Cocaine differentially inhibits neuronal differentiation and proliferation in vitro.

The outcome of in utero cocaine exposure is unclear. To determine if cocaine affects neuronal growth and differentiation, we used PC-12 cells, which have a mitogenic response to IGF-I and differentiate into neurons on exposure to nerve growth factor. Differentiation was quantified as neurite extension after a 72-h exposure to 20 ng/ml nerve growth factor (dosage at 50% maximal effectiveness) and cocaine doses ranging from 0.01 to 10 micrograms/ml. The results were 49 +/- 2, 40 +/- 3, 29 +/- 2, 23 +/- 2, and 12 +/- 2% differentiation with respective cocaine concentrations of 0, 0.01, 0.1, 1, and 10 micrograms/ml (P < 0.0001). Cocaine stability studies showed insignificant spontaneous hydrolysis under the conditions of this study. Cocaine did not affect cell viability or number, but had a relatively modest, statistically significant (P < 0.001) inhibitory effect on IGF-I-stimulated thymidine incorporation. The dose-response curves for differentiation vs mitogenic response differed significantly (P = 0.021). Therefore, cocaine inhibition of these processes is probably mediated by different mechanisms, and not caused by generalized toxicity. To our knowledge, this is the first demonstration of cocaine effects on neuronal multiplication and differentiation in vitro. The results suggest in utero exposure may directly impair brain development.

The effect of insulin on norepinephrine uptake by PC12 cells.

We have previously reported that insulin can enhance endogenous noradrenergic activity in vitro in the rat CNS. In the present study, we examined one potential mechanism underlying this effect: the ability of insulin to inhibit norepinephrine (NE) reuptake and secondarily increase its synaptic concentration. Acute (20 min) insulin treatment (0.1-10 nM) significantly inhibited specific 3H-norepinephrine uptake by rat hypothalamic slices. To ascertain whether this is a direct effect of insulin on cells that can synthesize and release norepinephrine, we studied NE uptake by the rat pheochromocytoma PC12 cell line. In PC12 cells, insulin (0.5-10 nM) inhibited NE uptake whereas the related peptide, insulin-like growth factor 1 (IGF-1), did not. Insulin did not compete with 3H-mazindol (a ligand for the NE reuptake transporter) binding to PC12 cell membranes. Thus, this effect of insulin is not due to interaction with either IGF-1 receptors or the norepinephrine transporter, but may be due to insulin interaction with its own receptor. Chronic (96-h) insulin treatment of PC12 cells also resulted in an inhibition of 3H-norepinephrine uptake, and membranes prepared from cells chronically treated with insulin bound less 3H-desipramine than control membranes. Thus, chronic insulin treatment may result in a decrease in the numbers of membrane-associated transporters. We conclude that insulin has a direct and physiological role in the modulation of synaptic norepinephrine levels by modulating reuptake by cells that synthesize and release norepinephrine.

Insulin-like growth factors (IGFs): implications for aging.

The insulin-like growth factors (IGF)-I and IGF-II are peptides with structural homology to insulin and potent mitogenic and anabolic actions in vitro and in vivo. IGF-I levels are growth hormone (GH)-dependent and vary strikingly with age. IGF-I levels are typically low in infancy and childhood, increase dramatically during puberty, and then gradually decline with advancing age. Whether age-associated changes in GH production or sex steroid secretion, or other unknown factors, cause diminished IGF production in the elderly remains to be determined. In the brain, IGF-II appears to be the most prevalent IGF, but a truncated form of IGF-I also has been recognized. IGF actions are mediated by binding to a family of receptors, which includes the insulin receptor, the structurally homologous type I IGF receptor, and the IGF-II/M-6P receptor, all of which are found in the central nervous system. Additionally, the IGFs bind with high affinity to a family of IGF-binding proteins (IGFBPs). Of the six known IGFBPs, IGFBP-2 appears to be the major one in the mammalian brain and is a major component of CSF. Immunoreactive IGFBP-2 has been identified in astrocytes, and its mRNA has been identified in fetal and adult brain and choroid plexus. The IGFBPs transport the IGFs in serum and other body fluids and appear to regulate IGF access to receptors. In vivo regulation of IGFBPs includes tissue-specific proteases, which cleave specific IGFBPs, altering their affinities for IGF peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Not all insulin-like growth factor-binding proteins (IGFBPs) are detectable by western ligand blotting: case studies of PC12 pheochromocytoma and rat anterior pituitary IGFBPs and proteolyzed IGFBP-3.

We studied the limitations of the Western ligand blot (WLB) for detecting insulin-like growth factor-binding proteins (IGFBPs). PC12 rat pheochromocytoma cells and rat anterior pituitary cells (AP) secrete IGFBPs that cannot be detected by WLB. We used affinity labeling, WLB, dot blotting, competitive binding, ion exchange chromatography, and deglycosylation to characterize these IGFBPs. These IGFBPs were compared with pregnancy protease-derived IGFBP-3 fragments that also bind insulin-like growth factors (IGFs), but are not detectable by WLB. We showed that PC12 IGFBP is cationic, not glycosylated, with 25,500 mol wt reduced (18,500 unreduced), with high affinity for IGF-II and low affinity for IGF-I. It cannot be detected by WLB and is not a proteolytic derivative of other IGFBPs or IGF-II receptors. Its binding activity is not destroyed by sodium dodecyl sulfate (SDS) and heating. It binds to nitrocellulose and IGF-II after dot blotting, but not to IGF-II during WLB. AP also secrete an IGFBP(s) that was not detectable by WLB. AP IGFBPs, unlike those of PC12, have a higher mol wt, and at least one component is glycosylated. The failure of WLB to detect these proteins remains unexplained. Pregnancy protease-derived IGFBP-3 fragments also bind IGFs and are not detectable by WLB. However, they do electrotransfer to nitrocellulose. The failure of WLB to detect these fragments is probably due to proteolysis rendering the binding site susceptible to irreversible denaturation (under conditions of WLB) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that WLB, while valuable, may have significant limitations in specific cases. Other techniques must complement WLB for detection of IGFBPs in conditioned media and other biological specimens.

Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.

Immunofluorescent cytometry and electron microscopic immunolocalization of insulin-like growth factor (IGF)-II receptors in infant rat choroid plexus.

The choroid plexus contains receptors for insulin-like growth factor (IGF)-II, it synthesizes IGF-II, and it secretes soluble IGF binding proteins with high affinity for IGF-II. Although the actions of IGFs in the choroid plexus are unknown, IGFs may participate in CNS growth and differentiation, CNS injury/repair mechanisms, regulation of satiety, and growth hormone secretion. Because the choroid plexus is a heterogeneous tissue containing several cell types, to study the role of IGF-II in the choroid plexus we need to know precisely which cells contain IGF-II receptors. Therefore, we studied the distribution of IGF-II receptors in infant rat choroid plexus by electron microscopic immunohistochemistry and immunofluorescent cytometry of dispersed choroid plexus cells using a well-characterized, highly specific rabbit anti-rat IGF-II receptor antibody. These studies show that IGF-II receptors are detectable in large quantities in both choroid plexus epithelial cells and in the fenestrated capillary endothelial cells. Results were confirmed by immunofluorescent cell sorting using the anti-IGF-II receptor antiserum. Other cell types in the choroid plexus contained lower levels of immunoreactivity, including red blood cells. Physiological studies of IGF-II effects on intact choroid plexus may, therefore, reflect effects on both of these cell populations. These data provide a compelling rationale for the development of choroid plexus epithelial cell lines or pure culture techniques to study the physiology of IGF-II in individual cell types.

Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment epithelium.

Insulin-like growth factors (IGFs) I and II are mitogenic polypeptides structurally homologous to insulin, which are thought to mediate important neurobiologic actions in the CNS. The purpose of this study was to determine if cultured bovine retinal pigment epithelial cells (RPE) express IGF receptors and secrete soluble IGF binding proteins, and to characterize these receptors and binding proteins. We also characterized the soluble IGF binding proteins present in juvenile and adult rat vitreous and serum, as well as those in fetal bovine vitreous and serum, in order to facilitate identification of the RPE IGF binding protein, and to determine potential destinations for this protein once produced. Affinity labeling was used to characterize insulin, IGF-I and IGF-II receptors. Western radioligand blotting and immunoprecipitation were used to characterize IGF binding proteins. We found that RPE cells in culture express virtually no insulin receptors, and only modest amounts of IGF-I receptors. IGF-II receptors were abundantly expressed. Additionally, RPE cells secrete a soluble IGF binding protein which is immunologically related to IGFBP-2, the primary IGF binding protein produced in the central nervous system. Bovine vitreous was found to contain a mixture of IGF binding proteins (IGFBP). The most prominent IGFBP in this mixture is immunologically related to IGFBP-2. Likewise, juvenile and adult rat vitreous contained only one IGF binding protein that was shown to be immunologically related to IGFBP-2. Juvenile rat vitreous contained more binding activity corresponding to IGFBP-2 than did adult vitreous, suggesting developmental regulation. These data suggest that IGF's and their binding proteins may have important, and as yet undefined, roles in retinal neurophysiology.

Characterization of insulin-like growth factor binding proteins produced in the rat central nervous system.

Insulin-like growth factor binding proteins (IGFBP) are thought to modulate the biological actions of the insulin-like growth factors (IGF), including possible regulatory roles in the growth and differentiation of the central nervous system. Extracellular fluids usually contain a mixture of IGFBPs, three of which have been cloned, sequenced, and designated IGFBP-1, -2, and -3. We used Western ligand blotting, immunoprecipitation, and competitive binding analysis to characterize IGFBPs found in fetal and adult rat cerebrospinal fluid (CSF) and IGFBPs produced by cultures of neonatal rat choroid plexus, astrocytes, and C6 glial cells. Pooled rat CSF contains primarily IGFBP-2 (a narrow band at Mr = 29,000), lesser quantities of IGFBP-3 (a multicomponent broad band at Mr = 37,500-43,000), and trace amounts of low mol wt IGFBPs. Conditioned medium from cultures of choroid plexus cells contained a single binding protein corresponding to IGFBP-2, whereas C6 cells made predominately an IGFBP corresponding to IGFBP-3. Astrocytes secreted two IGFBPs corresponding to IGFBP-2 and -3, primarily IGFBP-3. Neonatal CSF contained substantially more binding activity corresponding to IGFBP-2 than did adult CSF. In all samples showing Western ligand binding profiles corresponding to IGFBP-2, identification was established by immunoprecipitation. Competitive binding analysis performed on choroid plexus IGFBP showed preferential high affinity binding for IGF-II compared with that for IGF-I. In conclusion, CSF contains a mixture of distinct IGFBPs, primarily IGFBP-2. The other IGFBPs found in CSF are capable of being synthesized locally within the central nervous system by glial cells and neurons, suggesting that they are not derived from plasma by transport across the blood-brain barrier.

Developmental expression of insulin-like growth factor-II receptor immunoreactivity in the rat central nervous system.

A polyclonal antiserum raised against the insulin-like growth factor-II (IGF-II) receptor has been used to map the distribution of this receptor in the developing rat central nervous system (CNS). Transiently high levels of receptor immunoreactivity were found in the developing brain, particularly in the cortex and hypothalamus. The amount of receptor immunostaining in these areas decreases toward the time of birth, and levels are approximately equivalent to those in the adult by postnatal day 7. The choroid plexus, cerebral vasculature, ependymal cells, retina, and pituitary contain high levels of receptor immunoreactivity throughout embryogenesis and adulthood. Some mesodermally derived tissues, such as bone, also demonstrate transient expression of IGF-II receptor during fetal development. These data are consistent with potential roles for IGF-II in CNS development, in the development of specific mesodermal tissues, and in specific regions of the postnatal CNS.

Localization and structural characterization of insulin-like growth factor receptors in mammalian retina.

Insulin-like growth factors (IGFs) are peptide mitogens, structurally related to insulin, whose biological actions in the CNS are incompletely known. The retina is largely uncharacterized with respect to IGF receptors. We, therefore, studied IGF receptors in bovine and murine retinal tissues by immunohistochemistry, autoradiographic localization, and affinity labeling. Notable IGF-II receptor immunoreactivity was found in retinal pigment epithelium (RPE), with intermediate levels in choroid, low levels in the inner and outer plexiform layers and outer nuclear layer, and very low levels in other regions. Autoradiographic localization using [125I]IGF-II confirmed the IGF-II receptor immunohistochemistry. Autoradiographic localization using [125I]IGF-I labeled the nuclear layers and the photoreceptor region. Affinity labeling disclosed differences in the apparent mol wt of IGF-I and IGF-II receptors from bovine eye tissues and those from liver and brain. IGF-I receptor alpha-subunits (the IGF-binding subunit) migrated at: liver, 139,000; brain, 125,000; RPE, 125,000 and 135,000 (two sizes); and retina, 125,000 and 135,000. IGF-II receptors migrated at: liver, 245,000; brain, 235,000; RPE, 240,000; and retina, 230,000. We conclude that mammalian retina contains both IGF-I and -II receptors, which differ from those found in other tissues and have a characteristic spatial distribution within the retina.

Expression of the BRL-3A insulin-like growth factor binding protein (rBP-30) in the rat central nervous system.

The expression of the BRL 3A insulin-like growth factor binding protein (rBP-30) was characterized in rat brain, hypothalamus, and pituitary tissue and cultured neuronal and astroglial cells. The 27K BP expressed by BRL 3A cells (rBP-30) was found to also be expressed in conditioned media from newborn rat astrocytes and fetal neurons, but not in the medium from the neuroblastoma cell line, B104. Moreover, a polyclonal antibody, anti HEC1, specifically immunoprecipitated the BRL 3A BP from the same conditioned media, as well as from rat cerebrospinal and amniotic fluid and from conditioned medium of cells isolated from the neurointermediate lobe of adult rat pituitary. The same antibody also immunoprecipitated hBP-31 from human CSF. Northern blot analyses showed that rBP-30 mRNA was expressed in adult rat brain and pituitary, fetal brain and liver, and in fetal neurons and newborn astrocytes maintained in culture. We conclude that the BRL 3A BP (rBP-30) is the major insulin-like growth factor binding protein in the rat CNS and may be the rat analog of hBP-31, the predominant BP in human CSF.

Characterization of insulin-like growth factor-binding proteins in cultured rat pituitary cells.

Binding proteins (BPs) for the insulin-like growth factors (IGFs) produced by cultured rat anterior pituitary (AP) and neurointermediate lobe (NI) cells were studied by competitive binding, affinity cross-linking, and Western ligand blot techniques. Conditioned medium from AP cultures contained specific high affinity IGF BPs with apparent mol wt of 35K, 27K, and 24K, while the 27K BP predominated in NI conditioned medium. Treatment of AP and NI conditioned media with endoglycosidase-F did not alter the 27K BP, but significantly reduced the apparent mol wt of the 35K BP into the 27-29K range, suggesting that the 35K BP may be a glycosylated form of the 27K BP. This 27K pituitary BP appeared similar to the BP produced by BRL-3A cells in both size and apparent lack of glycosylation. Although type 2 IGF receptors could be identified in conditioned medium from NI and GH3 pituitary cells, binding of [125I]IGF to pituitary BPs could not be inhibited, nor could the cross-linked BPs be immunoprecipitated, by antibody against the type 2 receptor. We conclude that cultured AP and NI cells produce a variety of related IGF BPs that are structurally distinct from the type 2 IGF receptor.

Somatic and intellectual development in a patient with 47,XX,psu dic(X)(p11.2) chromosome constitution.

An unusual form of X chromosome aneuploidy, 47,XX,psu dic(X)(p11.2), was found during an evaluation for short stature of a prepubertal girl. Unlike 45,X, 47,XXX, 48,XXXX, and 49,XXXXX females, this patient is phenotypically normal except for her short stature, which appears to be unrelated to her chromosome abnormality. X chromosome inactivation studies disclosed inactivation (late replication) of one normal X and the abnormal X chromosome in all cells examined from this patient. Therefore, she is disomic for early-replicating distal Xp loci, found in inactivated X chromosomes, and thought to remain active after lyonization. These data suggest that the presence of three or more copies of the early-replicating, active Xp loci may be responsible for the cognitive deficits and other phenotypic abnormalities seen in and other phenotypic abnormalities seen in polysomy X females.

Structural characterization and immunohistochemical localization of receptors for insulin-like growth factor II in the rat pituitary gland.

Insulin-like growth factor II (IGF-II) receptors were detected, localized, and structurally characterized in rat pituitary tissue sections and cultures of dispersed pituitary cells by immunohistochemistry, and affinity labeling with gel electrophoresis. Using highly specific antisera against IGF-II receptors (type 2 IGF receptors) and somatotropin (GH), intense type 2 receptor immunoreactivity was detected in both anterior and intermediate pituitary lobes. In anterior pituitary sections and cultures, type 2 receptor immunoreactivity colocalized to most GH-immunoreactive cells (somatotropes), as well as cells which did not contain GH. Intermediate lobe immunoreactivity was uniformly distributed throughout the parenchyma. In both anterior and intermediate pituitary lobes, type 2 receptor immunoreactivity was predominately localized to the plasma membrane of labeled cells, with little or no cytoplasmic labeling. GH immunoreactivity, on the other hand, was intracellular. Affinity labeling of microsomal membranes from anterior and neurointermediate pituitary tissues with 125I-IGF-II disclosed classical 230k type 2 receptors. The magnitude of affinity cross-linking from both lobes was similar to that of rat liver, indicating pituitary tissues, like liver tissue, are rich sources of type 2 receptors. These results suggest possible roles for IGF-II and the type 2 receptor in the regulation of synthesis or secretion of pituitary trophic hormones, including GH and pro-opiomelanocortin gene products.

Characterization of insulin-like growth factor binding proteins of cultured rat astroglial and neuronal cells.

Insulin-like growth factor binding proteins produced by cultured rat neurons, astrocytes, and rat cell lines BRL-3A and B104 were compared to binding proteins found in rat serum, using affinity labeling, deglycosylation, and Western ligand blotting studies. Each source elaborated an unique pattern of heterogeneous binding proteins. Some of the binding proteins from different sources behaved similarly in each experimental system suggesting that subsets of these binding proteins may be structurally related. In particular, our data suggest that cultured astrocytes and neurons make the major binding protein produced by BRL-3A cells.

Structural and immunohistochemical characterization of insulin-like growth factor I and II receptors in the murine central nervous system.

The description of the cellular localization of insulin-like growth factor (IGF) receptors in the central nervous system (CNS) remains incomplete, as do the descriptions of changes in their characteristics with respect to different developmental stages. We, therefore, performed affinity labeling studies in microsomal membrane preparations of adult and fetal rat brain and liver tissues with [125I]IGF-I and [125I]IGF-II. These studies demonstrated tissue- and developmental stage-specific structural variants of type I receptor alpha-subunits as well as type II receptors. The adult rat brain type I alpha-subunit had an apparent mol wt (Mr) of 127,000, whereas those of adult and fetal rat liver measured 140,000. Fetal rat brain microsomes, however, had two types of type I receptor alpha-subunits measuring 130,000 and 120,000 Mr. The larger subunit from fetal brain consistently migrated at an apparent Mr of 3,000, greater than subunits from adult brain. Both type I and II receptors were more abundant in fetal liver and brain than in adult tissues. Affinity labeling was also performed directly to monolayers of cultured fetal brain neurons and newborn astrocytes. These studies detected both type I and II receptors on the surfaces of both types of cells. However, only the high Mr (140,000) form of the type I alpha-subunit was detected in cultured CNS cells, suggesting that expression of low Mr variant receptors is altered in vitro. Type II receptors were demonstrated by immunohistochemistry in adult rat hypothalamic neurons. However, the majority of neurons did not react with type II receptor antibody. This finding implies that only a minority of hypothalamic neurons are capable of responding to IGF-II via type II receptors. On the other hand, all astrocytes had striking type II receptor immunoreactivity. This signifies a more general biological role for this receptor in astrocytes compared with neurons. These results suggest that different tissue-, developmental stage-, and cell-specific processes are mediated by IGF receptors and suggests new directions in which to explore potential biological actions for these receptor-ligand systems in the CNS.