A human prostatic growth factor (hPGF): partial purification and characterization

A growth factor capable of stimulating DNA synthesis of BALB/3T3 cells was purified about 1,000-fold from the cytosol of human benign hypertrophic prostates by heparin-Sepharose chromatography; the growth factor bound to the column in the presence of 0.5 M NaCl was eluted with 1.5-1.7 M NaCl. Its molecular weight and isoelectric point were estimated to be 11,000-13,000 and 10.5, respectively. It was sensitive to heat- and acid-treatments but resistant to disulfide-reducing agent. The final preparation was able to stimulate DNA synthesis at 10 ng/ml. The degree of stimulation was dependent on serum concentration in the assay system; the degree of maximum stimulation increased about 5 times as serum concentration increased from 0.2 to 2%.     

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.     

Characterization of a sheep pituitary-derived growth factor for rat and human mammary tumor cells

A growth factor for rat and human mammary tumor cells (MTGF-Pit) was isolated from lyophilized powders of whole sheep pituitaries by a rapid four-step procedure utilizing acetic acid extraction, heating at 93 degrees C, and sequential chromatography in 0.10 M acetic acid on sulphopropyl Sephadex and Sephadex G-50. From 10 g of pituitary powder, 8-10-mg amounts of MTGF-Pit were isolated. By 8 M urea, 0.1% SDS-12.5% polyacrylamide gel electrophoresis analysis followed by Coomassie blue staining, this preparation was shown to be one major stained band. When assayed for growth effects on cells maintained in serum-free medium, 5.1-19.2 nM MTGF-Pit half replaced the growth of MTW9/PL rat and MCF-7 and T-47D human mammary tumor cells in response to 2% to 10% serum. MTGF-Pit shows mitogenic activity toward normal human diploid fibroblasts only at concentrations in excess of 2.5 X 10(-4) M, while rat fibroblasts are unresponsive even at this high concentration. From data available, we conclude that a mitogenic activity for epithelial-type mammary cells has been isolated, and this growth factor appears to be a previously undetected acid- and heat-stable activity that is highly abundant (estimated at 0.16% or more of the total dry weight of the pituitary powder). The isolated ovine MTGF-Pit (3,900 +/- 200 daltons) does not share the molecular weight of native prolactin (24,000 daltons), "cleaved" prolactin (16,000 daltons), or growth hormone (22,000 daltons), and by all tests applied cannot be replaced with other known hormones and purified growth factors. We conclude a potent new mammary tumor cell mitogenic activity has been identified from sheep pituitaries.     

Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

Atrial natriuretic factor inhibits vasopressin secretion from rat posterior pituitary

The effects of synthetic atrial natriuretic factor (ANF) were studied in superfused rat posterior pituitary gland. ANF (10(-6)M, 10(-10)M) significantly inhibited basal as well as KC1 (50 mM) or angiotensin II-stimulated immunoreactive arginine vasopressin secretion. The magnitude of inhibition was greater at 10(-6)M than at 10(-10)M. ANF also decreased cAMP secretion and increased cGMP secretion from the posterior pituitary. These results suggest that ANF directly acts on the posterior pituitary to inhibit arginine vasopressin secretion and that this effect is, at least, partly mediated by the changes in cyclic nucleotide production.     

Identification of estrogen-responsive genes in the GH3 cell line by cDNA microarray analysis

To identify estrogen-responsive genes in somatolactotrophic cells of the pituitary gland, a rat pituitary cell line GH3 was subjected to cDNA microarray analysis. GH3 cells respond to estrogen by growth as well as prolactin synthesis. RNAs extracted from GH3 cells treated with 17beta-estradiol (E2) at 10(-9) M for 24 h were compared with the control samples. The effect of an antiestrogen ICI182780 was also examined. The array analysis indicated 26 genes to be up-regulated and only seven genes down-regulated by E2. Fourteen genes were further examined by real-time RT-PCR quantification and 10 were confirmed to be regulated by the hormone in a dose-dependent manner. Expression and regulation of these genes were then examined in the anterior pituitary glands of female F344 rats ovariectomized and/or treated with E2 and 8 out of 10 were again found to be up-regulated. Interestingly, two of the most estrogen-responsive genes in GH3 cells were strongly dependent on E2 in vivo. #1 was identified as calbindin-D9k mRNA, with 80- and 118-fold induction over the ovariectomized controls at 3 and 24 h, respectively, after E2 administration. #2 was found to be parvalbumin mRNA, with 30-fold increase at 24 h. Third was c-myc mRNA, with 4.5 times induction at 24 h. The levels were maintained after one month of chronic E2 treatment. Identification of these estrogen-responsive genes should contribute to understating of estrogen actions in the pituitary gland.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

Desensitization of rat pituitary somatotrophs to growth hormone-releasing factor occurs in vitro

Desensitization of rat pituitary somatotrophs to human growth hormone-releasing factor (hGHRF) was investigated using cultured rat anterior pituitary cells. Growth hormone (GH) release decreased but the production of cAMP was still induced in response to subsequently added 10(-9) M hGHRF from cells pretreated with hGHRF at concentrations ranging from 10(-11) to 10(-7) M for 4 h. Desensitization to 10(-9) M hGHRF was also observed in cells pretreated with 10(-9) M hGHRF for 4 h in the presence of 2 mM EGTA, 10 ng/ml nifedipine or 10(-9) M somatostatin-28, which decreased GH release during pretreatment. Forskolin and A23187, at concentrations of 10(-6) M and 10(-4) M, respectively, stimulated GH release from cells pretreated with hGHRF to the same extent as that from the control cells. These results, therefore, suggest that desensitization to GHRF occurs regardless of the presence of releasable GH pool and that some changes such as uncoupling of GHRF receptors with adenylate cyclase and decreased sensitivity to cAMP of cAMP-dependent protein kinase of the secretory mechanism of GH, in addition to the decrease in releasable GH pool and down regulation of GHRF receptors, may be involved in the desensitization mechanism.     

Purification and characterization of a human pituitary growth factor

A growth factor has been purified to homogeneity from human pituitary glands. The pituitary growth factor (PGF) is trypsin-sensitive and acid- and heat-labile and has a molecular weight of 18,000 and an isoelectric point of 7.5. PGF was purified by heparin and copper affinity chromatography followed by carboxymethylcellulose 52. The amino-terminal amino acid sequence of PGF was established as PALPEXGGXGA and is identical with that of basic fibroblast growth factor at the identified amino acid residues. PGF was mitogenic for rabbit fetal chondrocytes and bovine corneal endothelial cells in the range of 0.015-15 ng mL-1. Heparin alone at low concentrations (0.5 microgram mL-1) was found to be weakly mitogenic for rabbit fetal chondrocytes. In combination with PGF a marked increase in cell growth was observed, which was inhibited by protamine sulfate. These data demonstrate the presence of a potent mitogen in human pituitaries that is structurally related to basic fibroblast growth factor and synergizes with heparin to promote cell growth.     

Growth hormone-releasing peptide (GHRP) binding to porcine anterior pituitary and hypothalamic membranes.

The synthetic hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP, Growth Hormone-Releasing Peptide), has no structural similarities with any of the GH-releasing peptides known and its action in releasing GH is by a complementary but yet not clearly defined action on the pituitary as well as hypothalamus. Therefore, in vitro studies have been performed to demonstrate and characterize GHRP binding sites on peripheral membranes of both porcine pituitary and hypothalamus. The membrane binding sites were specific, reversible, saturable and time, temperature, pH and concentration dependent under optimum binding assay conditions. The maximum specific binding was observed between pH 5.0 and 6.0. In the presence of Ca2+ and Mg2+ ions, with or without chelating agents there was a significant reduction in the specific binding. Scatchard analysis of these binding sites using increasing doses of unlabeled GHRP revealed a single low affinity site with a 2.1 x 10(-5) M and 1.7 x 10(-5) M and a maximum number of sites of 10 nmol/mg protein and 5 nmol/mg protein for pituitary and hypothalamus, respectively. It is also observed that (D-Lys3)-GHRP, substance P antagonists and growth hormone-releasing factor analog were potent inhibitors of GHRP binding in both tissues.     

A permissive role for extracellular Ca2+ in regulation of prolactin production by 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

A clonal strain of rat pituitary tumor cells (GH3) that spontaneously synthesizes and secretes prolactin (PRL) and growth hormone (GH) was used as model system to study the mechanism of action of 1,25-(OH)2D3. We have previously demonstrated that these cells possess specific cytosol binding proteins for 1,25-(OH)2D3 (Haug and Gautvik, 1985). When the GH3 cells were incubated in a serum-free, chemically defined medium of low extracellular Ca2+ concentration, 1,25-(OH)2D3 stimulated PRL production in a dose-dependent manner. The stimulation was detectable at 10(-11) M, and the maximum effect (2-fold increase) was observed at 10(-9) M (ED50 = 2 x 10(-11) M). The dose-response curve was bell-shaped, and at 10(-6) M 1,25-(OH)2D3 even suppressed PRL production to about 75% of controls. The stimulatory effect was first seen after 2 days and was maximal after 4 days. On a molar basis 25-OHD3 and 1-OHD3 were at least 100 times less potent than 1,25-(OH)2D3, while 24,25-(OH)2D3 had no effect on PRL production. At an extracellular concentration of Ca2+ as low as 4 x 10(-5) M the stimulatory effect of 1,25-(OH)2D3 was small (1.3-fold). Increasing extracellular Ca2+ to 1.5 x 10(-4) M increased the 1,25-(OH)2D3-induced PRL response to 2.1-fold. In contrast to the biphasic effect of 1,25-(OH)2D3 on PRL production, GH production was decreased to about 60% of controls at 10(-8) M and above. These findings indicate that in serum-free medium the stimulatory effect of 1,25-(OH)2D3 on PRL production is critically dependent on the concentration of extracellular Ca2+.     

Angiotensin II stimulation of the rat pituitary tumoral cell proliferation in vitro.

The effect of angiotensin II (AT II) on proliferation of rat pituitary tumoral cells was investigated in vitro. The tumoral cells were isolated from the prolactin-secreting pituitary tumors induced by stilboestrol implantation. The incorporation of [3H]-thymidine into DNA was used as an index of cell proliferation. It was found that AT II significantly enhanced the [3H]-thymidine incorporation into pituitary tumoral cells in the concentrations of 10(-10) and 10(-8) M. The stimulatory effect disappeared at the concentration of 10(-6) M. The possible involvement of pituitary renin-angiotensin system in pituitary tumorigenesis was discussed.     

Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands

Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.     

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.     

Endothelial cell mitogens derived from retina and hypothalamus: biochemical and biological similarities

Bovine retina and hypothalamus contain anionic endothelial cell mitogens that display unusual affinities for the negatively charged glycosaminoglycan heparin. Both growth factor activities are acidic polypeptides (pl's of 5.0) as determined by isoelectric focusing and DEAE-affinity chromatography. In spite of their anionic nature, the factors bound to heparin-Sepharose columns with high affinity and could be eluted only at high salt concentrations (0.9-1.1 M NaCl). The affinity of the retina-derived growth factor (RDGF) for heparin permitted a 15,000-fold purification of the mitogen in two steps: heparin-affinity chromatography and size exclusion high-performance liquid chromatography. RDGF and the anionic hypothalamus-derived factor (aHDGF) exhibit three major biochemical similarities including isoelectric point, (pl's of 5.0), heparin affinity (elution at 0.9-1.1 M NaCl) and molecular weight (18,000). Additionally, the two factors display similar biological activities, stimulating the proliferation of capillary and human umbilical vein endothelial and 3T3 cells but not vascular smooth muscle cells. We suggest that RDGF and aHDGF are related if not identical growth factor molecules.     

Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells

A procedure is described for purification of a factor (D-factor)-inducing differentiation of mouse myeloid leukemic cells (M1) into macrophages from serum-free mouse L929 cell-conditioned medium. The procedure included ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-200 and phenyl-Sepharose column chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel-filtration column. The purified factor gave a single band of protein with a molecular weight of 62,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with biological activity. Its half-maximal concentration for inducing differentiation of M1 cells into macrophages was 1.7 X 10(-11) M. Even at 2.6 X 10(-9) M, it did not induce colony formation of normal bone marrow cells, suggesting that it was distinct from the growth factor for normal precursors of macrophages and/or granulocytes.     

Tumor necrosis factor-alpha affects growth hormone secretion by a direct pituitary interaction.

Administration of 50, 250, and 1,250 ng/kg iv of recombinant bovine tumor necrosis factor-alpha (RBTNF) did not affect basal plasma concentrations of growth hormone (GH) or thyroid-stimulating hormone in male calves. However, when administered 30 min before challenge with 1 microgram/kg iv of thyrotropin-releasing hormone (TRH), 250 ng/kg of RBTNF increased the subsequent incremental GH response. At 1,250 ng/kg of RBTNF, GH response to TRH was significantly blunted. For each dose of RBTNF administered, the incremental change in plasma thyroid-stimulating hormone following TRH was not significantly different from control. To examine direct effects of RBTNF on pituitary function, fresh bovine pituitaries were sliced into 1-mm cubes and incubated with 0 or 10(-8), 10(-9), or 10(-10) M RBTNF. Additional cultures were treated with 10(-8) or 10(-9) M GH-releasing factor or 10(-8) M TRH and 0 or 10(-8) M RBTNF. Media GH increased in cultures with 10(-10) M RBTNF and declined linearly as RBTNF concentration increased. RBTNF blocked GH release from GH-releasing factor- and TRH-challenged pituitary slices. Membranes prepared from homogenized bovine pituitaries had specific saturable binding characteristics for monomeric 125I-RBTNF. Membranes treated with 4 M MgCl2 for 10 min and washed free of Mg2+ produced Scatchard plots fit to a two-site model (high affinity site Kd = 6.6 nM), while Scatchards of non-Mg(2+)-treated membranes fit a single site (Kd = 8.9 nM). Polyacrylamide gel electrophoresis separation of 125I-RBTNF cross-linked pituitary membranes showed specific binding of monomeric 125I-RBTNF to protein components ranging in molecular weight from 19,000 to 77,000. The data suggest that RBTNF has modulatory effects on the regulation of GH secretion acting directly at the pituitary through specific receptors.