Regulation of somatostatin and growth hormone-releasing factor by gonadal steroids in fetal rat hypothalamic cells in culture.

The mechanism underlying the sexually dimorphic pattern of growth hormone (GH) secretion in the rat has not been clearly elucidated. In the present study, we assayed the possible direct effect of gonadal steroids on both somatostatin (SS) and growth hormone-releasing factor (GRF) in fetal rat hypothalamic cells in culture. Hypothalamic cells, obtained by mechanical dispersion, were maintained as monolayer cultures in serum-supplemented medium. After 20 days in culture, cells were incubated with serum free medium containing testosterone (T, 10, 20, 40 ng/dl) or estradiol (E, 0.1, 1, 10 ng/dl) for 48 h. At the end of the experiments, immunoreactive SS (IR-SS) and immunoreactive GRF (IR-GRF) were measured by specific radioimmunoassays (RIAs) in media and cell extracts. After 48 h of incubation with testosterone, somatostatin in both media and cells was significantly reduced. On the contrary, this treatment lead to a dose-dependent increase in media and cell GRF content. When cells were incubated with estradiol for 48 h, a significant inhibition in medium SS release was observed, whereas intracellular SS slightly increased at the highest concentration of 10 ng/dl. Estradiol treatment resulted in an inconsistent decrease in media and cells IR-GRF. Our results indicate that both SS and GRF are under the influence of testosterone and estradiol acting at the hypothalamic level, and furthermore suggest that at this stage of brain development, gonadal steroids may regulate GH secretion through their ability to modulate hypothalamic SS and GRF.     

The effects of bumetanide and furosemide on lung liquid secretion in fetal sheep

Thirty-four experiments were carried out on the effects of loop diuretics on lung liquid secretion in 20 fetal sheep (128-145 days gestation) with indwelling catheters. Bumetanide placed in the lung liquid at 2.19 +/- 0.52 X 10(-4) M produced immediate reabsorption of fluid, and effects lasted 3 hr (n = 6). Bumetanide at 1.1 +/- 0.17 X 10(-5) M reduced secretion significantly for 2 hr (n = 4), but at 1.07 +/- 0.06 X 10(-6) M there was no clear effect (n = 6). Controls showed no significant change (n = 6). Furosemide was less effective. At 3.1 +/- 0.07 X 10(-3) M it produced an immediate reabsorption, which lasted 3 hr, but at 1.0 +/- 0.04 X 10(-4) M it increased secretion slightly (n = 4); controls showed no significant change (n = 6). The results are consistent with the presence of a chloride transport system, perhaps with sodium cotransport, as the major factor in fetal lung liquid secretion.     

Divergent Effects of Acute Depolarization on Somatostatin Release and Protein Synthesis in Cultured Fetal and Neonatal Rat Brain Cells

The influence of membrane depolarization on somatostatin secretion and protein synthesis by fetal and neonatal cerebrocortical neurons was studied. Cortical cells obtained by mechanical dispersion were maintained as monolayer cultures for 8 days. The ability of fetal cerebrocortical and hypothalamic cells to release immunoreactive somatostatin (IR-SRIF) was confirmed. Total protein synthesis was determined by the incorporation of [3H]phenylalanine into trichloroacetic acid-precipitable proteins. To study the effect of acute depolarization on protein synthesis, cells were incubated for 30 min with [3H]phenylalanine or [3H]leucine and the depolarizing agent. In fetal cerebrocortical cells, potassium (30 and 56 mM) decreased protein synthesis and RNA levels and increased IR-SRIF release. Depolarization by veratridine, a sodium channel activator, induced a similar effect. The effect of veratridine on IR-SRIF and protein synthesis was reversed by tetrodotoxin, a sodium channel blocker, or verapamil, a calcium channel blocker. These findings suggest that protein synthesis by cerebrocortical cells is decreased in fetal brain cells by membrane depolarization and is dependent on Na+ and Ca2+ entry into cells. In postnatal (day 7) cerebrocortical cells, depolarization induced by high potassium concentrations led to a concomitant increase in protein synthesis, RNA content, and somatostatin release. These findings indicate that depolarization of the cellular membrane is coupled to an increase in protein synthesis in neonatal, but not in fetal, dispersed brain cells.     

Control of interferon-tau secretion by in vitro-derived bovine blastocysts during extended culture and outgrowth formation

A series of experiments was conducted to examine the pattern of interferon-tau (IFN-tau) secretion by bovine blastocysts during extended culture in vitro. In the first experiment, blastocysts were cultured individually for three 48-hour periods. The day of blastocyst formation affected how much IFN-tau was produced during the first two culture periods, but not during the third period. The overall secretion of IFN-tau during the 6-day period increased significantly and well beyond what could be accounted for by the concomitant increase in cell numbers. In the second experiment, blastocysts were initially cultured in individual droplets for 48 hr, then plated into 48-well plates. Medium concentrations of IFN-tau were determined after 48 hr and again after 6 and 12 days of culture. Initial IFN-tau secretion did not affect the ability to form outgrowths or their final size, and initial differences in secretion between groups of blastocysts had disappeared by the second and third analyses. In the third experiment, blastocysts were cultured individually for 48 hr in droplets containing the medium that had been flushed through the uteri of non-pregnant sheep on days 10, 12, and 15 of the estrous cycle. Culture in the medium obtained from the Day 15 flush significantly increased the number of cells that blastocysts contained, as well as IFN-tau secretion.     

3H-cyclosporine internalization and secretion by human fetal pancreatic islets

Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude. It is concluded that human fetal pancreatic islets during 48 hr of culture in the presence of pharmacologically relevant concentrations of CsA can internalize the drug, which is compartmentalized and concomitantly secreted with insulin following maximal stimuli. Transplanted human fetal islets utilized as delivering units for CsA could be beneficial for the induction of immunotolerance to allografted fetal islets.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.     

Regulation of insulin synthesis in an insulin-producing cell line (RINm5F): Long-term experiments

Summary The insulin-producing cell line RINm5F, has been used in short-term experiments to evaluate insulin secretion. We sought to maintain the responsiveness of these cells to stimuli for up to 2 days. We examined the course of new insulin synthesis over this period by measuring at intervals immunoreactive insulin (IRI) in two parts: IRI in the medium (M) and IRI extracted from the cells (C). Control cells were incubated in RPMI 1640/2.8 mM glucose/10% fetal bovine serum/200 μg/ml bacitracin (to prevent insulin degradation). The addition of dibutyryl cAMP 10 mM to the experimental dishes significantly increased total (M+C) IRI at 48 hr to 37% above the insulin content of the control dishes (p<0.01). Theophylline 10 mM increased total (M+C) IRI by 24% over control (p<0.05) after 24 hrs. Glucose, glyceraldehyde, leucine, arginine, glucagon and tolbutamide, other stimulants of insulin production, had no effect. Under the experimental conditions reported here, including the use of bacitracin, IRI synthesis can be studied for up to 48 hr. Portions of this study have been published in abstract form for the 47th Annual Meeting of the American Diabetes Association, Indianapolis, Indiana, 1987. Supported in part by the American Diabetic Association, Maryland Affiliate.     

The influence of dehydroepiandrosterone on basal and gonadotrophin-stimulated testosterone secretion by Mongolian gerbil interstitial cells incubated or superfused in vitro

1. Testosterone secretion by Mongolian gerbil interstitial cells incubated in the absence of HCG linearly increased with cell concentration (1 x 10(5) cells: 0.6 ng/4 hr, 10 x 10(5) cells: 8.0 ng/4 hr). Addition of 100 mIU HCG resulted in a drastic increase of testosterone secretion which was linear between concentrations of 1 x 10(5) and 4 x 10(5) cells. 2. Compared to HCG-stimulated testosterone release, secretion was significantly higher by cells incubated with 60-100 ng DHEA. 3. During the 4-hr incubation period, 53-69% of added progesterone and 72-88% of added dehydroepiandrosterone (DHEA) were converted to testosterone by cells freshly prepared or stored for 1-3 days at 4 degrees C. On the other hand, prolonged storage at 4 degrees C resulted in a marked decrease of HCG-stimulated testosterone secretion. 4. Testosterone secretion by interstitial cells superfused in vitro increased with the length of HCG (100 mIU/ml) application from 0.08 to 0.22 ng/10(6) cells/min (10 and 60 min, respectively). A much faster and pronounced elevation was found when cells were stimulated with DHEA (200 ng/ml: 0.06-0.80 ng/10(6) cells/min, 0 and 20 min, respectively). 5. After interstitial cells have been stimulated with a DHEA (200 ng/ml) pulse for 30 min and then superfused with medium only for an additional 30 min, testosterone secretion remained significantly elevated and could not be further stimulated by superfusing medium which contained as much as 100 mIU/ml HCG.     

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.     

Depolarizing influences regulate somatostatin synthesis and processing in cultured cerebral cortical cells

There is increasing evidence that persistent depolarization plays a critical role not only in excitation-secretion coupling, but also in the mechanisms linking excitation of neuronal cells to long-term adaptative changes in biosynthesis of neuropeptides. Somatostatin (SRIF) release and synthesis are affected by numerous agents, such as high concentrations of potassium that cause depolarization of cellular membrane. In the present work, we tried to determine whether prolonged exposure to veratridine (VTD) regulates SRIF synthesis. We found that exposure to VTD (100 microM) resulted in the stimulation of total (cell content + media) immunoreactive SRIF (IR-SRIF). This effect was calcium- and sodium-dependent, since it was prevented when verapamil (VPM) 20 microM or tetrodotoxin (TTX) 1 microM were added simultaneously with VTD. Cerebral cortical cells were exposed to high potassium concentrations, and the nature of the IR-SRIF was characterized by high-pressure liquid chromatography (HPLC) or gel filtration. It was evident that chronic exposure to high potassium concentrations modified the elution profile of medium IR-SRIF on HPLC and gel filtration, causing an increase in somatostatin-28 (S-28) and a decrease in somatostatin-14 (S-14). The results indicate that chronic exposure to VTD or high potassium concentration increases immunoreactive somatostatin and augments synthesis of its high-molecular-weight forms. This suggests that chronic membrane depolarization activating sodium and calcium channels initiates the entry of calcium ions, which triggers somatostatin release and causes a depletion of its intracellular stores. The stimulation of somatostatin secretion could be coupled to synthesis of the peptide.     

Fetal adrenal VIP: distribution and effect on medullary catecholamine secretion

Vasoactive intestinal peptide (VIP) was found in the adrenal gland of ovine fetuses at 130-135 days gestation and was shown to stimulate catecholamine secretion. VIP was demonstrated by immunocytochemistry using the indirect antibody-enzyme method. VIP-immunoreactive nerve fibers were observed in the capsule, zona glomerulosa and inner layer of the cortex as well as in the medulla; furthermore small clusters of VIP-containing cell bodies were found at the corticomedullary border. To study the direct effect of VIP on catecholamine release, fetal adrenal medulla was dispersed into single cells and incubated in vitro with VIP for 6 hours. Catecholamine release into the medium was measured at 1, 3 and 6 hours. At 6 hours of incubation, VIP stimulated total catecholamine release from fetal adrenomedullary cells in a dose-dependent manner at concentrations ranging from 10(-8) to 10(-4) M. The release of norepinephrine and epinephrine, but not dopamine, was significantly enhanced. The presence of VIP in the fetal adrenal cortex and medulla, and the ability of VIP to stimulate catecholamine release from fetal adrenomedullary cells in vitro suggest that VIP may be an important modulator of medullary catecholamine secretion during fetal life.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

Interleukin 3-dependent mouse mast cells express the cholera toxin-binding acidic glycosphingolipid, ganglioside GM1, and increase their histamine content in response to toxin

The acidic glycosphingolipid, ganglioside GM1, which is the binding site for cholera toxin on many cell types, was identified by chemical and by flow cytometric analyses of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC). Ganglioside GM1 and other acidic glycosphingolipids were isolated from BMMC by chloroform/methanol extraction and chromatography on DEAE-Sephadex and were analyzed by thin layer chromatography. The presence of ganglioside GM1 in the BMMC extract was demonstrated by its co-migration with ganglioside GM1 standard in thin layer chromatography and by the binding of peroxidase-labeled cholera toxin B subunit to both molecules. As assessed by fluorescence flow cytometric analysis of the binding of fluorescein-conjugated cholera toxin B subunit, the majority of BMMC expressed ganglioside GM1 on their surface, and the total presentation per cell increased as cells progressed from the G1 to S to G2 + M phases of the cell cycle. The addition of increasing amounts of cholera toxin starting with 0.08 microgram/ml to BMMC cultured in 50% WEHI 3-conditioned medium containing IL 3 for 48 hr caused the adhesion of BMMC to the tissue culture flasks to increase in a dose-related manner, from less than 1% adherent cells in cultures without toxin to a plateau value of approximately 17% adherent in the presence of 1.25 micrograms/ml of toxin. The histamine content of BMMC increased from 26.7 +/- 3.59 ng/10(6) cells (mean +/- SD, n = 4) for control cultures to 201 +/- 17.4 ng/10(6) cells (mean +/- SD, n = 4) for nonadherent cells and to 588 +/- 89.4 ng/10(6) cells (mean +/- SD, n = 4) for adherent cells after 48 hr of culture in 0.31 microgram/ml cholera toxin, which was the optimal dose for nonadherent and adherent populations. The content of another preformed intragranular mediator, beta-hexosaminidase, did not increase appreciably in the presence of cholera toxin (n = 3). The increase in the histamine content of BMMC after the addition of 0.31 microgram/ml cholera toxin was detectable at 4 hr, plateaued by 24 to 48 hr, and gradually declined over the next 6 days. Cholera toxin also augmented the histamine content of BMMC in the presence of purified synthetic IL 3. Preincubation of whole cholera toxin with purified ganglioside GM1 inhibited the histamine-augmenting effects of cholera toxin on BMMC, indicating that the effect was not due to a contaminant, and neither the A nor B subunit of cholera toxin alone increased the histamine content of BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-l-glutamine

Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 × 10^–6 M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10^–6, 10^–5, or 10^–4M) or glycyl-L-glutamic acid (10^–6 M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradully increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t_1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.Abbreviations AChE acetylcholinesterase - NG108-15 cell neuroblastoma-glioma 108-15 cell - DMEM Dulbecco's modified Eagles minimal essential medium - FBS fetal bovine serum - GLGA glycyl-L-glutamic acid - L-GA L-glutamic acid - GLG glycyl-L-glutamine - GD soman The opinions or assertions contained herein are the private views of the authors and are not to be construed as reflecting the view of the Department of the Army or the Department of the Army or the Department of Defense.     

Reconstituted basement membrane influences prolactin, LH, and FSH secretion from adult and fetal adenohypophyseal cells in vitro.

These investigations tested the hypothesis that secretion of prolactin (PRL), LH, and FSH in vitro is influenced by the substratum on which adult or fetal adenohypophyseal cells are cultured. Adenohypophyses were removed from adult male Golden Syrian hamsters and from fetal hamsters on day 16 of gestation. The glands were dissociated and cultured in a 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 medium containing 10% fetal bovine serum (FBS), 25 mM Hepes, and antibiotics. The cells were cultured on three substrata: glass, laminin, and the reconstituted basement membrane of the Engelbreth-Holm-Swarm (EHS) tumor (Matrigel). Medium was collected and replaced every 48 h for 14-22 days. Concentrations of PRL, LH, and FSH in medium were measured by RIA. The substratum influenced hormone secretion. PRL concentrations were elevated in cultures of adult cells on Matrigel in each of four experiments. Adenohypophyseal cells on Matrigel maintained a rounded shape longer than cells on glass or laminin. In studies using fetal adenohypophyseal cells, PRL concentrations were elevated significantly in medium from cultures on Matrigel at and after 2 days as were concentrations of LH and FSH after 6 days. Additional experiments showed that the higher PRL concentrations in medium surrounding adult cells plated on Matrigel were not due to the release of soluble factors from Matrigel, differential cell attachment on Matrigel, the differential presence of adenohypophyseal fibroblasts, nor differential rates of cell proliferation. The results show that Matrigel maintains the secretion of PRL from adult adenohypophyseal cells in vitro more effectively than glass or laminin substrata and support the hypothesis that cell-matrix interactions mediate the observed differences. The results also show that in long-term cultures (14-22 days), fetal adenohypophyseal cells secrete significantly more PRL, LH, and FSH on Matrigel than they secrete when cultured on glass or laminin. Thus, Matrigel influences the function and possibly the maturation of adenohypophyseal cells in vitro. Furthermore, although laminin is the most abundant component in Matrigel, the effects of Matrigel on lactotrophs and gonadotrophs in vitro are probably not attributable solely to its laminin content.     

Leukocyte complement: interleukin-like properties of factor Bb

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".     

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

Forskolin has biphasic effects on osteoprogenitor cell differentiation in vitro

Cells isolated from fetal rat calvaria (RC) and maintained in vitro in medium containing ascorbic acid and B-glycerophosphate form three-dimensional, mineralized nodules having the histological, immunohistological, and ultrastructural characteristics of woven bone. We have studied the effects of forskolin (FSK), a diterpene that activates adenylate cyclase, in this system. While 10(-7)-10(-5) M FSK significantly stimulated cAMP levels in RC cells, lower concentrations did not. cAMP levels with 10(-5) M FSK reached a maximum by 30 min at 37 degrees C and returned to basal level in 2-3 hr. Changes in cAMP levels correlated with changes in cellular shape: cells treated with 10(-5) M FSK assumed a stellate morphology, lost microfilament bundles, and reduced their substrate adhesiveness, while cells treated with 10(-9) M were not affected. Exponential growth and saturation densities of FSK-treated cultures were similar to untreated cultures, indicating that FSK was neither toxic nor stimulatory to the population. The effect on bone nodule formation of FSK present continuously depended on concentration: 10(-5) M FSK significantly inhibited the number of nodules formed, while 10(-9) M FSK significantly stimulated bone nodule formation. Single short treatments with either 10(-5) M or 10(-9) M FSK had no effect on nodule formation, but repeated short duration treatments (1 hr every 2 days for 21 days) gave results similar to continuous exposure. These results indicate that intermittent elevations in intracellular cAMP have an inhibitory effect on bone formation. In addition, our work indicates that low concentrations of FSK stimulate differentiation of osteoprogenitor cells possibly through a non-cAMP-dependent process.     

Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line

A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.