells. The Ca2+ source for this angiotensin II-mediated [Ca2+]i transient appeared to be internal stores, and a pertussis toxin-sensitive guanine nucleotide binding protein was implicated in this receptor-mediated Ca2+ rise. Our single-cell studies also revealed a striking functional heterogeneity among granulosa cells, since follicle-stimulating hormone-responsive cells were not angiotensin II responsive. We conclude that single swine granulosa cells are targets of specific angiotensin II action on intracellular pools of Ca2+.     

Differential expression of aminopeptidase-N on human ovarian granulosa and theca cells.

The expression of aminopeptidase-N and neutral endopeptidase in human ovarian tissue was examined using specific monoclonal antibodies for each of these peptidases and histochemical staining for enzyme activity. Aminopeptidase-N is a membrane-bound metalloprotease catalyzing the removal of N-terminal amino acids from peptides and was detected by immunofluorescence staining on theca interna cells in secondary follicles and on luteinized thecal cells in preovulatory follicles and corpora lutea. However, aminopeptidase-N was not detected on granulosa cells. Peptidase activity was also detected by histochemical staining on theca interna cells and luteinized thecal cells. Luteinized granulosa cells showed peptidase activity, despite the lack of aminopeptidase-N. Neutral endopeptidase was not detected in ovarian granulosa and thecal cells. These observations indicate that aminopeptidase-N can be a useful surface marker for thecal cells.     

Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.     

Actions of gonadotropin-releasing hormone antagonists on steroidogenesis in human granulosa lutein cells

OBJECTIVE: GnRH antagonists have recently been introduced for the prevention of premature LH surges during controlled ovarian hyperstimulation (COH). We have here investigated whether the GnRH antagonists cetrorelix and ganirelix exert effects on ovarian steroidogenesis. Since there is some controversy about the action of GnRH agonists in the human ovary we also tested the effect of triptorelin on steroid production in cultured human granulosa lutein cells. METHODS: Cells were obtained from patients treated with different protocols of COH. In addition to gonadotropins they received triptorelin, cetrorelix, ganirelix or no GnRH analogue. RESULTS: Such in vivo treatment did not result in significant effects of triptorelin or the two GnRH antagonists on spontaneous or human chorionic gonadotropin (hCG)-stimulated steroidogenesis. To exclude the possibility that the in vivo treatment might not affect in vitro steroid production because of low or absent peptide activity, we performed in vitro treatments with triptorelin, cetrorelix and ganirelix for up to 96 h. However, these treatment paradigms did not influence basal or hCG-stimulated steroid production. CONCLUSIONS: We conclude that GnRH antagonists do not exert any significant effects on ovarian steroidogenesis in vitro and therefore their introduction into protocols of COH is unlikely to impair ovarian function.     

Bipotential actions of estrogen on progesterone biosynthesis by ovarian cells. II. Relation of estradiol's stimulatory actions to cholesterol and progestin metabolism in cultured swine granulosa cells

Although both inhibitory and stimulatory actions of estradiol on swine granulosa cells have been described, the bases for these inconsistent effects are not clear. We have tested properties of ovarian follicles and in vitro culture conditions that result in consistently stimulatory effects of estradiol on progesterone biosynthesis. Stimulatory actions of estradiol (in contrast to inhibitory effects) were critically dependent upon the density of granulosa cells in culture and the size and maturational status of the parent Graafian follicles. Granulosa cells isolated from small, rather than medium or large sized, swine follicles exhibited the greatest peak response to estradiol, although half-maximally stimulatory concentrations (ED50) of estradiol were similar (mean, 81 ng/ml). Granulosa cells from atretic follicles also secreted increased quantities of progesterone in response to estradiol, but the ED50 for estrogen stimulation was significantly higher (ED50 = 322 ng/ml estradiol) than that of comparable healthy follicles (ED50 = 109 ng/ml). This estrogen-responsive system was used to test the mechanisms subserving estrogen's trophic actions on granulosa cells. Estradiol significantly enhanced the activity of 3 beta-hydroxysteroid dehydrogenase with consequently increased production of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Estrogen also augmented functional cholesterol side-chain cleavage activity in a dose- and time-dependent fashion with a resultant increase in pregnenolone biosynthesis. Moreover, parallel observations documented concordant dose responses for the synthesis of all three major progestins by pig granulosa cells. The trophic actions of estrogen on the steroidogenic pathway were associated with enhanced hydrolysis of endogenous cholesteryl ester stores but were not significantly antagonized by inhibition of de novo cholesterol biosynthesis. We conclude that suitable follicle selection and appropriate in vitro culture conditions provide a consistently estrogen-responsive granulosa-cell system, in which estradiol modulates certain key aspects of progestin and cholesterol metabolism. These trophic actions of estrogen are likely to prepare granulosa cells for the increased rates of progesterone biosynthesis ultimately required by fully differentiated luteal cells.     

Activin signaling through type IB activin receptor stimulates aromatase activity in the ovarian granulosa cell-like human granulosa (KGN) cells

In addition to a stimulatory effect on FSH production by the pituitary gland, activin is thought to have a paracrine or autocrine role in follicular development in the ovary, where it is produced. Recently, we established a human ovarian granulosa tumor cell line, KGN, which possesses in vivo characteristics of granulosa cells, namely the expression of functional FSH receptors and cytochrome P-450 aromatase. Here, we have demonstrated the activin signaling pathway and its role in KGN cells. A series of transient transfection experiments revealed that activin type IB receptor (ActRIB) is an essential component of the activin signaling pathway in KGN cells. Smad2 was found to act downstream of ActRIB as an intracellular signal transmitter. Smad7, but not Smad6, was an inhibitory Smad in the pathway. Finally, we show that FSH receptor expression and cytochrome P-450 (P-450) aromatase activity was up-regulated by activin stimulation through ActRIB in KGN cells. These results show that we have clarified the signaling mechanisms and the roles of activin in the human granulosa cell line, KGN. Activin signaling mediated by ActRIB-Smad2 system in the ovary may thus be essential for the regulation of follicular differentiation.     

Insulin regulates low density lipoprotein metabolism by swine granulosa cells

Insulin synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells. The mechanisms subserving this facilitative interaction included the following: 1) insulin's synergism with LDL was profoundly attenuated by covalent modification of arginine residues in LDL by 1,2-cyclohexanedione treatment; 2) insulin increased by 2- to 6-fold the number of specific high affinity LDL receptors on granulosa cells, with no change in apparent binding affinity; 3) insulin augmented rates of [125I]iodo-LDL internalization and degradation without enhancing nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrollidone); 4) insulin increased by 2.5- to 3-fold granulosa cell content of free and esterified cholesterol (measured by fluorometry) in response to treatment with unlabeled LDL; 5) insulin stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester, and amplified [3H]progesterone secretion by granulosa cells exposed to [3H]cholesteryl linoleate-labeled LDL; and 6) insulin action was specific in that it was not mimicked by desoctapeptide insulin, epidermal growth factor, fibroblast growth factor, or relaxin. We conclude that insulin and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via specific mechanisms that depend upon 1,2,-cyclohexanedione-sensitive residues within LDL apoprotein. Insulin action results in significantly augmented binding, internalization, and degradation of LDL, which is accompanied by increased effectual delivery of cholesterol substrate into cellular sterol pools that participate in enhanced steroidogenesis.     

Production of insulin-like growth factors by ovarian granulosa cells

Evidence for ovarian secretion of somatomedins or insulin-like growth factors (IGF's) was generated by two approaches. First, porcine granulosa cells were shown to produce IGF's and an IGF-binding protein under serum-free conditions in vitro. The ovarian IGF's were recognized in two competitive binding assays specific for IGF's, a RIA using antibodies to human IGF-I and a radioreceptor assay using rat liver plasma membranes. IGF secretion was maintained for at least 10 days in culture. Second, ovarian production of IGF's in vivo was suggested by studies which showed that IGF levels in follicular fluid from preovulatory follicles were significantly greater than those in either serum or immature follicles. In contrast, similar low levels of insulin were observed in the follicles and serum. In conjunction with previous evidence of IGF action on granulosa cells, the present studies suggest the possibility of an autocrine role of IGF's in regulating follicular growth and development.     

Human endometrial adenocarcinoma cells express endothelin-1.

The expression of endothelin-1 (ET-1) in five human endometrial adenocarcinoma cell lines was studied. Using specific radioimmunoassay, immunoreactive ET-1 was detected in conditioned medium from two of the cell lines (RL 952 and HEC 1A). In reverse-phase high-performance liquid chromatography (HPLC), synthetic ET-1 and immunoreactive ET-1 from conditioned media revealed the same elution profile. By amplification of cDNA using the polymerase chain reaction, normal human endometrium as well as cell lines RL 952 and HEC 1A were shown to express ET-1 mRNA. In addition, cell line HEC 1B and KLE, which did not produce measurable amounts of immunoreactive ET-1, contained ET-1 specific mRNA whereas cell line AN3CA had no detectable ET-1 mRNA and did not secrete immunoreactive ET-1.     

Ovarian actions of tumor necrosis factor-alpha (TNF alpha): pleiotropic effects of TNF alpha on differentiated functions of untransformed swine granulosa cells.

We have examined interactions between tumor necrosis factor-alpha (TNF alpha), a product of the immune system, and ovarian cells using serum-free monolayer cultures of untransformed swine granulosa cells. Recombinant human TNF alpha, a potent cytoactive product of activated macrophages, bound specifically and with high affinity to intact granulosa cells. Binding sites had an apparent Kd of 0.17 nM (95% confidence interval, 0.065-0.31), and a binding capacity of 80 nmol/micrograms DNA (95% confidence interval, 52-110). The binding capacity of granulosa cells for TNF alpha (but not the binding affinity) was increased approximately 2-fold by treatment with FSH and insulin. The biological effects of TNF alpha on pig granulosa cells were expressed after 48 and 96 h in culture. At the latter time, TNF alpha significantly suppressed insulin- and insulin- plus FSH-stimulated progesterone accumulation, with respective ID50 values of 0.08 +/- 0.008 and 0.06 +/- 0.014 nM, but did not affect basal progesterone accumulation or DNA content. TNF alpha also significantly attenuated the stimulatory effect of combined treatment with FSH and insulin on cAMP generation during 48-96 h of culture. TNF alpha inhibited the stimulatory effects of forskolin, cholera toxin, and the cAMP analog 8-bromo-cAMP on progesterone accumulation, indicating multiple sites of action of this immune modulator. Inhibition of progestin biosynthesis was observed even in the presence of 25-hydroxycholesterol, a soluble oxygenated sterol substrate for the cholesterol side-chain cleavage reaction, and was accompanied by decreased concentrations of specific cellular mRNA encoding cholesterol side-chain cleavage enzyme. There were no changes in the amounts of a constitutively expressed enzyme, phosphoglyceraldehyde dehydrogenase. Inhibitory actions of TNF alpha were specific to de novo steroid hormone biosynthesis, since nanomolar concentrations of this cytokine stimulated accumulation of prostaglandin E2 and prostaglandin F2 alpha basally and during treatment with FSH, cholera toxin, or 8-bromo-cAMP. In contrast, prostaglandin accumulation was not enhanced by interferon-gamma or interleukin-2. In summary, untransformed porcine granulosa cells exhibit specific, high affinity, low capacity saturable binding sites for TNF alpha, and the number of such binding sites can be regulated by combined treatment with insulin and FSH. Granulosa cells are susceptible to the inhibitory actions of TNF alpha on FSH- and insulin-supported progesterone biosynthesis and cAMP accumulation. One important locus of TNF alpha action is blockade of hormonally stimulated increases in specific mRNA encoding the cholesterol side-chain cleavage cytochrome P450 enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of low density lipoprotein binding by cultured swine granulosa cells

The properties of low density lipoprotein (LDL) binding to ovarian cells were investigated in cultured swine granulosa cells in serum-free monolayer cultures. Swine and human LDL bound with high affinity and specificity, with apparent dissociation constant (Kd) values for swine and human LDL of 1.1 and 3.6 micrograms/ml at 4 C, and 2.7 and 4.8 micrograms/ml at 37 C. In contrast, high density lipoprotein competed sparingly with [125I]iodo-LDL with an apparent half-maximally inhibitory concentration of 650 micrograms/ml. Binding of LDL was dependent upon arginine residues within the apoprotein B moiety, since covalent modification of LDL with 1,2-cyclohexanedione markedly reduced its ability to compete for binding or degradation and to support progesterone biosynthesis. This specific, high affinity saturable binding of LDL to pig granulosa cells exhibited a maximal binding capacity of 0.7 micrograms LDL protein/mg DNA, which corresponds to approximately 5500 LDL receptors per cell. The relative time course of LDL binding, internalization, and degradation by swine granulosa cells was assessed by examining trypsin-releasable (surface-bound) and trypsin-resistant (internalized) [125I]iodo-LDL. At 37 C, granulosa cells exhibited a rapid increase in surface-bound lipoprotein, followed by delayed but subsequently progressive increases in internalized and degraded LDL. LDL degradation by swine granulosa cells was a saturable, temperature- and time-dependent process, with half-maximal degradation occurring at a concentration of 16 micrograms/ml LDL. This correlates closely with the half-maximal concentration of LDL that stimulates progesterone secretion. Degradation of [125I]iodo-LDL was not attributable to bulk fluid-phase pinocytosis, since the cellular ingestion of an impermeant probe, [125I]iodo-polyvinylpyrrolidone, occurred at 1/40 the rate of lipoprotein degradation. In addition, degradation of [125I]iodo-LDL was specifically inhibited by excess unlabeled LDL, decreased by prior exposure of granulosa cells to the soluble sterol, 25-hydroxycholesterol, and antagonized by the lysosomotrophic agent, chloroquine. Moreover, in separate experiments, rates of degradation of LDL were found to be significantly correlated with progesterone one production (r = +0.88, P less than 0.01). In summary, swine granulosa cells exhibit specific, high affinity, saturable, and low capacity receptors for homologous and heterologous LDL. These LDL recognition sites on ovarian cells depend upon cyclohexanedione-sensitive arginine residues with the apoprotein B moiety for effective binding, internalization, and functional (steroidogenic) responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of inhibitor and activator of ghrelin receptor (GHS-R1a) on porcine ovarian granulosa cell functions

It was previously shown, that ghrelin and its agonistic analogue, ghrelin 1-18, can be a stimulator of ovarian cell functions (promoter of proliferation, inhibitor of apoptosis and stimulator of hormones release). The aim of our studies was to compare the action of two ghrelin analogues - ghrelin 1-18, activator of ghrelin receptors (GHS-R1a), and (d-Lys3)-GHRP-6, its inhibitor, on porcine ovarian granulosa cell functions. Effects of (d-Lys3)-GHRP-6 added at doses of 0, 1, 10 or 100 ng/ml on the expression of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2), apoptosis (bax, p53, caspase 3) and release of steroid hormones (progesterone, testosterone, estradiol) were examined. In addition, some effect of ghrelin 1-8 on some of these parameters (expression of MAPK/ERK1,2, bax, p53) were verified. It was shown, that (d-Lys3)-GHRP-6 promotes all markers of granulosa cell proliferation, inhibits all markers of apoptosis and stimulates the release of all three steroid hormones. Similar effects of (d-Lys3)-GHRP-6 (inhibitor of GHS-R1a) and ghrelin 1-18 (its stimulator) suggest that the examined effects of these substances on porcine ovaries are not mediated by GHS-R1a. Both chemical analogues could be potentially useful for stimulation of reproductive processes, at least in in vitro conditions.     

The influence of di-(2-ethylhexyl) phthalate on steroidogenesis by the ovarian granulosa cells of immature female rats

Phthalate esters are known to exert harmful effects on mammalian reproduction and fertility, but their potential adverse effects on the hormonal functions of the ovary have not yet been elucidated in detail. Here, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the hypothalamic-pituitary-gonadal axis of young developing female rats, as well as on ex vivo steroidogenesis by granulosa cells (GCs) and secretion of LH by gonadotropes. Exposure of 20-day-old female rats to 500 mg DEHP by oral gavage once daily for 10 days reduced their serum levels of progesterone and estradiol, while tending to enhance levels of LH. Furthermore, primary cultures of GCs isolated from these rats exhibited an attenuated capacity to produce progesterone in response to stimulation by LH and FSH, as well as a lower degree of transport of endogenous cholesterol into mitochondria. Moreover, the ability of primary cultures of pituitary cells isolated from DEHP-treated rats to produce and secrete LH in response to GnRH was significantly enhanced. In addition, 2-ethylhexanoic acid, a metabolite of DEHP, significantly potentiated GnRH-stimulated production of LH by cultures of pituitary cells isolated from untreated 20-day-old female rats. Together, these data indicate that DEHP exerts dual effects on the pituitary-gonadal axis, stimulating the hormonal function of the pituitary and, at the same time, by inhibiting steroidogenesis by GCs.     

Phosphoramidon blocks the pressor activity of porcine big endothelin-1-(1-39) in vivo and conversion of big endothelin-1-(1-39) to endothelin-1-(1-21) in vitro.

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.     

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary.