Receptors for insulin and insulin-like growth factor-I in the human adrenal gland

Human adrenal glands contain high-affinity receptors for insulin and insulin-like growth factor I (IGF-I). Comparative studies with rat, hamster and human adrenal membranes confirmed that IGF-I receptors are most abundant in rat and hamster adrenals, whereas insulin and IGF-I receptors are present in equivalent numbers in human adrenal glands. Covalent crosslinking studies revealed that the human adrenal gland IGF-I receptor binding subunit migrated on dodecyl sulfate polyacrylamide gels with Mr = 135,000, which is identical to the migration of IGF-I receptor binding subunits isolated from other tissues. Autoradiography of frozen human adrenal slices incubated with [125I]insulin showed prominent, displaceable binding of this radioligand to the zona reticularis, zona glomerulosa, vasculature and medulla; in contrast, [125I]IGF-I binding to human adrenal tissue was most prominent in the zona reticularis and negligible in the medullary region.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.     

Distribution of receptors for insulin and insulin-like growth factor I (somatomedin C) in the adrenal gland

Rat adrenal glands contain cell surface high-affinity receptors for several peptide hormones. Receptors for IGF-I were abundant in this tissue, but receptors for insulin were relatively scarce. The behavior of adrenal membrane IGF-I receptors in radioligand binding assays was similar to the behavior of IGF-I receptors from other tissues, with a KD congruent to 6.2 x 10(-9) M. Covalent cross-linking studies with [125I]IGF-I revealed an IGF-I receptor alpha-subunit with Mr congruent to 135,000 on dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, as well as a smaller radiolabeled peptide, Mr = 116,000. In contrast, little binding of [125I]insulin to adrenal membranes was observed and no labeling occurred in cross-linking studies using [125I]insulin. These results contrast with the findings of whole-body autoradiographic studies that indicated substantial binding of [125I]insulin to adrenal glands and suggest that IGF-I, rather than insulin, may play a critical role in the growth and development of the adrenal gland.     

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.     

125I]EXP985: a highly potent and specific nonpeptide radioligand antagonist for the AT1 angiotensin receptor.

[125I]EXP985 is the first nonpeptide radioligand with high specific activity for the AT1 angiotensin receptor. The biochemical and pharmacological profiles of this ligand were determined using either ligand-receptor binding techniques in rat adrenal cortical microsomes or cellular Ca2+ mobilization in rat smooth muscle cells. Specific binding with 0.1 nM [125I]EXP985 increased slowly with time reaching an equilibrium at 60 min of incubation (22 degrees C). Scatchard analysis of the inhibition/binding data revealed a single class of binding sites having a Kd of 1.49 +/- 0.06 nM and a Bmax of 3.6 +/- 0.1 pmol/mg protein. These sites were saturable and the ligand-receptor complex dissociated with a t1/2 of 58 min. The binding was inhibited by Ang peptides with the following order of potency and IC50 (nM): Ang II (3.7) > Ang III (69) > Ang I (3650), and by the nonpeptide AT1 receptor antagonist, losartan, with an IC50 of 3.2 nM. PD123177, an AT2 selective antagonist, showed minimal inhibitory effect. Specific binding of [125I]EXP985 was found on rat aortic smooth cells. Ang II-induced Ca2+ mobilization in these cells was blocked by EXP985 in a noncompetitive manner. These data show that [125I]EXP985 (or its unlabeled) is a potent and highly specific radioligand or noncompetitive antagonist which represents a novel tool to further our understanding of the biochemistry of AT1 receptors.     

Fetal and adult sheep adrenal cortical cells contain glucocorticoid receptors

Specific receptors for glucocorticoids were identified in the fetal and adult sheep adrenal cortex by a whole-cell binding assay using [3H]triamcinolone acetonide ([3H]TA) as the radiolabelled ligand. [3H]TA binding sites were saturable and of high affinity, with dissociation constant (Kd) of 2-3nM. Scatchard analysis revealed a single class of binding sites with a binding capacity (Bmax) of 207 and 5 fmol/10(6) cells for d100 fetuses and adults, respectively. By single point analyses at saturating [3H]TA concentration, we found that glucocorticoid receptors (GR) were present in the fetal adrenal cortex as early as d60. Highest concentrations were found at d100-110. GR decreased to d125, then increased to term (approx. d145) before declining again in newborn and adult animals. This demonstration of glucocorticoid receptors in ovine fetal adrenal cortical cells provides a mechanistic basis for the concept that glucocorticoids may act, perhaps in a paracrine or autocrine fashion, to influence adrenocorticotropin (ACTH)-induced activation of fetal adrenal function and the events leading to parturition.     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.     

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When [125I]IGF-I or [125I]IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. The dissociation constants were determined to be 0.106 nM for IGF-I and 0.209 nM for IGF-II binding. Cross-linking of [125I]IGF-I or [125I]IGF-II to the CMs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range 24-32 kDa. The binding was competitively inhibited by addition of cold IGF-I and IGF-II but not by insulin. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.     

Angiotensin II binding sites in frog kidney and adrenal.

Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [125I]-[Val5]AII binds to a single class of binding sites with a dissociation constant (Kd) of 548 +/- 125 pM in glomeruli and 593 +/- 185 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 2.48 +/- 0.71 and 3.05 +/- 1.02 fmol/mm2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar1]AII greater than human AII greater than [125I]-[Val5]AII = [Val5]AII = human AIII much greater than human AI. The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog.     

Insulin-like growth factor I (IGF-I) and insulin binding to erythrocytes of normal prepubertal children and adults.

Erythrocyte insulin-like growth factor I (IGF-I) and insulin receptors were characterized in 10 normal prepubertal children (5 girls and 5 boys) aged 4-11 yrs and 10 normal adults (4 women and 6 men) aged 32-47 yrs. erythrocytes were purified from 5 ml of blood by Ficoll-Paque gradient centrifugation. Reticulocytes count in the erythrocyte suspensions were lower than 1%. Insulin and IGF-I binding assays were performed simultaneously. Maximal percent binding of [125I] labelled IGF-I was significantly higher in prepubertal children than in adults (8.7 +/- 0.7% versus 6.2 +/- 0.5% at a concentration of 5 x 10(9) erythrocytes/ml). Scatchard analysis revealed the high affinity constant was better in prepubertal children (Ka = 4.6 +/- 1.3 nM-1 versus 1.8 +/- 0.2 nM-1), whereas the binding capacity was similar (5.8 +/- 1.1 versus 7.7 +/- 0.8 high affinity binding sites/cell). In both groups, unlabelled IGF-I inhibited tracer-binding half maximally at about 1 nM. Insulin was 100-fold less potent. In adults, specific binding of [125I] labelled IGF-I was higher in women (7.6 +/- 0.7%) than in men (5.3 +/- 0.4%). No significant difference was observed in maximal specific binding of [125I] labelled insulin between prepubertal children (8.2 +/- 0.5%) and adults (7.2 +/- 0.7%). In both groups, competition by unlabelled insulin for [125I] labelled insulin binding gave 50% displacement for approximately 0.25 nM and IGF-I was about 80-fold less potent. Both IGF-I and insulin binding parameters were not significantly correlated with plasma hormone levels. In prepubertal children, the high-affinity IGF-I receptors number decreased with increasing high-affinity insulin receptors number.(ABSTRACT TRUNCATED AT 250 WORDS)     

IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes.

Recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) and human insulin promoted the differentiation of spermatogonia into primary spermatocytes in newt testes fragments cultured in a chemically defined medium. The biological potency for promoting differentiation was dose-dependent for all the ligands with the highest potency displayed by IGF-I, followed by IGF-II, and the least by insulin. The difference in potency was larger between IGF-II and insulin than that between IGF-I and IGF-II. This order of biological potency was in good accordance with the order of affinity in binding specificity of [125I]IGF-I to the testicular membrane fractions: IGF-II and insulin competed the binding of [125I]IGF-I only at concentrations 20-fold and 100-fold higher, respectively, than IGF-I. Specific binding was observed in both somatic cells (mostly Sertoli cells) and germ cells (spermatogonia and primary spermatocytes), though the binding to somatic cells was about 2.7 times higher than that to germ cells. These results indicate that (1) specific binding sites for IGF-I are present in the newt testes, (2) IGF-II and insulin also bind to these receptors but to a lesser degree, and (3) IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor.     

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of insulin receptors in the bovine adrenal cortex and medulla.

In order to identify insulin receptors in the bovine adrenal cortex and medulla, we have studied 125I-porcine insulin binding to the membrane preparations from the bovine adrenal cortex and medulla. 125I-porcine insulin bound not only to the bovine adrenal cortex but to the medulla in time-, temperature-, and pH-dependent manners. The maximum levels of 125I-porcine insulin binding in the two tissues were observed at 4 degrees C for 24 h of incubation, and its optimum pH ranged from 7.6 to 8.0. Under these conditions, at tracer concentration of porcine insulin (200 pg/ml), 10.4% and 6.6% of 125I-porcine insulin added to each reaction tube bound specifically to 10(5) x g-pellet fractions (microsomal membrane) from the cortical tissue (0.3 mg of protein) and from the medullary tissue (2 mg of protein), respectively. 125I-porcine insulin binding was observed predominantly in the microsomal membrane from the bovine adrenal cortex, and in a 15,000 x g- pellet fraction (synaptosomal membrane) from the bovine adrenal medulla. Scatchard analysis of binding data yielded curvilinear plots in each tissue. Analysis of curvilinear plots based on two sites model revealed similar affinity constant between the cortex and medulla. Receptor concentration of the cortex was several times higher than that of the medulla. In the two bovine adrenal tissues, human proinsulin and insulin-like growth factor I (IGF-I) had about 1/100 potency compared to porcine insulin in displacing 125I-porcine insulin binding. Porcine glucagon added with concentration up to 10(-6) M did not inhibit 125I-porcine insulin binding to both the cortex and the medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

The alpha7 nicotinic receptors in human fetal brain and spinal cord

The alpha7 nicotinic acetylcholine receptor subtype is believed to be involved in the regulation of neuronal growth, differentiation and synapse formation during the development of the human brain. In this study the expression of the alpha7 nicotinic acetylcholine receptor was investigated in human fetal brain and spinal cord of 5-11 weeks gestational age. Both the specific binding of [125I]alpha-bungarotoxin to prenatal brain membranes and the expression of alpha7 mRNA were significantly higher in the pons, medulla oblongata, mesencephalon and spinal cord of 9-11 weeks gestational age compared with cerebellum, cortex and subcortical forebrain. A significant positive correlation between gestational age and the expression of alpha7 mRNA was observed in all brain regions except cortex. A positive correlation was also observed between the gestational age and the [125I]alpha-bungarotoxin binding in the pons, medulla oblongata, mesencephalon, and cerebellum. Consequently, a significant relationship between the alpha7 mRNA levels and the binding sites for [125I]alpha-bungarotoxin was found in the fetal brain. The increasing levels of the alpha7 nicotinic acetylcholine receptor during the first trimester support the important role of nAChRs for the development of the central nervous system.     

Delayed appearance of liver growth hormone binding sites and of growth hormone-induced somatomedin production during rat development

The present study was designed to determine whether the apparent paradox of high circulating growth hormone levels in the fetus and the minimal effect of this hormone on growth might reflect a diminished responsiveness of fetal target organs to GH. Specific uptake by rat liver of [125I] bGH was very low in fetuses as compared to suckling and adult rats. Also, liver uptake of the iodinated hormone decreased proportionally with the simultaneous injection of increasing amounts of growth hormone, but was not modified by the simultaneous injection of unlabelled chemically-related hormones. Since the water content is significantly greater in fetal than adult tissues, results were expressed by liver dry weight and again, [125I] bGH liver uptake continued to increase with age. After bovine growth hormone administration to adult rats, plasma somatomedin C concentrations increased significantly, while they had no effect in fetuses. These results suggest that reduced liver somatogenic binding sites in the fetus prevents growth hormone from inducing growth-promoting effects during intrauterine life.     

Specific binding of vascular permeability factor to endothelial cells

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor, has recently been purified from guinea pig, human, and bovine sources. We show that various fetal or adult endothelial cell strains originating from either capillary or large vessels possess specific high affinity and saturable binding sites for guinea pig tumor-derived [125I]VPF. Two classes of sites with KDs of approximately 10 pM and 1 nM were detected for all endothelial cell types examined. Guinea pig [125I]VPF binding to endothelial cells was inhibited by human VPF (ID50 = 0.8 ng/ml) and by suramin (ID50 = 75 micrograms/ml) but not by heparin. Cross-linking experiments revealed specific [125I]VPF-receptor complexes of two types. Most of the complexes migrated very slowing in SDS-PAGE, indicating that they were of very high molecular weight and probably highly cross-linked. A portion of the molecules migrated as 270 kDa complexes, indicating that the molecular weight of the endothelial cell VPF receptor is about 230 kDa.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.