Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A.

Recombinant expression of human alpha- and beta A-inhibin subunit cDNAs in mammalian 293 cells results in the secretion of 20-53K free alpha-subunit-derived products, 30-105K alpha beta A-inhibin dimers, and 24-110K beta A-activin dimers. The present study verifies that the wide variation in the size of these products is due to incomplete cleavage of the proteolytic processing sites and the differential glycosylation of the N-linked glycosylation site at amino acid number 302 in the alpha C-subunit. The identity of each of these products was established by mutagenesis of proteolytic processing sites and N-linked glycosylation sites, combined with the analysis of transfection products by immunoprecipitation and one- and two-dimensional SDS-PAGE (SDS/SDS-beta-ME). Transient expression of processing site mutants of the alpha- and beta A-subunits in 293 cells was used to generate microgram quantities of noncleavable 55K and 65K inhibin dimers, and noncleavable 110K activin A dimers. The 55K and 65K inhibin A forms were purified and found to be fully biologically active in a rat pituitary cell bioassay. The 110K high molecular weight (HMW) form of human activin A failed to show any FSH-releasing activity in the pituitary assay. Since radioactively labeled 55K and 65K inhibin A and 110K activin A remained intact after incubation with rat pituitary cells for 72 h, there appears to be no conversion of these dimers to lower molecular weight forms by proteolytic cleavage at additional sites. These results show for the first time that 55K and 65K inhibit A are intrinsically biologically active and do not require cleavage to the 32K form for activation. In contrast, cleavage of the 110K activin A precursor to the 24K form would appear to be necessary for activity.

Interaction between activin and follicle-stimulating hormone-suppressing protein/follistatin in the regulation of basal inhibin production by cultured rat granulosa cells.

There is evidence that FSH-suppressing protein (FSP) antagonizes the action of activin on the differentiation of rat granulosa cells by binding activin in vitro. We tested the interaction of activin and FSP in this in vitro system by examining the effects of FSP on activin dose-related stimulation of immunoreactive inhibin release by rat granulosa cells. Granulosa cells (2 x 10(5) viable cells/well) from diethylstilbestrol-treated immature rats were cultured for 48 h in McCoy's 5a serum-free medium with additives and increasing doses of bovine FSP (0-30 nM) and human recombinant activin (0-20 nM). Inhibin was measured in the medium by RIA. Activin caused a dose-related increase in basal inhibin production, which was maximal between 4-10 nM activin (ED50, 0.6 nM). With the addition of FSP, an apparent increase in the ED50 of the activin dose-response curves was observed, but there were no changes in the maximum response. This pattern closely resembled that of chemical antagonism of an agonist by an agent that binds with relatively high affinity to form a biologically inactive complex. Based on this premise, apparent high affinity activin binding to FSP was determined by Scatchard analysis to have a Kd of 0.13 +/- 0.07 nM (mean +/- SD) and to occur in a 2:1 or greater FSP/activin molar ratio. These data support the proposition that the antagonistic effect of FSP on activin is due to the formation of an inactive complex.

Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture.

The aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 M HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62-0.77 and an iodine incorporation ratio of 1.7-2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nM and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (<2%), transforming growth factor-beta (<0.2%), or follistatin (<<0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.

Ovine anterior pituitary production of follistatin in vitro.

Rat pituitary cells express messenger RNA for the activin-binding protein, follistatin (FS), and rat and bovine pituitary cell cultures secrete FS into the medium. In the present study, a previously validated, heterologous RIA for ovine FS was employed to investigate FS synthesis, secretion, and regulation in cultures of ovine anterior pituitary cells. The validity of the RIA was confirmed by the finding that FS immunoreactivity in ovine pituitary cell culture-conditioned medium diluted in parallel with purified bovine FS, and fractionation of the conditioned medium resulted in the coelution of activin-binding activity with the FS immunoactivity. The concentration of endogenous ovine FS achieved in the culture medium (0.08-0.6 nM) was in the range over which bovine FS suppresses FSH secretion in these cultures (IC50 = 0.5 nM). To characterize the relationship between endogenous FS and FSH secretion, dispersed ovine pituitary cells were preincubated with 10% fetal bovine serum for 2 days, then cultured between days 2-5 in the presence of a chemically defined serum substitute. Under these conditions, FS was continuously secreted at a rate of 12.1 +/- 1.8 ng/10(6) cells.day (mean +/- SEM; n = 18), whereas FSH was secreted at 64 +/- 13 ng/10(6) cells.day (n = 7). The secretion of FS and FSH changed in a reciprocal way as culture conditions were altered either by maintaining exposure of the cells to fetal bovine serum or by plating the cells at a 6- to 10-fold higher seeding density. Under the latter circumstance, for instance, FS secretion during the 3-day test period decreased to 47 +/- 14% (n = 10) and FSH secretion increased to 137 +/- 6% (n = 6) of the respective values in cultures of dispersed cells. FS secretion was increased nearly 3-fold (P < 0.05) in a dose-dependent manner by continuous exposure of ovine pituitary cells between days 2-5 to recombinant human activin A (1-10 nM), which concomitantly increased FSH secretion. Recombinant human inhibin A (0.003-10 nM); the synthetic glucocorticoids, RU28362 and dexamethasone (each 1-100 nM); the sex steroids, testosterone (1-100 nM), 17 beta-estradiol (0.001-5 nM), and progesterone (4-2500 nM); and the vitamin A derivative, retinoic acid (0.3-32 microM), each inhibited FSH secretion from these cultures, but only the last agent significantly (P < 0.05) increased FS secretion. Inhibin prevented the stimulation of FSH secretion by activin A without affecting its stimulation of FS secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of purified 31K bovine inhibin on the specific binding of gonadotropin-releasing hormone to rat anterior pituitary cells in culture.

The mechanism by which inhibin decreases the responsiveness of the pituitary gonadotroph to GnRH in terms of secretion of gonadotropins is largely unknown. We studied the effect of pure 31K bovine inhibin on the specific binding of GnRH to rat anterior pituitary cells in culture using iodinated Buserelin as tracer. Results showed that treatment of cultured anterior pituitary cells from adult male rats with inhibin (0-30 U/ml) for 72 h decreased Buserelin binding in a dose-dependent manner. In the presence of a maximally inhibiting dose of manner. In the presence of a maximally inhibiting dose of inhibin, Buserelin binding decreased progressively with time, reaching a minimum of 42% of the control value after 3 days. Exposure of pituitary cells for 3 days to the inhibin-related peptides transforming growth factor-beta (up to 400 pM) and Müllerian inhibitory substance (up to 100 nM) did not decrease binding of Buserelin, suggesting that the effect was specific to inhibin. Inhibin did not compete with iodinated Buserelin for GnRH-binding sites when they were added to the assay tube simultaneously. In addition, treatment with inhibin halved the number, but did not change the affinity, of GnRH-binding sites and had no effect on either cell number of cell viability. It is concluded that the reduction by inhibin of rat gonadotroph responsiveness to GnRH may be partly related to a decrease in the number of GnRH receptors on the cell surface.

Inhibitory effect of pure 31-kilodalton bovine inhibin on gonadotropin-releasing hormone (GnRH)-induced up-regulation of GnRH binding sites in cultured rat anterior pituitary cells.

Primary cultures of enzymatically dispersed rat anterior pituitary cells were used to examine the effect of pure 31 kilodalton bovine inhibin on GnRH-induced up-regulation of GnRH binding sites. After 2 days in culture, the cells were exposed to stimuli with or without test substances for 10 h, followed by evaluation of GnRH binding sites using iodinated GnRH-A (Buserelin) as tracer. Inhibin suppressed GnRH-induced up-regulation of GnRH binding sites in a dose-dependent manner with an IC50 of 0.13 U/ml (5.5 pM). The inhibin-related peptides transforming growth factor-beta, and Müllerian inhibitory substance had no detectable effect (stimulatory or inhibitory), suggesting that the action is specific to inhibin. In addition, inhibin inhibited the calcium ionophore A23187-induced up-regulation of GnRH binding sites, indicating that this effect of inhibin can occur, at least in part, at a stage subsequent to Ca2+ mobilization. Inhibin did not compete with iodinated GnRH-A for GnRH binding sites. In conclusion, pure 31 kilodalton bovine inhibin suppressed GnRH-induced up-regulation of GnRH binding sites in cultured rat anterior pituitary cells, providing direct evidence that inhibin modulates delayed actions of GnRH.

Chronic inhibitory effect of follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin on activin- and gonadotropin-releasing hormone-stimulated FSH synthesis and secretion in cultured rat anterior pituitary cells.

The effects of bovine FSH-suppressing protein (FSP) or follistatin on activin- and GnRH-stimulated FSH synthesis and secretion have been studied using cultured pituitary cells from adult male Sprague-Dawley rats. Exposure to FSP (0.001-10 nM) for 3 days dose-dependently suppressed basal FSH secretion (IC50 = 146 +/- 21 pM., mean +/- SE), cellular content (IC50 = 269 +/- 8 pM) and total FSH (IC50 = 181 +/- 25 pM), with no effect on LH. Activin (0.3 nM) increased FSH secretion 2.1-fold, cellular content 1.3-fold, and total FSH 1.9-fold during a 3-day incubation, but these increases were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP (0.1-3 nM), with complete inhibition occurring at concentrations between 1 and 3 nM. The 31- and 39-kDa forms of bovine FSP also antagonized the actions of activin. GnRH (1 nM) increased FSH secretion 1.8-fold and total FSH 1.6-fold during a 3-day incubation, effects that were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP. The highest tested concentration of FSP (3 nM) suppressed GnRH-stimulated FSH secretion and total FSH to 59 and 57%, respectively, of the levels found in untreated cultures. All three forms of bovine FSP produced a significant inhibition of FSH secretion and total FSH stimulated by GnRH. FSP also suppressed FSH secretion and total FSH in response to activators of protein kinase C including 100 nM phorbol 12-myristate 13-acetate (43 and 59%, respectively) and 100 nM mezerein (40 and 60%, respectively). Finally, treatment of cultured pituitary cells with 35-kDa FSP at 1 and 3 nM for 3 days resulted in 21 and 24% decreases in GnRH binding sites, respectively. It is concluded that (i) FSP inhibits not only the secretion but also the synthesis of FSH induced by activin and GnRH in long-term culture, and (ii) FSP may cause its inhibitory effects on GnRH by suppression of the protein kinase C system, and possibly by reduction of GnRH binding sites.

Effect of inhibin on activators of protein kinase-C and calcium-mobilizing agents which stimulate secretion of gonadotropins in vitro: implication of a postgonadotropin-releasing hormone receptor effect of inhibin on gonadotropin release.

To further characterize the subcellular mechanisms by which inhibin suppresses GnRH-stimulated gonadotropin release, anterior pituitary cells from adult male Sprague-Dawley rats were treated on day 2 of culture with or without purified 31-kDa bovine inhibin (1-300 pM) for a further 3 days. On day 5, the pretreated cells were washed and incubated in the absence or presence of various secretagogues for 4 h. At the end of the stimulation, the media were saved, and cells were lysed for measurement of both extracellular and intracellular FSH and LH by specific RIAs. Released hormone was expressed as the proportion of total (released plus intracellular) hormone that was available for release in each case. This manipulation of the data corrects for the differential effect of the inhibin pretreatments to suppress intracellular FSH before the stimulation period. Pretreatment for 3 days with inhibin suppressed the proportions of FSH and LH released during 4 h in response to 1) phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase-C, by maxima of 48% and 53% with inhibin median inhibitory concentrations (IC50) of 17 and 18 pM, respectively; 2) mezerein (100 nM), another type of activator of protein kinase-C, by maxima of 49% and 50% with inhibin IC50 of 19 and 20 pM, respectively; 3) high extracellular K+ (60 mM) by 42% (P less than 0.01) and 38% (P less than 0.01), respectively, with 130 pM inhibin; 4) the calcium ionophore, A23187 (100 microM) by maxima of 54% and 56% with IC50 of 18 and 17 pM, respectively; and 5) GnRH (10 nM) by maxima of 52% and 53% with IC50 of 18 and 19 pM, respectively. However, inhibin had no effect on the proportional release of gonadotropin induced by melittin, an activator of phospholipase-A2. Finally, inhibin had no effect on ACTH release either under basal conditions or in response to CRF (10 nM), phorbol 12-myristate 13-acetate (100 nM), or A23187 (100 microM). We conclude that inhibin suppresses the stimulated release of hormones from gonadotrophs in part by a mechanism common to both gonadotropins that is independent of the previously described inhibitory effect of inhibin on the GnRH receptor. The results are consistent with an action at a site(s) beyond the GnRH receptor, such as protein kinase-C and calmodulin.

Regulation of the synthetic rate of gonadotropin-releasing hormone receptors in rat pituitary cell cultures by inhibin.

Regulation of steady state levels of plasma membrane receptors for GnRH is the arithmetic result of processes that contribute to the appearance of receptors (synthesis, recycling, and unmasking) less those that contribute to the loss of receptors (degradation, internalization, and inactivation). We have adapted the density shift technique to evaluate specifically the rate of synthesis of GnRH receptors in rat pituitary cell cultures. Recently, it has been shown that inhibin can decrease the steady state levels of GnRH receptors in rat pituitary cell cultures and can block homologous up-regulation of GnRH receptors. In the present study we have evaluated the ability of purified inhibin to affect the synthesis rate of GnRH receptors under basal conditions and after exposure of cultured gonadotropes (from female weanling rats) to GnRH. Cells were exposed to inhibin alone (4 or 12 ng/ml) or to GnRH (10(-10) M) plus inhibin (0.4, 4, or 12 ng/ml) in the presence of densely labeled amino acids. GnRH was administered as a 20-min pulse, but inhibin treatment was continued for up to 2 days. After these treatments, GnRH receptors were covalently linked to a radio-labeled photoaffinity probe (125I- Tyr5-[azido-benzoyl-D-Lys6] GnRH) and solubilized with 1% sodium dodecyl sulfate. Newly synthesized GnRH receptors (those that had incorporated the dense amino acids) were separated from previously synthesized receptors (those containing normal amino acids) by velocity sedimentation through sucrose gradients (O-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After velocity sedimentation, gradients were fractionated, and the radioactivity in each fraction was quantified. Treatment with inhibin alone had no effect on the synthesis rate of GnRH receptors compared to that of control cultures (t1/2, 23.5 +/- 0.3 vs. 23.3 +/- 0.3 vs. 22.9 +/- 0.9 h for control, 4 ng/ml inhibin, and 12 ng/ml inhibin, respectively). In contrast, inhibin blocked the stimulation of homologous receptor synthesis by GnRH in a dose-dependent manner (t1/2, 12.2 +/- 0.7 vs. 14.0 +/- 0.7 vs. 19.2 +/- 1.5 vs. 20.0 +/- 2.9 h for GnRH alone and GnRH plus 0.4, 4, or 12 ng/ml inhibin, respectively). These data indicate that in rat pituitary cell cultures, inhibin does not decrease basal levels of GnRH receptors by affecting the synthesis rate of receptors, but prevents up-regulation of GnRH receptors by blocking stimulation of GnRH receptor synthesis by homologous hormone.

Effects of 31 kilodalton bovine inhibin on follicle-stimulating hormone and luteinizing hormone in rat pituitary cells in vitro: actions under basal conditions.

The effects of purified 31 kilodalton (kDa) bovine inhibin on the basal release and cell contents of FSH and LH in pituitary cells from adult male Sprague-Dawley rats have been investigated for periods up to 28 days in tissue culture. A constant rate of basal FSH release (1.90 +/- 0.24 ng/10(6) cells.h) was observed during the first 10 days of culture and this release was blocked by inhibin in a concentration-dependent manner (IC50 = 0.16 U/ml, IC100 greater than 2.5 U/ml). The constant basal LH release between days 2 and 12 of culture (0.15 +/- 0.02 ng/10(6) cells.h) was also blocked by inhibin in a concentration-dependent manner to a minimum of 25% of control (IC50 = 0.66 U/ml, ICmax greater than 6 U/ml). FSH and LH cell contents decreased exponentially in the presence and absence of inhibin over the first 20 days of culture permitting the calculation of decay half-times (t1/2). Inhibin reduced the FSH cell content t1/2 from 5.0 to 1.8 days and the LH cell content t1/2 from 5.8 to 3.6 days (IC50 = 0.81-0.83 U/ml, ICmax greater than 6 U/ml for both FSH and LH). The sum of the FSH released and FSH cell content (i.e. total FSH) increased with time in culture and this increase was blocked by inhibin. In contrast, total LH decreased with time in all cultures. The data suggest that in pituitary cells separated from hypothalamic and gonadal inputs 1) inhibin primarily suppresses tonic FSH synthesis coupled to its basal release; 2) at higher concentrations inhibin partially suppresses basal LH release and promotes intracellular degradation of FSH and LH. Thus, based on differences in sensitivity, inhibin acts at two or more sites, one associated with FSH tonic synthesis-basal release and the other(s) with FSH/LH mobilization and/or degradation.

Identification of specific inhibin A-binding proteins on mouse Leydig (TM3) and sertoli (TM4) cell lines.

The binding of human inhibin A to cell surface binding proteins of mouse Leydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysis identified two classes of inhibin A-binding sites on TM3 (K(d(1)) = 85 pM and 4,160 sites/cell; K(d(2)) = 520 pM and 12,500 sites/cell) and TM4 (K(d(1)) = 61 pM and 2,620 sites/cell; K(d(2)) = 520 pM and 10,400 sites/cell) cells. Compared with inhibin A, inhibin B only partially competed [(125)I]inhibin A binding (6-8%), whereas activin A competed weakly (<0.01%). Chemical cross-linking of [(125)I]inhibin A to both cell lines identified five [(125)I]inhibin A binding complexes with apparent molecular masses of 70, 95, 145, 155, and more than 200 kDa. Inhibin A displacement of [(125)I]inhibin A from each of these cross-linked species (ED(50) = 60-110 pM) closely resembled displacement from intact TM3 (ED(50) = 97 +/- 32 pM) and TM4 (ED(50) = 75 +/- 28 pM) cells, suggesting that all of these proteins are involved in the high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human betaglycan identified protein complexes of more than 200, 145, and 95 kDa. It is concluded that the high affinity binding complex for inhibin A found in these cell lines consists of betaglycan and several proteins of unknown identity and may represent the putative inhibin receptor complex.

Pregnant mare's serum gonadotropin stimulates inhibin subunit gene expression in the immature rat ovary: dose response characteristics and relationships to serum gonadotropins, inhibin, and ovarian steroid content.

We have investigated the temporal changes and dose responses of ovarian inhibin gene expression, circulating inhibin, and ovarian estradiol content in PMSG-primed immature female rats. The relative levels of ovarian inhibin alpha- and beta A-subunit messenger RNA (mRNA), serum inhibin, and ovarian steroid content were measured after stimulation of immature female rats with 20 IU PMSG. Each of these variables rose in parallel and peaked at 48 h (relative ovarian inhibin alpha- and beta A-subunit mRNA increased 3.8-fold and 2.8-fold, respectively, serum inhibin 269 U/ml, vs. control 31, P less than 0.01, ovarian estradiol content 114 pmol/pair of ovaries vs. controls 4.7 P less than 0.001), corresponding to the time of maximal follicular development in this animal model. Subsequently each fell to reach a nadir at 96 h. The data are consistent with the hypothesis that PMSG stimulation of inhibin secretion occurs by a mechanism involving inhibin gene transcription. The increase in ovarian inhibin and estradiol synthesis during folliculogenesis and their simultaneous decline before ovulation suggest that 1) the circulating levels of inhibin and estradiol reflect follicular maturation and 2) these two hormones are regulated via a common mechanism during follicular development in the rat.

Gonadotrophin secretion revisited. How many ways can FSH leave a gonadotroph?

In summary, gonadotrophs make and secrete many biologically active proteins, some of which participate in autocrine regulation of gonadotrophin, and particularly FSH, secretion. This essay focuses on different vehicle(s) for secretion of protein products as one way that the gonadotroph might control secretion of the two gonadotrophins. Gonadotrophs phasically secrete LH and (proportionately less) FSH via secretory granules in response to an increase in intracellular Ca2+, but also secrete FSH without LH by a distinct pathway. The latter is tonically regulated by not only activin, but also inhibin and follistatin, at least partly at the level of FSH synthesis. Thus GnRH-independent secretion of FSH may masquerade as a second form of 'regulated' secretion through its linkage with FSH synthesis. Three major types of vacuole that possibly mediate gonadotrophin secretion have been identified in gonadotrophs: the small, dense core secretory granule that is rich in LH; a larger, more diffuse granule that is rich in FSH; and the much smaller, synaptic vesicle-like vacuole that contains no identifiable gonadotrophin. Which of these subserves the tonically regulated, GnRH-independent pathway for secretion of FSH without LH has not yet been determined. It is unlikely to be the small granule, because of its preponderance of LH. It may be a form of synaptic vesicle-like vacuole, which is immunocytochemically 'silent' with respect to FSH, or the FSH-rich larger form of secretory granule, for which a specialized, constitutive-like secretory function has not yet been assigned.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 31 kDa bovine inhibin on FSH and LH in rat pituitary cells in vitro: antagonism of gonadotrophin-releasing hormone agonists.

The effects of 31 kDa bovine inhibin on the release of FSH and LH stimulated by gonadotrophin-releasing hormone (GnRH) or its agonist analogue buserelin have been studied using 5-day-old cultures of pituitary cells prepared from adult male Sprague-Dawley rats. Exposure of cultures to increasing concentrations of inhibin for 3 days before and during a 4-h stimulation with GnRH resulted in the progressive suppression of both basal and stimulated gonadotrophin release. At the highest inhibin concentrations FSH release was abolished (inhibin median inhibitory concentration (IC50) = 0.15 U/ml) whereas LH release was suppressed by 75% (IC50 = 0.93 U/ml). To correct for the reduced size of the FSH pool resulting from inhibin pretreatment, the amount of FSH or LH released by an agonist was expressed as a proportion of the total hormone available for release in each case. Following this adjustment, concentrations of inhibin producing maximal effects increased the GnRH median effective concentration for FSH release 4.1-fold and that for LH release 2.2-fold, with inhibin IC50 values of 0.45 and 0.32 U/ml respectively. Inhibin also suppressed the maximum proportion of both FSH and LH that excess GnRH released in 4 h by 36%, with IC50 values of 0.53 and 0.76 U/ml respectively. These effects were not changed by reduction of the inhibin pretreatment period from 3 days to 1 day or by exclusion of inhibin during the stimulation period. After a 3-day pretreatment, inhibin inhibited gonadotrophin release by buserelin less effectively than that by GnRH, but the pattern of antagonism was the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of bovine 35 kDa FSH-suppressing protein on FSH and LH in rat pituitary cells in vitro: comparison with bovine 31 kDa inhibin.

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3-9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC50) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC50 = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC50 values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC50 values = 142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute inhibitory effect of follicle-stimulating hormone-suppressing protein (FSP) on gonadotropin-releasing hormone-stimulated gonadotropin secretion in cultured rat anterior pituitary cells.

Follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin, a novel gonadal glycoprotein hormone, has been shown to have chronic inhibitory effects on the secretion of both FSH and luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) in vitro. The present study was designed to investigate the acute effects of bovine FSP on GnRH-stimulated gonadotropin secretion and to examine the potential subcellular sites of this action of FSP using cultured pituitary cells. Anterior pituitaries from adult male Sprague-Dawley rats were enzymatically dispersed and cultured for 48 h, after which the cells were treated with bovine FSP for 6 h, followed by a 4 h stimulation with secretagogues in the continued presence of FSP. Results showed that the 35 kDa form of bovine FSP (0.1-3 nM) dose-dependently suppressed GnRH-stimulated FSH and LH secretion, with inhibition of 38 and 25%, respectively, at 3 nM. In addition, FSP suppressed gonadotropin secretion in response to activators of protein kinase C (phorbol 12-myristate 13-acetate (PMA) and mezerein) and a calcium ionophore (A23187). However, FSP had no effect on gonadotropin secretion evoked by melittin, an activator of phospholipase A2. Furthermore, 35 kDa bovine FSP did not compete with GnRH for GnRH binding sites in a direct competition study and treatment of cultured pituitary cells with FSP (0.1-3 nM) for 10 h did not alter the number of GnRH binding sites on the cell membranes. Finally, similar inhibitory effects on gonadotropin secretion in response to GnRH, PMA and mezerein were obtained with the 31 and 39 kDa forms of bovine FSP, each at a concentration of 1 nM. We conclude from the present study that FSP acutely inhibits GnRH-stimulated gonadotropin secretion in cultured pituitary cells, and that FSP exerts its action beyond the GnRH receptor, possibly by affecting the protein kinase C and/or the calcium-calmodulin systems.

Inhibin binding sites and proteins in pituitary, gonadal, adrenal and bone cells.

Activin signals via complexes of type I (50-55 kDa) and II (70-75 kDa) activin receptors, but the mechanism of inhibin action is unclear. Proposed models range from an anti-activin action at the type II activin receptor to independent actions involving putative inhibin receptors. Two membrane-embedded proteoglycans, betaglycan and p120, have recently been implicated in inhibin binding, but neither appears to be a signalling receptor. The present studies on primary cultures of rat pituitary and adrenal cells, and several murine and human cell lines were undertaken to characterise inhibin binding to its physiological targets. High affinity binding of inhibin to the primary cultures and several of the cell lines, like that previously described for ovine pituitary cells, was saturable and reversible. Scatchard analysis revealed two classes of binding sites (K(d) of 40-400 and 500-5000 pM, respectively). Affinity labelling identified [125I]inhibin binding proteins with apparent molecular weights of 41, 74, 114 and >170 kDa in all cell types that displayed high affinity, high capacity binding of inhibin. Additional labelling of a 124 kDa species was evident in gonadal TM3 and TM4 cell lines. In several cases, activin (> or =20 nM) competed poorly or not at all for binding to these proteins. The 74, 114 and >170 kDa inhibin binding proteins in TM3 and TM4 cells were immunoprecipitated by an anti-betaglycan antiserum. These three proteins correspond in size to the activin receptor type II and the core protein and glycosylated forms of betaglycan, respectively, that have been proposed to mediate anti-activin actions of inhibin, but the identity of the 74 kDa species is yet to be confirmed. Studies of [125I]inhibin binding kinetics and competition for affinity labelling of individual binding proteins in several cell lines suggest these three species and the 41 and 124 kDa proteins form a high affinity inhibin binding complex. In summary, common patterns of inhibin binding and affinity labelling were observed in inhibin target cells. Novel inhibin binding proteins of around 41 and 124 kDa were implicated in the high affinity binding of inhibin to cells from several sources.

Bombesin regulation of adrenocorticotropin release from ovine anterior pituitary cells.

Mammalian members of the bombesin-like peptide family (gastrin releasing peptides; GRP) have been localized in the ovine median eminence and in hypophysial-portal blood, suggesting a role in the regulation of anterior pituitary function. In this study we have shown that although bombesin cannot stimulate ACTH secretion alone, it potentiates release by ovine CRF, an effect blocked by the GRP receptor antagonist D-Tyr6bombesin (6-13) propylamide. Bombesin did not potentiate AVP-stimulated ACTH release; instead release was attenuated when bombesin was given at a 10-fold or greater molar excess over AVP, with no interaction seen at lower concentrations. We conclude that ovine corticotrophs express bombesin receptors, and that GRP may act in concert with other hypothalamic releasing factors to regulate ACTH secretion.

Action of 1,10-phenanthroline transition metal chelates and their constituents on the rat isolated diaphragm preparation.

The actions of two related series of fully co-ordinated, divalent 1,10-phenanthroline transition metal chelates have been investigated on the rat isolated diaphragm muscle-phrenic nerve preparation and, where possible, compared with those of their constituent metal ions and ligands. Each member of both series of chelates produced blockade of neuromuscular transmission, although mechanistically not of a uniform type, and several elicited varying degrees of muscle contracture. The kinetic reactivity of the metal chelate appeared to be an important factor determining the nature of the biological response and profound differences in response were observed between labile and inert chelates differing in some cases by only one electron in the 3d shell.

Metallothionein induction in mouse tissues by cis-dichlorodiammineplatinum(II) and its hydrolysis products.

cis-Dichlorodiammineplatinum(II) (cis-DDP) doubled the amount of metallothioneins (MTs) in the livers and kidneys of BALB/c mice when injected i.p. in a single high dose of 30 mumol/kg (9 mg/kg). Two such doses given 17 h apart increased hepatic MTs 5-fold and also increased the relative rate of incorporation of radiolabelled cyst(e)ine into hepatic MTs. Hydrolysed cis-DDP was more effective than cis-DDP, increasing MT-bound zinc 27-fold and [3H]cysteine incorporation 6-fold in liver while doubling each of these in kidney. The MTs from the livers of mice treated with cis-DDP bound zinc, copper and platinum in ratios of 5:1:0.3, respectively, similar to those in whole liver and its soluble fraction, indicating that MTs do not selectively sequester platinum under these circumstances. The effects of cis-DDP on zinc and copper levels in serum, liver and kidney suggest that induction of MTs by cis-DDP is not mediated by displacement of endogenous zinc. Indirect induction by corticosteroids secreted in a stress response to cis-DDP is also an unlikely cause. cis-DDP, probably in a hydrolysed form, can therefore induce and bind to MTs in normal tissues, particularly when given at repeated high dosage.