Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta

Specific binding site for atrial natriuretic factor (ANF), a potent natriuretic and vasorelaxant polypeptide recently isolated from mammalian atria, was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. Binding studies of 125I-labeled-synthetic alpha-human natriuretic peptide (alpha-hANP) revealed the presence of a non-interacting, single class of high affinity binding sites for alpha-hANP on VSMC in culture: the apparent dissociation constant (Kd) was approximately 1-2 X 10(-9)M and the number of maximal binding sites was approximately 200,000-300,000 sites/cell. A variety of vasoactive substances and other polypeptide hormones did not affect the binding of 125I-labeled-alpha-hANP to its binding sites. alpha-hANP significantly increased the concentrations of intracellular cyclic GMP in VSMC in a dose-dependent manner (3.2 X 10(-9)-1.6 X 10(-7)M). These data indicate that the specific receptor for ANF is present in VSMC and suggest that intracellular cyclic GMP may be involved in its vasorelaxant effect.     

Identification and initial characterization of an autocrine pheromone receptor in the protozoan ciliate Euplotes raikovi

The polypeptide pheromone Er-1, purified from the ciliate Euplotes raikovi of mating type I and genotype mat-1/mat-1, was iodinated with 125I-Bolton-Hunter reagent to a sp act of 0.45-0.73 mu Ci/microgram of protein. This preparation of 125I-Er-1 bound specifically to high affinity binding sites on the same cells of mating type I. Binding of 125I-Er-1 occurred with an apparent Kd of 4.63 +/- 0.12 X 10(-9) M in cells in early stationary phase. It was estimated that these cells carry a total number of approximately 5 X 10(7) sites/cell, with a site density that falls in the range of 1,600-1,700/microns 2 of cell surface. Unlabeled Er-1, other homologous pheromones such as Er-2 and Er-10, antibodies specific for Er-1, and human IL-2 were shown to act as effective inhibitors of specific binding of 125I-Er-1 to mating type I cells. The "autocrine" nature of the identified specific high affinity binding sites for Er-1 was further substantiated by cross-linking experiments. These experiments revealed that mating type-I cell membranes contain one protein entity of Mr = 28,000 that is capable of reacting specifically with the homodimeric native form of Er-1.     

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

Specific endothelin binding sites in renal medullary collecting duct cells: lack of interaction with ANP binding and cGMP signalling.

The diverse biological actions of endothelins (ET) appear to be mediated by specific cell-surface receptors. Autoradiography and membrane binding studies have shown abundant ET binding sites in the kidney. However, their expression in specific types of renal cells is unclear. We studied the binding of 125I-labelled endothelin-1 in freshly isolated cell suspensions from canine inner medullary collecting duct. Competition binding experiments revealed the presence of specific high-affinity binding sites: unlabelled ET-1 and ET-2 compared with the radioligand with an IC50 of 135 and 83 pM, respectively, while the IC50 of ET-3 and big ET-1 were 2 and 4 orders of magnitude higher, indicating the presence of ETA-type receptor. Angiotensin II, vasopressin, and atrial natriuretic peptide (ANP) did not compete for ET binding even at a concentration of 10(-6) M. Saturation binding experiments showed a single class of binding sites of high density (Bmax = 56.7 +/- 10.3 fmol/10(6) cells) and high affinity (Kd = 69.8 +/- 10 pM). In contrast, ANP receptors in the same cell preparations appeared as two classes of binding sites with widely different affinity and density. The high-affinity ANP site (Kd = 311 +/- 48 pM) was compatible with ANP-B (guanylate cyclase-coupled) receptor. ET-1 did not compete for this receptor. ET-1 (10(-7) M) did not alter ANP-induced cGMP generation in these cells (3.8-fold increase at 10(-7) M ANP), nor basal levels of cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.     

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of 125I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.     

Purification and characterization of cytostatic lymphokines produced by activated human T lymphocytes. Synergistic antiproliferative activity of transforming growth factor beta 1, interferon-gamma, and oncostatin M for human melanoma cells

Supernatants from activated human T lymphocytes were highly growth inhibitory for A375 human melanoma cells. Three growth inhibiting polypeptides, transforming growth factor beta 1 (TGF-beta 1), interferon-gamma (IFN-gamma), and oncostatin M, were isolated from the acid-soluble fraction of serum-free T cell-conditioned medium and purified by gel permeation chromatography and reverse-phase high performance liquid chromatography in volatile solvents at acid pH. The purification was monitored in a growth inhibition assay. The release of TGF-beta 1 biologic activity by and the purification of IFN-gamma from the medium of activated human peripheral blood T lymphocytes have been reported. We now describe the isolation of oncostatin M from the conditioned medium of activated human T cells. The concentration of oncostatin M required for half-maximal inhibition of A375 melanoma cells was approximately 4 pM when assayed in the presence of 10% fetal bovine serum. The purified oncostatin M had an apparent m.w. 28,000 and an amino-terminal sequence that was identical with the sequence of oncostatin M isolated from supernatants of macrophage-like cells. Suboptimal concentrations of TGF-beta 1 in combination with suboptimal concentrations of IFN-gamma or oncostatin M resulted in synergistic antiproliferative responses for A375 cells (1.9 and 3.1 times the expected additive responses, respectively). Combinations of oncostatin M and IFN-gamma added simultaneously to A375 cells caused an additive growth inhibitory response. These results demonstrate that oncostatin M is a novel lymphokine, and its interaction with other cytostatic polypeptide growth inhibitors may play a role in the immune regulation of tumor cell growth.     

Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

We demonstrated bradykinin receptors in human endothelial cells and studied whether bradykinin receptors might be regulated by cyclic AMP. Messenger RNA for bradykinin B(1) and B(2) receptors was detected with real-time PCR and B(2) receptor protein was confirmed by immunoblotting. Saturation binding experiments with increasing concentrations of (125)I-[Tyr(8)]-bradykinin (25-700 pM) were made to determine maximal binding capacity and dissociation constant. However, saturation binding experiments suggested one class of binding sites, maximal binding capacity of 39.3 +/- 1.3 fmol/mg protein and dissociation constant of 352 +/- 27 pM. Competition studies with bradykinin B(1) and B(2) receptor antagonists showed that binding was competed by a B(1) antagonist, and when internalization was inhibited with hypertonic buffer, by both B(1) and B(2) antagonists. Stimulating cells with dibutyryl-cAMP, cholera toxin and forskolin for 24 h increased (125)I-[Tyr(8)]-bradykinin (90 pM) binding with approximately 50%. Saturation binding experiments with dibutyryl-cAMP stimulated cells showed, that the dissociation constant was altered from 352 +/- 27 pM in non-stimulated cells, to 203 +/- 18 pM (P < 0.001) in stimulated cells, while maximal binding capacity remained unchanged. Binding was competed similarly by the B(1) antagonist in stimulated and control cells. These results suggest, that the dibutyryl-cAMP stimulated increase in (125)I-[Tyr(8)]-bradykinin binding is probably due to increased B(1) receptor affinity with no change in receptor capacity. In conclusion, bradykinin B(1) and B(2) receptor mRNA was shown in human endothelial cells. Binding studies suggest that bradykinin receptors are competable with bradykinin antagonists. Adenylate cyclase activators probably increase bradykinin B(1) receptor affinity, without changing capacity, and thus increase bradykinin binding.     

Chronic desensitization to bombesin by progressive down-regulation of bombesin receptors in Swiss 3T3 cells. Distinction from acute desensitization

Prolonged exposure (40 h) of Swiss 3T3 cells to bombesin induced homologous desensitization to bombesin and structurally related peptides including mammalian gastrin releasing peptide (GRP). The ability of bombesin to mobilize intracellular Ca2+, inhibit epidermal growth factor binding, and stimulate DNA synthesis was profoundly and selectively inhibited. In contrast, Ca2+ mobilization by either vasopressin or bradykinin was unaffected, indicating that chronic desensitization is mechanistically distinct from acute desensitization of Ca2+ mobilization. Prolonged (24 or 40 h) pretreatment with bombesin also induced a 78 +/- 5% loss of bombesin receptor binding sites in both intact and plasma membrane preparations of Swiss 3T3 cells without an apparent change in receptor affinity (Kd = 1.9 +/- 0.1 x 10(-9) M and Kd = 1.8 +/- 0.2 x 10(-9) M for control and pretreated cells, respectively). Loss of 125I-GRP binding was slow and progressive with half-maximal loss of binding occurring after 7 h and maximal after approximately 14 h. Cross-linking of 125I-GRP to intact cultures and membrane preparations revealed an identical time-dependent loss of the Mr = 75,000-85,000 cross-linked band, previously identified as the bombesin receptor. Prolonged exposure of the cells to phorbol 12,13-dibutyrate, epidermal growth factor, cholera toxin, or mitogenic combinations of these agents did not alter 125I-GRP binding. Receptor down-regulation and loss of mitogenic responsiveness to bombesin were: (a) induced in a parallel dose-dependent manner by bombesin (ED50 = 1 nM), GRP (ED50 = 2 nM), and neuromedin B (ED50 = 20 nM), but not by the biologically inactive fragment GRP (1-16); (b) inhibited by the specific bombesin antagonist [Leu13-psi(CH2NH)-Leu14] bombesin, and (c) reversed upon removal of bombesin with a similar time course (full recovery after 15 h). On the basis of these observations, we propose that prolonged pretreatment of Swiss 3T3 cells with bombesin induces homologous desensitization to peptides of the bombesin family by down-regulation of cell surface bombesin receptors.     

Characterization of bombesin receptors on canine antral gastrin cells

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity binding of cholecystokinin to small cell lung cancer cells

The binding of 125I-Bolton Hunter-cholecystokinin octapeptide (125I-BH-CCK-8) to small cell lung cancer cell lines was investigated. 125I-BH-CCK-8 bound with high affinity (Kd = 2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific 125I-BH-CCK-8 binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease.     

Characterization of a neuronal subtype of insulin-like growth factor I receptor

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.