Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.     

Localization of basic fibroblast growth factor binding sites in the chick embryonic neural retina

We have investigated the localization of basic fibroblast growth factor (bFGF) binding sites during the development of the neural retina in the chick embryo. The specificity of the affinity of bFGF for its receptors was assessed by competition experiments with unlabelled growth factor or with heparin, as well as by heparitinase treatment of the samples. Two different types of binding sites were observed in the neural retina by light-microscopic autoradiography. The first type, localized mainly to basement membranes, was highly sensitive to heparitinase digestion and to competition with heparin. It was not developmentally regulated. The second type of binding site, resistant to heparin competition, appeared to be associated with retinal cells from the earliest stages studied (3-day-old embryo, stages 21-22 of Hamburger and Hamilton). Its distribution was found to vary during embryonic development, paralleling layering of the neural retina. Binding of bFGF to the latter sites was observed throughout the retinal neuroepithelium at early stages but displayed a distinct pattern at the time when the inner and outer plexiform layers were formed. During the development of the inner plexiform layer, a banded pattern of bFGF binding was observed. These bands, lying parallel to the vitreal surface, seemed to codistribute with the synaptic bands existing in the inner plexiform layer. The presence of intra-retinal bFGF binding sites whose distribution varies with embryonic development suggests a regulatory mechanism involving differential actions of bFGF on neural retinal cells.     

Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells

Leukemia inhibitory factor (LIF) is a glycoprotein that induces the differentiation of the monocytic leukemia cell line M1 but suppresses the differentiation of totipotent embryonic stem cells. In an attempt to define the normal cellular targets for LIF, the distribution of LIF receptors within hemopoietic and hepatic tissue was analyzed by binding cells with radioiodinated LIF (¹²⁵I-LIF) and subsequently carrying out autoradiography. Autoradiography demonstrated that in each he-mopoietic tissue examined cells of monocyte/macrophage lineage were the primary cell type labeled with ¹²⁵I-LIF. Moreover, both fetal and adult parenchy-mal hepatocytes displayed higher levels of labeling than either monocytes or macrophages. The number of receptors per positive cell varied from 150 for bone marrow monocytes to 2,000 for adult hepatocytes. In each case, however, binding was of high affinity, with an apparent K_D of 34–100 pM, and binding was specific, since labeling was competed for by unlabeled LIF but not a range of other structurally unrelated growth and differentiation factors. It is suggested that LIF may play a role in regulating macrophage function and hepatic acute phase protein synthesis in response to infection.     

Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.     

Identification of the two types of specific receptor for activin/EDF expressed on Friend leukemia and embryonal carcinoma cells

Activin and inhibin are polypeptide factors which control the release of follicle stimulating hormone(FSH) from pituitary cells. The recent finding that erythroid differentiation factor(EDF) is identical to activin showed the multifunctional feature of this protein. To identify the specific receptor for activin, the binding of 125I-labeled activinA was investigated for a number of culture cell lines. Friends leukemia cell, which can be differentiated by activin, and embryonal carcinoma(EC) cells(PCC3, P19 and F9), were found to express 3500-20,000 per cell of activin receptors. Scatchard plot analysis of the binding data shows that the receptors on those cells could be divided into two groups with different Kd values. The Kd values of high and low affinity receptors are 0.15-0.4 nM and 1.5-3.0 nM respectively. The proportion of the number of the high and low affinity receptors was varied in each cell. Inhibin was able to compete for activin binding to both types of receptors, although the binding affinity was about 50-200 fold lower than that of activinA. Transforming growth factor-beta had no binding ability to the activin receptors.     

Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells

Insulin and insulinlike growth factors I and II (IGF-I and IGF-II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I-labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF-I or -II binding occurred with induced HL60 cell differentiation. Insulin and IGF-I demonstrated cross competition for receptor binding and down-regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF-I and insulin increased HL60 cell proliferation, as assessed by 3H-thymidine uptake, IGF-I greater than insulin. IGF-I binding and mitogenic effects were blocked by the monoclonal anti-IGF-I receptor antibody IR3, indicating that IGF-I-induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti-IGI-I and anti-insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.     

Expression of high affinity receptors for murine interleukin 4 (BSF-1) on hemopoietic and nonhemopoietic cells

In this report a method for the affinity purification and radiolabeling of recombinant mouse interleukin (IL)-4 is described. It is shown on the basis of several criteria that IL-4 retains full biologic activity after radioiodination and can therefore be used as a valid model for measuring the binding characteristics of native IL-4. By using Scatchard plot analysis of equilibrium binding data, it is demonstrated that 125I-IL-4 binds to a high affinity cell surface receptor which is expressed by both hemopoietic and nonhemopoietic cells. The dissociation constant for 125I-IL-4 (Kd = 20 to 60 pM) corresponds to the concentration of IL-4 which gives 50% biologic activity (i.e., 10 to 30 pM). Binding of 125I-IL-4 is rapid (t1/2 of 2 min), whereas dissociation occurs at a slow rate (t1/2 approximately 4 hr). The IL-4 receptor shows a high degree of specificity. Whereas unlabeled mouse IL-4 competed with mouse 125I-IL-4 in an equimolar fashion for binding to IL-4 receptors, several other lymphokines, including mouse IL-2, IL-3, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and human IL-1, IL-2, and IL-4 were unable to inhibit, even at molar excesses of 400 to 800-fold. At 37 degrees C, 125I-IL-4 is rapidly internalized (approximately 200 molecules/cell/min) by HT-2 cells, with at least 85% of cell surface receptors being functional in this respect. Receptors for IL-4 were found to be expressed by subclasses of T and B cells, mast cells, macrophages, and by cells of the myeloid and erythroid lineages. This wide distribution of receptor expression closely matches the known spectrum of biologic activities of IL-4, including proliferation and/or differentiation of T and B cells, mast cells and granulocytes, and induction of macrophage antigen-presenting capacity. IL-4 receptors were also found on a variety of nonhemopoietic cells such as cloned stromal cell lines from the bone marrow, spleen, thymus, and brain, and on muscle, brain, melanoma, fibroblast, and liver cells. Indeed, only 5 of more than 90 cell types tested have undetectable numbers of IL-4 receptors. The biologic effects of IL-4 on nonhemopoietic cells have not yet been reported and await elucidation.     

Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate

We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.     

The role of benzodiazepine receptors in the induction of differentiation of HL-60 leukemia cells by benzodiazepines and purines

A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL-60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 microM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5-4864) was demonstrated in HL-60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation-inducing capabilities, but binding potencies were 1,000-fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL-60 membranes (KD = 28.6 microM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25-200 microM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL-60 cells to mature forms by 6-thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383-390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL-60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL-60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5-4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6-thiopurine and protein thiols, since the inhibition was reversed by the presence of 2-mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Insulin and IGF receptors are developmentally regulated in the chick embryo eye lens

We have previously reported that insulin-like growth factor (IGF) receptors appear to predominate over insulin receptors in early stages of embryogenesis in the chick (days 2-3 whole embryo membranes). Overall, [125I]IGF I and II binding to specific receptors was maximal when the rate of brain growth is highest. In the present study we used the embryonic chick lens, a well-defined tissue composed of a single type of cell, to analyse whether changes of insulin and IGF I binding are correlated with changes in growth rate and differentiation state of the cells. We show that both insulin receptors and IGF receptors are present in the lens epithelial cells, and that each type is distinctly regulated throughout development. While there is a direct correlation between IGF-binding capability and growth rate of the cells, there is less relation to differentiation status and embryo age. Insulin receptors, by contrast, appear to be mostly related to the differentiated state of cells, decreasing sharply in fibers, irrespective of their developmental age.     

Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.     

Glucocorticoid binding during the differentiation of 3T3-F442A fibroblasts into adipocytes. A possible regulatory effect of insulin

Until recently, few studies had been carried out on receptors for glucocorticoids in adipocytes, although the role of these steroids is considerable. In the present studies, we chose the pre-adipocyte line 3T3-F442A, which constitutes an excellent model for investigating the differentiation and function of adipocytes. Using a whole cell assay system, we showed the existence of a homogenous class of sites with the characteristics of glucocorticoid receptors, that is, high-affinity binding which is reversible, specific and saturable. Whatever the state of cellular differentiation, the affinity of the receptor for dexamethasone did not vary, although we observed an increase in the number of sites during differentiation. When cells were differentiated in the presence of insulin, there was a further increase in the binding capacity; moreover, insulin deprivation of such adipocytes caused a decrease in the number of sites. Our results therefore suggest that factors other than the glucocorticoids themselves influence dexamethasone binding. It is suggested that insulin plays a role in the regulation of the number of glucocorticoid receptors.     

Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes

Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function.     

Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen

Direct radioreceptor binding experiments and Scatchard analysis reveal CCK receptors on elutriator purified human peripheral blood monocytes, but not on purified human T cells. The monocyte receptors have a single class of high (0.1 nM) affinity binding sites. A structure-function analysis of monocyte binding by different CCK analogs correlates well with previously demonstrated chemotactic responses in monocytes and receptors in brain tissue. Biochemical cross-linking indicates that the monocyte CCK recognition molecule is comparable in molecular size to that in brain membranes. Utilizing a novel fluoresceinated Texas Red-CCK conjugate we have visualized that up to 20% of human peripheral monocytes bear receptors for CCK. A discrete and anatomically significant distribution of CCK receptors in rat spleen is shown by film autoradiography of tissue sections. A more detailed microscopic analysis identifies a dendritic population of monocyte-derived cells within the periarteriolar lymphocyte sheath (PALS) of the white pulp as the CCK receptor-bearing cell in spleen. The anatomical localization of receptor-bearing cells within the PALS region suggests a role for CCK in the antigen processing and sensitization phases of the immune response via regulatory effects of this peptide on a specific, local macrophage-related cell population.     

Autoradiographic Visualization of A1 Adenosine Receptors in Rat Brain with [3H]8-Cyclopentyl-1,3-Dipropylxanthine

A1 adenosine receptors were labeled in rat brain sections with the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and visualized at the light microscopic level using autoradiography. The specific binding of [3H]DPCPX to the sections showed the pharmacological characteristics of A1 adenosine receptors and was accompanied by very low levels of nonspecific binding. Whereas GTP had no significant effect on [3H]DPCPX binding to rat brain membranes, the addition of 100 microM GTP increased the apparent affinity of [3H]DPCPX to tissue sections fivefold (from 1.83 to 0.35 nM), enhancing it to the affinity measured in membranes. However, GTP altered neither the binding capacity nor the distribution of binding sites in tissue sections. It is suggested that a competitive antagonism with endogenous adenosine explains the lower affinity of [3H]DPCPX in the absence of GTP. The autoradiographic pattern of [3H]DPCPX binding was characteristic for A1 adenosine receptors. Distinct labeling of the different layers of the cerebellar cortex was shown by photomicrographs generated with the coverslip technique. In addition, several fiber tracts were found to be labeled. The high selectivity for A1 adenosine receptors and low nonspecific binding of [3H]DPCPX, the ability to produce high-resolution autoradiograms, together with the fact that the effects of endogenous adenosine can be eliminated by the addition of GTP make [3H]DPCPX a very useful tool in the autoradiographic study of A1 adenosine receptors.     

Visualization and characterization of interleukin 1 receptors in brain

Interleukin 1 (IL 1) is a polypeptide hormone produced by macrophages, keratinocytes, and brain glial cells which acts as a soluble mediator in immunological and inflammatory reactions. Although its best known effect on the central nervous system is its ability to cause fever, it has been found to influence cell growth, food intake, and slow-wave sleep. We have developed a binding assay for 125I-labeled recombinant murine IL 1 and show it to be highly specific. Additionally, affinity cross-linking studies indicate that the rat brain IL 1 receptor has a m.w. of approximately 80,000, which is similar to the previously described recognition molecule on T cells and fibroblasts. Using autoradiographic techniques, we visualized the distribution of 125I-IL 1 binding in sections of fresh frozen rat brain. IL 1 receptors were found to be widespread throughout the brain, forming a distinctive pattern of distribution. Areas especially dense in receptors were typically neuron-rich sites of the brain such as granule cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus, and the granule cell layer of the cerebellum as well as in the hypothalamus. The pattern of IL 1 receptor distribution indicates the presence of receptors on neuron cell bodies and the localization to numerous discrete brain areas other than those hypothalamic sites involved in temperature regulation, suggesting a broader role for IL 1 in brain functioning than previously recognized. IL 1, derived from local or systemic sources, may function in the brain to coordinate behavioral and neuroendocrine activities with immunological and inflammatory reactions throughout the body.     

Evidence that biological activity of NGF is mediated through a novel subclass of high affinity receptors.

Trophic factors, such as NGF, regulate survival and differentiation of many classes of neurons by binding specific receptors. Two types of NGF receptors have been identified, which bind NGF with low and high affinity. The latter mediates the major biological actions of NGF. To determine the relationship between these two receptor types, polyclonal antibodies to the low affinity receptor have been prepared and used in ligand-binding, ligand-cross-linking, and biological assays. These antibodies eliminated binding of NGF to low affinity receptors and to one class of high affinity receptors, but did not prevent binding to a second class of high affinity receptors. The same antibodies did not inhibit NGF-stimulated neuronal survival or neurite outgrowth. Thus, a biologically important class of high affinity NGF receptors is antigenically distinct from the low affinity receptor and may be encoded by a novel gene.     

State of GABA-benzodiazepine receptor complex in diabetic neuropathy: effect of nicotinamide and nicotinoyl-GABA

An increase in GABA uptake by isolated rat brain synaptic endings as well as a decrease of pharmacologically active GABA analogue muscimol specific binding have indicated a physiologically drastic failure in realization of GABA-mediated inhibitory effects in CNS induced by diabetic encephalopathy. In spite of the impairment of inhibitory function of GABAergic transmission in diabetes a crucial activation of benzodiazepine receptors was determined, as it is tested by the increase in specific binding of flunitrazepam by synaptic membranes. This increase may play an important role in endogenous control of neural activity associated with the factors undefined so far. Using the approach that GABA, and several synthetic GABA agonists, appear to increase the affinity of the benzodiazepine recognition sites for such ligands, presumably by some allosteric mechanism, the findings concerning the in vitro binding assay technique confirm at least some of the functional characteristics observed between GABA and benzodiazepine receptors in vivo under pathological conditions. Indeed, the absence of activating effect on the affinity of flunitrazepam specific binding in the presence of micromolar concentrations of exogenous GABA implicate diabetes-induced alterations in coupling GABA- and benzodiazepine receptors that might be linked to changes in conformantial state of this membrane-bound complex and could partially explain diabetes-induced impairments of GABAergic transmission evaluated in the present study. Our study suggests that nicotinamide and especially GABA play an important role in improving the functioning of brain GABA-benzodiazepine complex impaired in diabetes through specific ligand-mediated mechanism and can be useful in the management of diabetes-associated brain failures.     

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.