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.     

Receptor-mediated endocytosis and degradation of insulin-like growth factor I and II in neonatal rat astrocytes.

Receptor-mediated internalization and degradation of insulin-like growth factors, IGF-I and IGF-II, were studied in primary cultures of neonatal rat astrocytes. Surface-bound IGF-II was rapidly internalized, and 80% of cell-associated radioactivity was located intracellularly after 30 min. IGF-I was internalized at a slower rate, and only 40% of cell-associated radioactivity was inside the cell after 30 min. A pulse-chase experiment demonstrated that 55% and 70% of internalized IGF-I and IGF-II, respectively, was degraded to free amino acids after a 3-hr chase. Lysosomal and protease inhibitors had different effects on the binding, internalization, and processing of IGF-I and IGF-II. Inhibition of lysosomal acidification by chloroquine increased the amounts of surface-bound IGF-II and intracellular IGF-I and reduced the degradation of IGF-I. The chelating agent phenanthroline increased the surface binding of IGF-I and IGF-II and internalization of IGF-II and reduced the degradation of IGF-I and IGF-II. Finally, receptor-bound IGF-II on the cell surface was decreased with increasing cell density, whereas IGF-I binding was unaltered. Our data suggest that cell-surface expression of IGF-I receptors and IGF-II receptors is regulated by different mechanisms and that receptor-bound IGF-I and IGF-II are trafficked and processed by different intracellular pathways in neonatal rat astrocytes.     

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.     

N- and C-terminal substance P fragments: differential effects on striatal [3H]substance P binding and NK1 receptor internalization.

N- and C-terminal substance P (SP) fragments increase striatal dopamine outflow at nanomolar concentrations. This contrasts with their low affinity for NK1 receptors. To explore this discrepancy, we investigated the interaction of SP and SP fragments with NK1 sites in fresh striatal slices, the same model used in the functional studies on dopamine outflow. [3H]SP bound specifically to one site (Kd = 6.6 +/- 0.9 nM; Bmax = 12.6 +/- 0.7 fmol/mg protein). [3H]SP binding was displaced by SP (IC50 = 11.8 nM), but not by SP(1-7) or SP(5-11), up to 10 microM. In contrast, 10 nM SP(1-7) or SP(5-11) induced significant internalization of the NK1 receptor, similar to that induced by SP. We suggest that SP fragments have high affinity for an NK1 receptor conformer which is different from that labelled by [3H]SP.     

Insulin-like growth factor I: A mitogen for rat schwann cells in the presence of elevated levels of cyclic AMP

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulinlike growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective but in the presence of forskolin (5 μM) or dibutyryl cyclic AMP (dbcAMP, 10 μM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective, even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [¹²⁵I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (K_d = 1.5 nM). Competition studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [¹²⁵I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (- forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell). These results suggest that forskolin increases available receptors for IGF-I, which is consistent with the synergism between cAMP and IGF-I in stimulating Schwann cell growth. © 1993 Wiley-Liss, Inc.     

Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells

Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.     

Catecholaminergic cells and support cell precursors in neural crest cultures differentially express nerve growth factor receptors

Long-term neural crest cultures grown in the continuous absence of exogenous nerve growth factor (NGF) contain a subpopulation of cells with NGF receptors exclusively of the low affinity subtype (Kd of approximately 3.2 nM). The current studies combined immunocytochemistry, using GIN1 (a support cell marker) or tyrosine hydroxylase antibodies, with radioautography following exposure to iodinated nerve growth factor (125I-NGF). The majority of cells specifically binding 125I-NGF were found to be immunoreactive for GIN1, indicating that the primary cell phenotype expressing receptors for NGF appear to be support cell precursors, at least under these conditions. These cells are likely to be responsive to and/or dependent upon NGF; the nature of this response or dependency remains to be determined. Some cells exhibiting silver grains were not immunoreactive for GIN1, suggesting that other cell phenotypes in neural crest cultures also have NGF receptors. In addition, some neural crest cells were found that stained with GIN1 and lacked 125I-NGF binding. Tyrosine hydroxylase-like immunoreactive cells apparently did not bind 125I-NGF under these culture conditions. Catecholaminergic sympathetic and sensory neurons from embryonic ganglia, derived from the neural crest, express both the high and low affinity forms of the NGF receptor. In order to determine whether the microenvironment played a role in the type of catecholaminergic cells appearing in culture, neural crest cells were grown in the continuous presence of exogenous NGF. Under these conditions, many tyrosine hydroxylase-like immunoreactive cells were found that specifically bound 125I-NGF. In addition, silver grains were still detected on these cells following a chase with nonradioactive NGF, designed to eliminate 125I-NGF bound to low affinity sites. Therefore, the catecholaminergic cells possess both the low and high affinity forms of the receptor. NGF's ability to modulate tyrosine hydroxylase activity, as it does in mature catecholaminergic neurons, was tested in this system. Surprisingly, there was no statistically significant difference in tyrosine hydroxylase activity in cultures grown in the absence or presence of exogenous NGF. This raises the possibility that embryonic catecholaminergic cells are unable to respond to NGF in this specific way, even though the receptors for the factor are present.     

Murine mucosal T cells have VIP receptors functionally distinct from those on intestinal epithelial cells

Reports suggest that vasoactive intestinal peptide (VIP) binds to lymphocytes and modulates immune responses. The intestines are richly innervated with VIP-producing nerves. Thus, VIP from nerves or other sources may participate in mucosal immunoregulation. To explore this hypothesis further, murine intestinal mucosal inflammatory cells were scrutinized for functional VIP receptors. An [125I]VIP competitive binding assay characterized VIP receptors. Unfractionated lamina propria inflammatory cells bound [125I]VIP specifically. This binding was abrogated by T cell depletion. The VIP receptor on lamina propria T cells was of a single class with a Kd of 9.08 x 10(-9) M. It bound PHI and other peptide analogs poorly. The intestinal epithelial cell had a high-affinity VIP receptor (Kd 4.17 x 10(-10) M) that bound one VIP analog with moderate affinity. Both VIP and ConA stimulated mucosal inflammatory cells to release interleukin-5 (IL-5). Mucosal inflammatory cells depleted of T cells did not release IL-5 in response to VIP or ConA. It is concluded that: (1) some murine mucosal T lymphocytes have VIP receptors that may be distinct from those displayed on mucosal epithelial cells; (2) VIP affects mucosal T lymphocyte function.     

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.     

High molecular weight kininogen and factor XII binding to endothelial cells and astrocytes

We have quantitated the binding of high molecular weight kininogen (HK) to human microvascular endothelial cells of lung and dermal origin as well as to astrocytes and compared the results with those reported for human umbilical vein endothelial cells (HUVEC). We also reassessed parameters of binding to HUVEC employing cells in suspension as well as cells attached to the culture plate and report similar numbers of sites varying from 6.96x10(5) to 7.71x10(5) per cell. The present study shows that HK binds with high specificity and affinity to microvascular endothelial cells (Kd = 1.86 to 4.5 nM) compared to HUVEC (Kd = 10.35 nM) but with lower affinity to astrocytes (Kd = 23.73 nM). Human cytokeratin 1, urokinase plasminogen activator receptor and gC1qR were found to be HK binding proteins present at the surface of microvascular endothelial cells and astrocytes analogous to that seen in HUVEC, as assessed by inhibition of binding with antibody to each protein. Lung microvascular endothelial cells had approximately half the number of HK binding sites as HUVEC while dermal micro vascular endothelial cells and astrocytes had only 8-10% of the sites/cell. The affinity of binding to the microvascular endothelial cells was greater than HUVEC, the affinity of binding to astrocytes was considerably less, nevertheless binding to each cell type involves gC1qR, cytokeratin 1 and u-PAR to varying degrees. We also demonstrate, for the first time, that factor XII binds to all of these cell types in a saturable and Zn(+2) dependent manner. Given that factor XII accelerates the interactions among cell surfaces and proteins of the contact activation cascade to generate bradykinin, binding of factor XII (and the prekallikrein-HK complex) may serve as a mechanism by which these proteins are concentrated locally to facilitate their interactions.     

Insulin and insulin-like growth factor 1 receptors during postnatal development of rat brain

The binding of insulin and insulin-like growth factor 1 (IGF1) to high-affinity sites in the brain of rats aged 2-37 days was studied. Specific binding of insulin and IGF1 was assessed using tracer concentrations of 125I-insulin or 125I-IGF1. Sites for insulin and IGF1 were distinguished in these conditions as shown by competition experiments. The Kd were 3.6 nM (insulin) and 2.0 nM (IGF1). These values did not change significantly over the age range studied. The numbers of high-affinity binding sites for insulin and IGF1 were similar in adult animals. IGF1 binding was higher than the insulin binding in 2-day-old animals. The binding capacity for both insulin and IGF1 decreased from birth to age 15 and days remained stable thereafter. Tyrosine kinase activity, which is associated with these receptors, was measured using the artificial substrate poly (Glu, Tyr). It decreased over the first 15 days of life and remained stable thereafter. Autophosphorylation of the receptors confirmed this result. This decrease appears to be due to changes in the numbers of the two types of receptors, and is probably a reflection mainly of the variation in the number of IGF1 receptors. Similar results for insulin and IGF1 binding as well as tyrosine kinase activity were obtained with hypothyroid rats.     

Characterization of nicotinic receptors in chick retina using a snake venom neurotoxin that blocks neuronal nicotinic receptor function

Nicotinic receptor function has been described in the retinas of a variety of vertebrate species. Neuronal bungarotoxin (NBT, also known as bungarotoxin 3.1, toxin F, or kappa-bungarotoxin) blocks nicotinic receptors in several neuronal preparations, while the neuromuscular antagonist alpha-bungarotoxin (BGT) fails to block most of these receptors. NBT (100 nM), but not BGT (10 microM), substantially blocks nicotinic function on ganglion cells in intact chick retina. 125I-NBT binds to 2 sites in homogenates of chick retina; one site that is shared with BGT (Kd = 5-7 nM, Bmax approximately 500 fmol/retina) and one which is not (Kd = 2-3 nM, Bmax approximately 100 fmol/retina). 125I-NBT binding to the NBT-specific site (binding in the presence of 1 microM unlabeled BGT) is localized to 2 bands in the inner plexiform layer, corresponding to regions richly innervated by neurons containing immunoreactivity for choline acetyltransferase. Furthermore, this binding is blocked by competitive nicotinic agonists and antagonists, but nicotine or other nicotinic agonists do not displace 125I-NBT binding with very high affinity relative to the displacement of 3H-nicotine reported by others in brain. Thus, of the 2 NBT binding sites, the site not recognized by BGT most likely represents functional nicotinic receptors in the chick retina, but these receptors have relatively low affinity for nicotinic agonists, similar to nicotinic receptors found in autonomic ganglia.     

Glucocorticoid receptor properties and glucocorticoid regulation of glutamine synthetase activity in sensitive C6 and resistant C6H glial cells

The relationship between induction of glutamine synthetase activity by dexamethasone and binding of the steroid to cytosolic glucocorticoid receptors was examined in sensitive C6 and resistant C6H glial cell cultures. Glutamine synthetase activity increased 3-4-fold when C6 cultures were exposed to 7.6 x 10(-6) M dexamethasone. This inductive response was reversible, dose-dependent (ED50 approximately 2 x 10(-8) M), required de novo protein and RNA synthesis, and was elicited only by glucocorticoid steroids. Progesterone, but not epicortisol, antagonized the dexamethasone-induced enzyme increase. In contrast, only a slight inductive effect was observed in dexamethasone-treated C6H cells. Competitive binding assays demonstrated that specific binding of [3H]-dexamethasone to cytosolic receptors was also dose-dependent. The ED50 was approximately 10(-8) M for both C6 and C6H cells. Scatchard analysis revealed that each C6 cell contained approximately 10,800 receptor sites and that the equilibrium dissociation constant (Kd) was 4.5 x 10(-9) M. Each C6H cell possessed approximately 12,200 sites, and the Kd was 6.7 x 10(-9) M. Unlabeled dexamethasone and cortisol (but not epicortisol) competed effectively with [3H]-dexamethasone for binding to cytosolic receptor sites and nuclear sites of both cell types. These results suggest that induction of glutamine synthetase activity in dexamethasone-treated C6 cells is a glucocorticoid-directed response. Since C6H cells are refractory in this regard but contain functional cytosolic receptors which interact with cell nuclei, the basis for their resistance appears to involve some step beyond these cellular processes.     

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)     

Characterization and localization of glutathione binding sites on cultured astrocytes.

Glutathione (GSH) binding sites found in brain white matter in a previous study using biotinylated GSH (Third IBRO World Congress Neurosci. Abstr., 1991, P59.17) suggested that there might GSH receptors on glial cells. In the present study, radioligand receptor assays were performed on cultured astrocytes using [35S]GSH. Scatchard analyses of saturation binding of [35S]GSH revealed two binding sites: Kd1 = 2.0 +/- 0.1 nM, Bmax1 = 89.5 +/- 1.5 fmole/2.2 x 10(5) cells and Kd2 = 12.8 +/- 0.4 nM, Bmax2 = 187.7 +/- 2.4 fmol/2.2 x 10(5) cells. The saturable and displacible high affinity [35S]GSH binding we have observed suggests that this binding is not due to GSH sequestration by uptake sites or to the association of GSH with GSH S-transferases or GSH peroxidases which have Kds in the microM range. Colloidal gold and immunofluorescence double labelling were used to visualize the binding sites at the cellular level. Positive colloidal gold decoration further suggests that these labelled binding sites are membrane receptors on astrocytes.     

Insulin and IGF-II, but not IGF-I, stimulate the in vitro regeneration of adult frog sciatic sensory axons

We used the in vitro regenerating frog sciatic nerve to look for effects of insulin and insulin-like growth factors I and II (IGF-I, IGF-II) on regeneration of sensory axons and on injury induced support cell proliferation in the outgrowth region. In nerves cultured for 11 days, a physiological dose ( 10ng/ml, ≈ 2 nM) of insulin or IGF-II increased ganglionic protein synthesis (by 20% and 50%, respectively) as well as the level of newly formed, radiolabelled axonal material distal to a crush injury (both by 80%), compared to untreated, paired controls. In addition, insulin increased the outgrowth distance of the furthest regenerating sensory axons by 10%. The preparation was particularly sensitive to insulin during the first 5 days of culturing. Furthermore, both insulin and IGF-II were found to inhibit proliferation of support cells in the outgrowth region in a manner suggesting effects via their individual receptors. The inhibition, about 30%, was observable after 4 but not 11 days in culture. It is not clear if this reflects a stimulated differentiation of some cells. By contrast, IGF-I lacked effects on both regeneration and proliferation. In conclusion, the results suggest that insulin and IGF-II are involved in the regulation of peripheral nerve regeneration.     

Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site

Bovine and rat pineal benzodiazepine receptors were characterized using ligands with high affinities for either 'central-type' (CBR) or 'peripheral-type' (PBR) benzodiazepine receptors. The characteristics (Bmax = 83 +/- 10 fmol/mg protein, Kd = 3.88 +/- 0.46 nM) of benzodiazepine receptors in bovine pineal membranes measured with [3H]flunitrazepam (using flunitrazepam to define non-specific binding) were consistent with previously reported values. However, if non-specific binding was defined using Ro 15-1788 (a selective CBR ligand), the Bmax and Kd of [3H]flunitrazepam decreased 51 and 58%, respectively. In addition, when using PK 11195 to determine non-specific binding, the Bmax of [3H]flunitrazepam binding to bovine pineal decreased further (approximately 80%, Kd decreased approximately 39%). Together, these observations strongly suggested the presence of PBR in the bovine pineal. Bovine pineal PBR characterized with [3H]PK 11195 revealed a high density (relative to CBR) of high affinity binding sites (Kd = 1.08 +/- 0.30, Bmax = 776 +/- 33.0 fmol/mg protein). In contrast, when [3H]Ro 5-4864 (1-20 nM) was used to define PBR, no binding was detectable. These observations are in sharp contrast to the rat pineal gland, in which both [3H]Ro 5-4864 and [3H]PK 11195 bind to a large number of PBR with high affinity (Kd approximately equal to 1.9 nM, Bmax approximately equal to 26 pmol/mg protein). Bovine pineal PBR were further characterized with compounds structurally related to either Ro 5-4864 or PK 11195.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term exposure of cortical cell cultures to clonazepam reduces benzodiazepine receptor binding

To determine if reduced drug efficacy after long-term exposure to clonazepam may be a consequence of benzodiazepine receptor alterations, cerebral cortical cell cultures were exposed to the drug (200 nM) for 14 days. Receptor binding was assayed on living cells in situ. After drug exposure, binding in experimental cultures differed markedly from controls with respect to total, specific, and clonazepam-displaceable (neuronal) benzodiazepine binding (60%, 53%, and 6% of control values, respectively) but recovered within 96 h of drug removal. RO5-4864-displaceable (nonneuronal) binding was modestly reduced at 0 time (72% of control), but returned to control values in 24 h. The differences in binding could be attributable to a relatively reduced affinity of the high-affinity binding site (Kd approximately 18 nM for controls and approximately 30 nM for drug-exposed cultures) but not to changes in the low-affinity binding site or to reduced numbers of receptors.     

A single class of neurotensin receptors with high affinity in neuroblastoma X glioma NG108-15 hybrid cells that mediate facilitation of synaptic transmission

Receptor binding of [3H]neurotensin was examined on membrane preparations derived from neuroblastoma X glioma NG108-15 hybrid cells. The specific binding was saturable and reversible, and a dissociation constant (Kd) was calculated to be about 0.24 nM from the rate constants. Scatchard analysis of neurotensin binding at equilibrium revealed a single class of binding sites with a Kd of 0.86 nM and a maximal binding capacity (Bmax) of 250 fmol/mg of protein (7700 receptor sites/cell). [D-Arg9]-Neurotensin had a high affinity (IC50 = 0.5 nM) for the neurotensin receptors, but [D-Phe11]-neurotensin had a lower affinity (IC50 = 280 nM), while angiotensin II and bradykinin had almost no affinity for [3H]neurotensin-binding sites. Under similar conditions [3H]neurotensin binding to mouse and rat brain synaptosomal fractions showed two binding sites with high (0.86 and 0.44 nM) and low (13 and 19 nM) affinities. We have examined several possible physiological consequences of neurotensin receptor binding. Neurotensin (10 microM) exhibited no influence on adenylate cyclase activity, 45Ca uptake, or 32Pi incorporation into phosphatidylinositol fractions of NG108-15 cells. Electrophysiological study of isolated NG108-15 cells revealed neurotensin-induced transient hyperpolarization followed by sustained depolarization with enhanced membrane excitability. Application of neurotensin to NG108-15 cells that had formed synapses with cultured striated muscle cells caused a considerable increase in frequency of miniature endplate potentials from the muscle cells. These data show that NG108-15 cells possess a single class of neurotensin receptors similar to a high affinity site of synaptosomal membranes from the murine brains.(ABSTRACT TRUNCATED AT 250 WORDS)