Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Autoradiographic Localization of Insulin-Like Growth Factor I Binding Sites in the Bovine Adrenal Gland and on Cultured Bovine Adrenal Chromaffin Cells

Previous studies have reported the presence of binding sites for insulin-like growth factor I (IGF-I) in membranes prepared from isolated bovine adrenal medullary cells, and IGF-I was found to regulate the secretory function of bovine chromaffin cells. In the present study, binding sites for IGF-I have been localized in sections of bovine adrenal gland and on cultured bovine adrenal medullary cells, using [¹²⁵ l][Thr⁵⁹]-IGF-l as the ligand in conjunction with qualitative autoradiographic techniques. Binding sites were present throughout the adrenal gland and were distributed evenly over all cortical cell layers and over both adrenaline and noradrenaline cell types in the medulla. They were also present at lower density over blood vessels and nerve bundles and over the capsule. The binding of radioligand was to a single class of sites with K_d 0.61 nM, and was completely displaced by excess unlabelled [Thr⁵⁹]-IGF-l and by insulin (Actrapid, K_d 1.04μM). Binding sites were also identified on single cells in primary monolayer cultures of bovine adrenal medullary cells. More than 96% of the cells possessed binding sites, although only 85% of such cells were chromaffin cells, as previously determined from dopamine β-hydroxylase immunohistochemical staining. The results suggest that IGF-I may affect the maturation, growth or function not only of adrenal chromaffin cells but also of many others cell types in this tissue.     

Localization of [125I]IGF-I Binding on the Ovine Pars Tuberalis

In the sheep, it has been shown that the pars tuberalis of the pituitary may mediate the photoperiodic control of seasonal changes in prolactin secretion. High concentrations of melatonin receptors are present on the ovine pars tuberalis and melatonin is known to inhibit forskolin-stimulated cyclic AMP production in this tissue. Other hormonal inputs to the ovine pars tuberalis have not yet been identified. In the rat mRNA for the IGF-I receptor has been identified in the pars tuberalis using in situ hybridization. In order to define whether IGF-I may influence the function of the ovine pars tuberalis the presence of receptors for IGF-I has been investigated. Using in vitro autoradiography specific [¹²⁵I]IGF-I binding was found in high concentrations over the ovine pars tuberalis particularly associated with certain of the capillaries. Homogenate receptor assays showed saturable specific binding of [¹²⁵I]IGF-I with a mean dissociation constant (Kd) of 0.5 ± 0.1 nM (n=4). Competition studies revealed a rank order of potency of IGF-I>IGF-II> > >insulin, in displacing [¹²⁵I]IGF-I binding, indicative of a mixed population of IGF-I and IGF-II/rnannose-6-phosphate receptors and insulin-like growth factor binding proteins (IGFBPs). Cross-linking of [¹²⁵I]IGF-I to pars tuberalis membrane homogenates and analysis by SDS-PAGE under reducing conditions confirmed the presence of both IGF-I receptors and binding proteins. Autophosphorylation of a 97 kDa substrate, compatible with the β-sub-unit of the IGF-I receptor, was increased in the presence of IGF-I, indicating the existence of functional IGF-I receptors on the ovine pars tuberalis. In contrast in the rat [¹²⁵I]IGF-I binding was restricted to the median eminence region of the brain and was not detectable over the pars tuberalis.     

Insulin and insulinlike growth factor receptors regulating neurite formation in cultured human neuroblastoma cells

The functional role of brain insulin and insulinlike growth factor (IGF) receptors is being sought. Recently it has been found that these ligands are members of a newly identified family of neuritogenic polypeptides. We studied the relationship between 125I-insulin and 125I-IGF binding and their capacity to enhance neurite formation in cultured human neuroblastoma SH-SY5Y cells. The binding of 125I-insulin was temperature-dependent and heterogeneous. The Scatchard plot and dissociation rate were both consistent with the presence of two types of sites. There appeared to be about 900 high affinity sites per cell with a Kd of about 3 nM. This compared favorably with the half-maximal concentration of 4 nM for enhancement of neurite formation. The type I IGF sites were also present. Physiologic concentrations of insulin clearly enhanced neurite formation through the insulin sites, whereas physiologic concentrations of IGF-I and IGF-II enhanced through the IGF sites. Cross-occupancy of sites was observed at supraphysiologic concentrations, providing a reasonable explanation for the broad dose-response curves for these ligands. These results support the suggestion that one function of insulin and IGF receptors in neural tissues may be to modulate neurite formation.     

Triiodothyronine Regulates Insulin-Like Growth Factor-I Binding to Cultured Rat Pituitary Cells*

Triiodothyronine (T₃) stimulates the synthesis of growth hormone and enhances the growth of neoplastic rat pituitary somatomam-motrophs (GH cells) in culture. Moreover, T₃ has been shown to stimulate the production and secretion of an autocrine growth factor by these cells. We have previously demonstrated the presence of specific receptors for insulin-like growth factors (IGF) on GH cells. Since GH₃ cells contain mRNA encoding IGF-I, it has been suggested that IGF-I might act in an autocrine fashion in these cells. Therefore, it was of interest to learn how T₃ affects IGF-I binding to GH₃ cells. T₃ increased [¹²⁵I]IGF-I binding in a time - and dose-dependent manner. After 48 h of exposure to T₃, an increase in IGF-I binding was seen with 10^?11M T₃, maximizing with 10^?8M T₃. When cells were exposed to 10^?8 T₃, [¹²⁵I]IGF-I binding reached a maximum of 218 ± 20.8% of control (±SEM, P < 0.002) after 72 h of incubation. Scatchard analysis indicated that T₃ did not alter the K_d of IGF-I for its receptor, but that the total receptor number was increased. Dexamethasone (10^?7M) inhibited the T₃-induced increase in IGF-I binding, but glucocorticoid alone did not substantially alter receptor number. No significant change in insulin or IGF-II binding was seen after hormone treatment. 10^?8 M T₃ or IGF-I increased the growth of the GH₃ cells by ≥30%. Our data indicate that T₃ upregulates IGF-I binding in GH₃ cells without altering insulin binding and thereby provides a means for enhancing potential autocrine regulation in this cell line.     

Cultured rat Schwann cells express low affinity receptors for nerve growth factor

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.     

Nerve growth factor receptors on cultured rat Schwann cells

Neonatal rat Schwann cells were grown in tissue culture and assayed for NGF receptors with time in culture. NGF receptor levels on freshly prepared Schwann cells (day 0) were low but increased dramatically during the first week in culture. Characterization of 125I-NGF binding to resuspended cells grown for 4 d in culture revealed that binding was not saturable at high ligand concentrations (50-70 nM) and that a high-capacity, low-affinity NGF binding component existed on these cells as compared to PC12 cells. The monoclonal antibody, 192-IgG, which recognizes the rat NGF receptor, was used as an immunohistochemical tool to verify the presence of NGF receptors on the cultured rat Schwann cells. In contrast to radiolabeled NGF, 125I-192-IgG demonstrated saturable binding to Schwann cells in suspension, with Kd and Bmax values of 4 nM and 115 fmol/10(6) cells, respectively. Schwann cells showed no evidence of slow dissociation or internalization of NGF binding at any of several NGF concentrations. 192-IgG was used to immunoprecipitate 125I-NGF chemically crosslinked to cell membranes. SDS-PAGE and subsequent autoradiography of the immunoprecipitated NGF receptors revealed that 2 species of NGF receptors were precipitated from Schwann cells and PC12 cells. In PC12 cells, 2 bands with molecular weights of 90 and 210 kDa were identified. The Schwann cell NGF receptor species migrated slower on the gels, with apparent molecular weights of 95 and 220 kDa. Further analysis of glial cell NGF receptors showed that Schwann cells isolated from the vagus nerve of neonatal rats also expressed NGF receptors in culture; however, astrocytes cultured from neonatal rat cerebral cortex, cultured under conditions reported here, were devoid of detectable NGF receptors. These results show that NGF receptor levels on Schwann cells increase with time in culture, and this resembles what is observed in Schwann cells in vivo when adult peripheral nerve is injured. The data are discussed in terms of a supportive role for the Schwann cell in facilitating peripheral nerve development and regeneration.     

Interaction between cAMP elevation, identified growth factors, and serum components in regulating Schwann cell growth.

Most previous studies on Schwann cell proliferation in vitro have used serum-containing media. This complicates the analysis of agents required for cell division since serum contains an ill-defined mixture of hormones and growth factors. Serum-free medium has therefore been used to analyse the response of Schwann cell to previously identified Schwann cell mitogens. Serum factors were not necessary for DNA synthesis in response to platelet-derived growth factor, basic fibroblast growth factor, or glial growth factor, provided they were used in combination with forskolin to elevate intracellular cAMP. Transforming growth factor beta 1, a Schwann cell mitogen in serum, was not mitogenic under these conditions. Neither the growth factors nor forskolin were effective when used alone. Growth control was analysed further using long-term cultured Schwann cells that had spontaneously immortalized. Measurements of endogenous cAMP levels in short- and long-term Schwann cells revealed that long-term cells had two to three times higher basal cAMP levels. As predicted by these findings, platelet-derived growth factor, basic fibroblast growth factor, and glial growth factor stimulated DNA synthesis in long-term cells without requiring costimulation by agents which elevate cAMP (while transforming growth factor beta 1 had no effect).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of IGF-1 and IGFBP-1 and -3 in Axon-Schwann Cell Interactions

Insulin-like growth factor-I (IGF-I) promotes proliferation, differentiation and survival of Schwann cells (SC). Moreover, an effect of IGF-I on axon regeneration has been proposed. However, although IGF-I expression has been shown in postnatal SC and in SC precursors thus suggesting an autocrine regulation of SC survival, release of the trophic factor by SC has not been clearly demonstrated. To investigate the role of the IGF system, we studied the supernatant of primary SC cultures, purified sensory neuron cultures, and co-cultures of SC and sensory neurons. Moreover, since in its free form IGF-I interacts with specific binding proteins (IGFBPs) whose active role in modulating the effects of IGF-I is increasingly recognized, in the same culture systems we also evaluated levels of IGFBP-1 and -3. We established SC, sensory neuron and SC/sensory neuron cultures according to classical procedures. Purified SC (87,500 cells/dish) were grown in presence of serum, forskolin, cholera toxin and bovine pituitary extract. Purified sensory neurons were grown in the presence of serum and NGF; part of it was maintained alone and the rest was added with SC (87,500 cells/dish) and grown in a co-culture system. After 1 month all cultures were washed with PBS and grown for at least 4 days in a serum-free medium before collecting their conditioned media (CM) for chromatography assays. Similar IGF-I levels were found into the CM of SC and of sensory neurons when grown as purified cultures (94.40 vs 73.80 ng/5×10⁶ cell). Instead, higher amounts of IGF-I were detected into the medium of SC/sensory neurons co-cultures (277.70 ng/5×10⁶ cell). IGFBP-1 and -3 production was also found in both SC and sensory neuron purified cultures, but their expression was clearly higher in neurons compared to SC (0.74 vs 0.28 ng/5×10⁶ cell for IGFBP-1 and 4.48 vs 1.77 ng/5×10⁶ cell for IGFBP-3). Furthermore, SC/sensory neuron co-cultures maintained high levels of IGFBP-3 (3.93 ng/5×10⁶) but not of IGFBP-1 (0.28 ng/5×10⁶). In conclusion, IGF-1 is produced by both SC and pure sensory neurons, but co-culturing the two cell populations results in a possible reciprocal stimulation to synthesize this growth factor. Moreover, the decrease of IGFBP-1 in the medium of co-cultures could be due to the production of IGFBP proteases by SC. This action could be a mechanism to increase IGF-1 availability to the specific receptor.     

Characterization of oxytocin-binding sites in primary rat brain cell cultures

Detection and characterization of oxytocin-binding sites in dissociated brain cell cultures were performed, using a highly selective iodinated oxytocin antagonist [( 125I]OTA). Dissociated cells derived from hypothalamus and extrahypothalamic forebrain of 16-day-old fetal rats were maintained in chemically defined medium in order to enrich the cultures in neuronal cells. Specific binding of [125I]OTA, demonstrated in both hypothalamic and forebrain cell cultures, was temperature- and time-dependent; maximal binding was obtained by incubating the iodinated ligand for 60 min at 37 degrees C. The binding parameter were shown to be identical in both cell type cultures. The Scatchard plot analysis suggested the presence of a single class of binding sites of high affinity (Kd about 0.06 nM) and low binding capacity (Bmax about 4 fmol/dish). The specificity of these binding sites tested in competition experiments revealed that the unlabelled OT antagonist was the most potent in inhibiting specific [125I]OTA binding (Ki = 0.1 nM). A lower affinity, of the nM range was demonstrated for oxytocin (OT), arginine-vasopressin (AVP) and the V1 antagonist, whereas the V2 AVP agonist poorly competed for [125I]OTA binding sites (Ki about 250 nM). In conclusion, the [125I]OTA binding characteristics, identical in both hypothalamic and forebrain cultures, fulfil the classical conditions required for the existence of receptor sites since the binding was reversible, saturable and specific. As these characteristics were similar to those already described in the adult rat, at the central level in hippocampus, and at the periphery in the mammary gland, it could be postulated that [125I]OTA binds to an OT receptor site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of 2-[I]iodomelatonin by rat splenocytes: characterization and its role on regulation of cyclic AMP production

In the present paper we show that pineal hormone melatonin interacts with rat splenocytes through high-affinity binding sites. Binding of 2-[¹²⁵I]iodomelatonin ([¹²⁵I]MEL) by splenocytes fulfills all criteria for binding to a receptor site. Binding exhibited properties such as dependence on time and temperature as well as reversibility, saturability, high affinity, specificity, and increased under constant light exposure. Results suggest binding to a single class of binding sites without cooperative interactions. The dissociation constant (K_d) for the single site was 0.34 nM with a binding capacity of 2.25 fmol/107 cells. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived value of the dissociation constant was 0.20 nM. The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, pharmacological doses of melatonin also inhibited cyclic AMP production stimulated by forskolin, a potent activator of adenylate cyclase system. The demonstration of [¹²⁵I]MEL binding sites in the spleen, in addition to those described in blood mononuclear cells and thymus, provides evidence to support a direct mechanism of action of melatonin on immune system.     

Amygdala kindling decreases insulin-like growth factor-I receptor binding sites in the rat hippocampus

The neural excitability characteristic of kindling has been linked to structural alterations such as mossy fiber sprouting and synaptic reorganization within the hippocampus. Recent evidence suggests that growth factors may play a key role in kindling-related synaptic plasticity. Insulin-like growth factors-I and -II (IGF-I/-II) and insulin are structurally-related pleiotropic growth factors known to be involved in neural growth and differentiation. In the present study, we investigated the effect of kindling on [125I]IGF-I, [125I]IGF-II and [125I]insulin receptor binding in the hippocampus of adult rats. Our results indicate a progressive decrease in [125I]IGF-I (but not [125I]IGF-II or [125I]insulin) binding sites in the CA1, hilus and the granule cell layer of the kindled rats compared to sham-stimulated rats. These results, in keeping with the established neurotrophic effects of IGF-I, suggest a potential role for this growth factor in mediating the structural alterations associated with kindling.     

Mitogenic response of rat Schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels.

Rat sciatic nerve Schwann cells either do not proliferate, or proliferate very slowly, in medium containing 10% fetal bovine serum (FBS). They were previously shown to respond only to a limited number of mitogens associated with cells of central and peripheral nervous systems, which appeared to be distinct from FGFs and PDGF, and to agents that raise intracellular cAMP levels. In a basal medium consisting of 75% DMEM, 25% Ham's F-12, 5 nM sodium selenite, 50 microM 2-amino ethanol, and 2 mM histidine, supplemented with 5% FBS, we showed that aFGF, bFGF, and PDGF were all capable of stimulating Schwann cell growth and the stimulation was greatly potentiated by forskolin and dibutyryl-cAMP. In addition, pretreating culture surface with purified matrix proteins such as laminin, fibronectin, or type 1 collagen, was necessary for obtaining a better cellular response to the mitogenesis of these growth factors even in 10% FBS. Our results clearly indicated that providing a suitable medium and substratum, aFGF, bFGF and PDGF are mitogens for rat sciatic nerve Schwann cells in medium with and without forskolin or dibutyryl-cAMP.     

Growth promoting effects of IGF-I on fetal hypothalamic cell lines under serum-free culture conditions

Recent evidence indicates that the insulin-like family of peptides may act as endogenous trophic factors in the central nervous system. To further examine this possibility we have investigated the effects of three insulin-like peptides on the in vitro growth of fetal hypothalamic cell lines. Two virally transformed rat hypothalamic cell lines which have been developed in our laboratory (A-6 and F-12) were used. Cells were plated at varying densities and cultured in the presence or absence of either insulin-like growth factor I (IGF-I), insulin, or multiplication stimulating activity (MSA or IGF-II), in serum-free medium for 1 wk. Cell growth was assessed by counting or by measuring cellular incorporation of 3H-thymidine. Of the three peptides tested IGF-I was the most potent in eliciting cell growth. Insulin also stimulated growth of both cell lines, but was 100 times less potent for A-6 cells while it was equipotent with IGF-I in F-12 cells. MSA had no effect on either cell line. Both IGF-I and insulin showed dose-response effects in increasing cell growth. We also found that the two cell lines had the greatest response to IGF-I at low cell densities. Finally, time-course experiments suggested that a continued presence of the peptide is essential for the growth-promoting effects. We conclude that IGF-I is a potent growth factor for virally transformed cell lines derived from the rat fetal hypothalamus. Since both IGF-I immunoreactivity and IGF-I receptors have been located in this diencephalic area these results suggest that IGF-I may constitute a mitogenic signal for hypothalamic cells during neurogenesis.     

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.     

Insulin-like growth factor (IGF)-I,-II and IGF-binding proteins in the cyst fluid of a patient with astrocytoma

Insulin-like growth factor (IGF)-I,-II and IGF-binding proteins (IGFBPs) were demonstrated in the cyst fluid of a patient with a hypothalamic astrocytoma. The astrocytoma cyst fluid was subjected to gel chromatography at low pH and the IGF-I and IGF-II levels were measured by specific radioimmunoassays. Immunoreactive IGF-I and IGF-II levels were 19 ng/ml and 78 ng/ml respectively. Several-fold higher IGF-II values were obtained when cyst fluid was not extracted or was extracted with acid ethanol before radioimmunoassay analysis. The immunoreactive IGFBP-1 concentration was 26 ng/ml. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western ligand blotting with [¹²⁵I]IGF-II revealed bands at 200, 34.5, 29.5, 24 and 21 kD as visualized by autoradiography. Binding studies demonstrated that these binding proteins bind specifically [¹²⁵I]IGF-I and [¹²⁵I]IGF-II. These observations suggest that IGFs as well as IGF-binding proteins are produced by astrocytoma cells and may act in a paracrine or autocrine fashion capable of modulating the growth of astrocytoma tumours.     

Heregulin and forskolin-induced cyclin D3 expression in Schwann cells: role of a CCAAT promoter element and CCAAT enhancer binding protein

Heregulin, a polypeptide growth factor, and forskolin, an adenylyl cyclase activator, synergistically stimulate expression of cyclin D3 and cell division in Schwann cells. Heregulin induces expression in Schwann cells of a luciferase reporter gene linked to the cyclin D3 promoter. Forskolin markedly augments reporter expression in the presence of heregulin. Deletion analysis identified several promoter sites that contribute to high-level reporter expression in heregulin- and forskolin-treated Schwann cells. A promoter fragment that contains 103 bp of 5'-flanking sequence produced significant reporter expression in heregulin- and forskolin-stimulated cells. Deletion of a consensus CCAAT site within this promoter fragment caused a nearly complete loss of reporter expression. Similar results were obtained when CCAAT site mutations were introduced into the promoter. Heregulin and forskolin increased steady-state levels of CCAAT/enhancer binding protein-beta (C/EBPbeta) in Schwann cells. Mobility shift assays identified proteins in Schwann cell nuclear extracts that formed stable complexes with the cyclin D3 CCAAT promoter element and were disrupted by anti-C/EBPbeta antibody. Transfection of Schwann cells with C/EBPbeta cDNA increased cyclin D3 reporter expression. In contrast to these results, mutation of a cAMP response element in the cyclin D3 promoter had only a modest effect on heregulin- and forskolin-stimulated reporter expression. These findings demonstrate that C/EBPbeta plays a key role in the heregulin and cAMP-dependent regulation of cyclin D3 expression in Schwann cells.     

Chromaffin cells express two types of insulin-like growth factor receptors

The receptor binding, internalization and tyrosine kinase activation of insulin-like growth factors, IGF-I and IGF-II have been investigated in cultured adult bovine chromaffin cells. IGF-I receptor alpha-subunits (Mr approximately 130,000) bound IGF-I and IGF-II with identical affinity (Kd approximately 1 nM) and insulin with about 1000 times lower affinity. IGF-II receptors (Mr approximately 250,000) bound IGF-II with a Kd of 0.5 nM, IGF-I with about 10 times lower affinity and insulin with greater than 10,000 times lower affinity. The amounts of IGF-I and IGF-II receptors on the cell surface were 8 x 10(4) and 4 x 10(4) sites per cell, respectively. Insulin bound to a specific receptor with Kd approximately 2 nM and the amount of receptors was 1.5 x 10(4) sites per cell. IGF-I and IGF-II stimulated tyrosine kinase activity and autophosphorylation of the IGF-I receptor beta-subunit (Mr approximately 94,000) with equal potency (ED50 approximately 1 nM), whereas insulin was approximately 5 times less potent. Both IGF-I and IGF-II were internalized after their binding to cell surface receptors. Mannose-6-phosphate, which binds to the IGF-II receptor, did not alter the binding or internalization of IGF-II. It is concluded that IGF-I and IGF-II can exert their biological effects in chromaffin cells by activation of the IGF-I receptor tyrosine kinase or by interaction with the IGF-II receptor.