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.     

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.     

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.     

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.     

Uptake of circulating insulin-like growth factor-I into the cerebrospinal fluid of normal and diabetic rats and normalization of IGF-II mRNA content in diabetic rat brain

Brain injury has been prevented recently by systemic administration of human insulin-like growth factor-I (hIGF-I). It is widely believed that protein neurotrophic factors do not enter the brain from blood, and the mechanism by which circulating hIGF-I may be neuroprotective is uncertain. This investigation tested the hypothesis that hIGF-I is taken up into cerebrospinal fluid (CSF) from the circulation. (125)I-hIGF-I was injected subcutaneously into rats. The (125)I-IGF-I recovered from CSF and plasma were indistinguishable in size from authentic (125)I-hIGF-I on SDS-PAGE. An ELISA was used that detected immunoreactive hIGF-I, but not rat IGF-I, rat IGF-II, human IGF-II, or insulin. Osmotic minipumps were implanted for constant subcutaneous infusion of various hIGF-I doses. Uptake into CSF reached a plateau at plasma concentrations above approximately 150 ng/ml hIGF-I; the plateau was consistent with carrier-mediated uptake. The plasma, but not CSF, hIGF-I level was significantly reduced in streptozotocin diabetic vs. nondiabetic rats, and uptake of hIGF-I into CSF was nonlinear with respect to plasma hIGF-I concentrations. Nonlinear uptake excluded leakage or transmembrane diffusion of IGF-I from blood into CSF as a dominant route for entry, but the site and mechanism of uptake remain to be established. The IGF-II mRNA content per milligram brain (P < 0.02) as well as per poly(A)(+) RNA (P < 0.05) was significantly increased towards normal in diabetic rats treated by subcutaneous administration of hIGF-I vs. vehicle. This effect of circulating hIGF-I may have been due to regulation of IGF-II gene expression in the choroid plexus and leptomeninges, structures at least in part outside of the blood-central nervous system barrier. These data support the hypothesis that circulating IGF-I supports the brain indirectly through regulation of IGF-II gene expression as well as by uptake into the CSF.     

Axonal transport of neurotrophins by visceral afferent and efferent neurons of the vagus nerve of the rat

The receptor-mediated axonal transport of [¹²⁵I]-labeled neurotrophins by afferent and efferent neurons of the vagus nerve was determined to predict the responsiveness of these neurons to neurotrophins in vivo. [¹²⁵I]-labeled neurotrophins were administered to the proximal stump of the transected cervical vagus nerve of adult rats. Vagal afferent neurons retrogradely transported [¹²⁵I]neurotrophin-3 (NT-3), [¹²⁵I]nerve growth factor (NGF), and [¹²⁵I]neurotrophin-4 (NT-4) to perikarya in the ipsilateral nodose ganglion, and transganglionically transported [¹²⁵I]NT-3, [¹²⁵I]NGF, and [¹²⁵I]NT-4 to the central terminal field, the nucleus tractus solitarius (NTS). Vagal afferent neurons showed minimal accumulation of [¹²⁵I]brain-derived neurotrophic factor (BDNF). In contrast, efferent (parasympathetic and motor) neurons located in the dorsal motor nucleus of the vagus and nucleus ambiguus retrogradely transported [¹²⁵I]BDNF, [¹²⁵I]NT-3, and [¹²⁵I]NT-4, but not [¹²⁵I]NGF. The receptor specificity of neurotrophin transport was examined by applying [¹²⁵I]-labeled neurotrophins with an excess of unlabeled neurotrophins. The retrograde transport of [¹²⁵I]NT-3 to the nodose ganglion was reduced by NT-3 and by NGF, and the transport of [¹²⁵I]NGF was reduced only by NGF, whereas the transport of [¹²⁵I]NT-4 was significantly reduced by each of the neurotrophins. The competition profiles for the transport of NT-3 and NGF are consistent with the presence of TrkA and TrkC and the absence of TrkB in the nodose ganglion, whereas the profile for NT-4 suggests a p75 receptor-mediated transport mechanism. The transport profiles of neurotrophins by efferent vagal neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus are consistent with the presence of TrkB and TrkC, but not TrkA, in these nuclei. These observations describe the unique receptor-mediated axonal transport of neurotrophins in adult vagal afferent and efferent neurons and thus serve as a template to discern the role of specific neurotrophins in the functions of these visceral sensory and motor neurons in vivo. J. Comp. Neurol. 393:102–117, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US government work and, as such, is in the public domain in the United States of America.

     

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.     

Effects of aminopeptidase inhibition on the half-lives of [I]angiotensins in the cerebroventricles of the rat

Anesthetized Sprague-Dawley rats fitted with intracerebroventricular (i.c.v.) cannulas were infused with one of the aminopeptidase inhibitors, amastatin or bestatin, over a 5-min period. After infusion, 1–2 × 10⁶ cpm of [¹²⁵I]angiotensin II([¹²⁵I]AII) or [¹²⁵I]angiotensin III([¹²⁵AIII) was injected through the same cannula. The rats were subsequently killed 60 s later by focused microwave irradiation which instantaneously terminated further [¹²⁵I]angiotensin metabolism. HPLC analysis of the extracted [¹²⁵I]angiotensin and metabolic products allowed for the calculation of of disappearance for the parent peptides. Both inhibitors effectively lengthened the half-lives of [¹²⁵I]AII and [¹²⁵I]AIII. Bestatin, which is considered a selective aminopeptidase B blocker, had a more pronounced effect on [¹²⁵I]AIII metabolism, while amastatin, a selective aminopeptidase A inhibitor, was better at slowing [¹²⁵I]AII degradation. The results indicate that amastatin and bestatin are very effective blockers of the cerebroventricular metabolism of angiotensins but are only marginally selective with regard to AII and AIII.     

Amphetamine effects on dopamine release and synthesis rate studied in the Rhesus monkey brain by positron emission tomography

Summary Positron emission tomography (PET) was used in a multitracer protocol to evaluate D-amphetamine induced effects on dopamine biosynthesis rate and release in propofol anesthetized Rhesus monkeys.l-[-¹¹C]DOPA was used as biochemical probe to study the brain dopamine biosynthesis rate whilst dopamine release was followed by the binding displacement of the [¹¹C]-radiolabelled dopamine receptor antagonists, raclopride and N-methylspiperone. Studies were performed with either a constant rate intravenous infusion of D-amphetamine aiming at plasma concentrations of 0.2 to 25 ng/ml or with intravenous bolus doses of 0.1 and 0.4 mg/ kg. Decreased binding of the dopamine receptor antagonists was measured in both modes of D-amphetamine administration but notably [¹¹C]N-methylspiperone was less able to sense D-amphetamine induced release of dopamine. At plasma concentrations aimed above 1 ng/ml a levelling off of the binding of [¹¹C]raclopride at 68 ± 8.1% of the baseline value indicated that displacement was only possible from a fraction of the binding sites. Amphetamine was observed to increase the rate constant forl-[-¹¹C]DOPA utilization in the brain. This was most likely due to an acutely induced subsensitivity of presynaptic dopamine receptors.l-[-¹¹C]DOPA and [¹¹C]raclopride were found suitable to indicate changes in dopamine synthesis rate and release respectively using PET and can be used to mirror drug-induced changes of brain dopaminergic function.     

Interferon-γ inhibits DNA synthesis and insulin-like growth factor-II expression in human neuroblastoma cells

Interferon-γ (IFN-γ) is known to be an antiproliferative, differentiation agent in many cell types, including neuroblastoma. In this study, we determined the effects of IFN-γ on cellular growth and expression of insulin-like growth factor II (IGF-II) and IGF receptors in the human neuroblastoma cell line SH-SY5Y. Incubation of SH-SY5Y cells in IFN-γ (20–100 U/ml) induced the formation of long neuritic processes. IFN-γ treatment also induced decreases in [³H]TdR incorporation, as well as serum-dependent changes in cell number. Treatment with IFN-γ reduced cell number 33% in the presence of serum but had no effect on cell number in the absence of serum. IGF-II mRNA content was 60% inhibited by IFN-γ, and was not serum dependent. The concentration of immunoreactive IGF-II in SH-SY5Y conditioned medium was also reduced in the presence of IFN-γ, to less than half of control levels. In contrast, type I IGF receptor mRNA content was increased more than three-fold after treatment with IFN-γ and serum. Co-incubation in IFN-γ (20–100 U/ml) and IGF-II on (3–10 nM) prevented the inhibitory effects of IFN-γ on [³H]TdR ncorporation in serum-free media. Our results suggest that IFN-γ may inhibit DNA synthesis and cell growth by interfering with an IGF-II/type I IGF receptor autocrine growth or survival mechanism.     

Characterization of angiotensin binding in the African Green monkey

The observation that there are differences in the characteristics and distribution of angiotensin receptors in the central nervous system of mammalian species led to the analysis of angiotensin binding in a primate model, the African Green monkey. Initial studies using [¹²⁵I]angiotensin II ([¹²⁵I]AII) as the radioligand showed binding in peripheral tissues but little binding in the central nervous system. Conversely, binding studies using [¹²⁵I]AIII as the radioligand indicated more central nervous binding with diminished peripheral binding. Specific binding of [¹²⁵I]AIII is evident throughout the brain with high binding in the circumventricular organs, striatum, caudate nucleus, olfactory bulb and localized areas of the thalamus and cerebral cortex. This binding was found to posses many of the properties commonly associated with binding to membrane-bound receptors. The specifically bound radioligand extracted from incubations of [¹²⁵I]AIII and central nervous tissue appears to be a product of the metabolism of [¹²⁵I]AIII rather that the peptide itself. Binding of [¹²⁵I]AII does occur in peripheral tissues and to a limited extent in the cerebellum, but to a different receptor from that characterized using [¹²⁵I]AIII. These results are similar to those seen in the gerbil and raise questions concerning the utilization of the rat as the primary model for studying the biochemistry of the brain-angiotensin system in humans.     

On the appearance of acetylcholine receptors in denervated rat diaphragm,and its dependence on nerve stump length

Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to 10 μg/ml of ACh and extrajunctional binding of [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]α-BTX) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level of ACh contracture and [¹²⁵I]α-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [¹²⁵I]α-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [¹²⁵I]α-BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.     

Characterization of the retrograde transport of nerve growth factor (NGF) using high specific activity [I]NGF

The process of the retrograde transport of nerve growth factor (NGF) has been recharacterized using a high specific activity preparation of [¹²⁵I]NGF. Most of the general conclusions reached in the previous studies of Hendry, Thoenen and co-workers have been confirmed. However, significant quantitative differences were noted. Intraocular (anterior eye chamber) administration of [¹²⁵I]NGF (< 10 ng) resulted in accumulation in the superior cervical ganglia beginning at about 4 h. The ratio of radioactivity in the ipsilateral contralateral ganglia was 15–30:1. Maximal accumulation was seen at about 12 h in the hamster and 16 h in rats. This pattern was quite different from that seen in other tissues. The uptake system from the eye of the rat was saturable (half-maximal at 15 ng) with maximal accumulation of 35–40 pg/ganglion. Systemic administration of [¹²⁵I]NGF (200 ng) to adult rats resulted in no accumulation in SGG or celiac ganglion prior to 3 h, with subsequent rapid accumulation by 6 h and a rapid fall in radioactivity after 12 h. A similar time course was seen in 5-day-old rats, although the time curve was shifted slightly toward shorter time. The radioactivity in ganglia co-migrated with native NGF by SDS gell electrophoresis. Cytochrome c of comparable specific activity was not transported, and NGF did not stimulate the uptake and transport of cytochrome c. The retrograde transport of [¹²⁵I]NGF was inhibited by the co-administration of biologically active, but not inactive, oxidized derivatives of NGF.By any route of administration, a significant percentage of the transported [¹²⁵I]NGF was found in a purified nuclear fraction of the ganglia. Coupled with previous observations of specific nuclear NGF receptors in embryonic chick and sympathetic ganglia, this suggests that, after internalization and retrograde transport, NGF may directly act on the nucleus to produce at least some of its effects on the responsive cell.     

Insulin-like growth factor II receptors in human brain and their absence in astrogliotic plaques in multiple sclerosis

Insulin-like growth factor (IGF) II receptors were studied in human adult brain by using autoradiography with [125I]IGF-II. Receptors were found to be widely distributed throughout all neuronal regions. The highest densities were found in plexus choroideus, granular layer of the cerebellar cortex, gyrus dendatus and pyramidal layer of the hippocampus, striatum, and cerebral cortex. White matter was devoid of IGF-II receptors. We also examined [125I]IGF-II binding in six plaques of multiple sclerosis, which were characterized by a dense network of astrocytes. We were unable to detect IGF-II receptors in any of the astrogliotic plaques, suggesting that IGF-II receptors in human brain are not involved in astrogliosis. The regional variations in neuronal distribution of IGF-II receptors suggest involvement of IGF-II in functions associated with specific neuronal pathways.     

Development and evaluation of [125I]IPPI for Tau imaging in postmortem human Alzheimer's disease brain

Objective: Alzheimer's disease (AD) is a neurodegenerative disease characterized by aggregation of Tau protein into paired helical filaments causing neurofibrillary tangles (NFT) in the brain. The aim of this study was to develop and evaluate the effectiveness of a novel radioiodinated tracer, 6‐[¹²⁵I]iodo‐3‐(1H‐pyrrolo[2,3‐c]pyridine‐1‐yl)isoquinoline ([¹²⁵I]IPPI), for binding to Tau protein (Ki = 0.75 nM) in postmortem human brain (AD and cognitively normal (CN). Methods: Radiosynthesis of [¹²⁵I]IPPI was carried out by radioiododestannylation and purified chromatographically. Computational modeling studies of IPPI and MK‐6240 binding on Tau fibril were evaluated. In vitro autoradiography studies were carried out with [³H]PIB for Aβ plaques and [¹²⁵I]IPPI for Tau in AD and CN brains and evaluate drug effects. Results: [¹²⁵I]IPPI was produced in >95% purity. Molecular modeling of IPPI revealed binding energies of IPPI (?7.8, ?8.1, ?8.2, ?7.5 Kcal/mol) at the four sites were comparable to MK‐6240 (?8.7, ?8.5, ?8.3, ?7.5 Kcal/mol). Ratio of average grey matter (GM) [¹²⁵I]IPPI in AD versus CN was found to be 7.31 (p = .07) and AD GM/ white matter (WM) = 4.35 (p = .09). Ratio of average GM/WM [¹²⁵I]IPPI in CN was 1.21. Binding of [¹²⁵I]IPPI correlated with the presence of Tau, confirmed by anti‐Tau Dako A0024. Specifically bound [¹²⁵I]IPPI to Tau in AD brains was displaced by MK‐6240 and IPPI (>90%). Monoamine oxidase inhibitors (MAO) inhibitors deprenyl and clorgyline effected [¹²⁵I]IPPI binding at >1 µM concentrations. Conclusion: [¹²⁵I]IPPI exhibited high binding in human AD frontal cortex and anterior cingulate and is a suitable radioiodinated ligand for Tau imaging.     

Altered Calcitonin Gene-related Peptide, Substance P and Enkephalin Immunoreactivities and Receptor Binding Sites in the Dorsal Spinal Cord of the Polyarthritic Rat

The dorsal horn of the spinal cord, which forms the locus of first synapses in pain pathways, is an important site of interaction between calcitonin gene-related peptide (CGRP), substance P and enkephalin—the neuropeptides considered to be especially involved in the regulation of pain perception. Since adjuvant-induced arthritic rats provide a suitable model for peripheral inflammation and hyperalgesia, the possible alterations of immunoreactive CGRP, substance P and enkephalin as well as the binding sites for [¹²⁵I]hCGRPα, [¹²⁵I]substance Plneurokinin-1, (NK1) and [¹²⁵I]FK-33-824/μ-opioid receptors were studied in the dorsal horn of the spinal cord receiving projections from the inflamed limbs. In arthritic rats compared to control animals, a bilateral increase in CGRP- and substance P-immunoreactive fibres and the presence of enkephalin-immunoreactive cell bodies were noted in the dorsal horn of the spinal cord. As for receptors, while a significant decrease in [¹²⁵I]hCGRPα and [¹²⁵I]substance P/NK1 binding sites was observed in selective layers, no measurable alteration in [¹²⁵I]FK-33-824/μ-opioid binding sites was noted in any regions of the arthritic rat dorsal horn compared to the unaffected control rats. Following unilateral section of the peripheral nerve prior to induction of arthritis, CGRP- and substance P-immunoreactive fibres were markedly depleted and no enkephalin-positive neurons were observed in the ipsilateral dorsal horn. Analysis of receptor binding sites in denervated arthritic rats, however, exhibited differential responses, i.e. a significant increase in [¹²⁵I]hCGRPα, a marked decrease in [¹²⁵I]FK-33-824/μ-opioid and apparently no alteration in [¹²⁵I]substance P/NK1 receptor binding sites were observed in the ipsilateral dorsal horn compared to the intact contralateral side. These results taken together provide anatomical evidence for a concerted role of these peptides in the regulation of adjuvant-induced hyperalgesia accompanying peripheral inflammation.     

Characterization of Desamino-5-[125I]Iodo-3-Methoxy-Zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain

• 1.1. Antagonists at 5-HT₃ receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT₃ ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT₃ antagonist, zacopride, binds with high affinity (1.3–1.5 nM) to CNS 5-HT₃ sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates.
• 2.2. Ninety-seven percent of total specific binding of [₁₂₅I]MIZAC (0.1 nM) of was displaced by bemesetron (3 μM), a selective 5-HT₃ antagonist. Competition studies using ligands with known affinities for 5-HT₃ sites give a high correlation with reported pK_i values (r² 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT₃ Receptor I.E. highest in cortex and hippocampus, and lowest in striatum and cerebellum.
• 3.3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [¹²⁵I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT₄, 5-HT₆, and 5-HT₇ receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [¹²⁵I]MIZAC for the 5-HT₃ receptor.
• 4.4. [¹²⁵I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [¹²⁵I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT₃ receptor. Given its high selectivity for 5-HT₃ binding sites, [¹²⁵I]MIZAC appears to be a promising ligand for labeling 5-HT₃ receptors in vitro and in vivo.

     

Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells

This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UC11. Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture. Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor. Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNFα) or interleukin-1β (IL1β), but not interleukin-2 or interleukin-6, decreased the density of SP receptors. This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation. The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines. The decrease in SP receptor density was readily reversible on washout of the cytokines. The EC₅₀ for TNFα was approximately 0.5 ng/ml, the EC₅₀ for IL1β was approximately 0.1 ng/ml. Radioligand binding studies with [¹²⁵I]TNFα indicated the presence of a low density of high affinity binding sites for this ligand: K_d = 2.5 ± 0.6ng/ml, B_max = 14.8 ± 2.7fmol bound/mg protein (assuming trimeric form of ligand bound). The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors.     

Diagnostic value of neuron specific enolase (NSE) in the cerebrospinal fluid of malignant brain tumor with intrathecal metastasis

Neuron specific enolase (NSE) in the cerebrospinal fluid (CSF) and serum of 54 cases of brain tumors, in the tumor tissues of 10 brain tumors and in the cyst fluids of 7 brain tumors was measured by radioimmunoassay with NSE measurement kit (Eiken Chemistry co.) NSE values in the cerebrospinal fluid of 35 malignant brain tumors showed abnormal increase higher than 10 ng/ml in 17 cases (about 50%) of them, whereas all of benign cases were lower than 10 ng/ml. The means and standard deviations of NSE in the cerebrospinal fluid of malignant tumors were 20.63 +/- 20.78 ng/ml in the astrocytoma grade 3 and 4, 19.73 +/- 15.5 ng/ml in the medulloblastoma and 12.4 +/- 8.9 ng/ml in the germ cell tumor. NSE values in the CSF of 12 brain tumors with intrathecal metastasis were 28.0 +/- 18.9 ng/ml (mean +/- SD) showing about three times as much as those without intrathecal metastasis. There was significant difference between these groups (p less than 0.01). All cyst fluids including in 7 cases of brain tumors demonstrated high values of NSE even if they were benign tumor and their NSE values in CSF were normal. NSE values of brain tumor tissues had no correlation with malignancy, but were higher in the periphery of the tumors than in the center. NSE values of CSF in brain tumors might be changed in relation with the improvement or deterioration on clinical state and CT. It might be suggested that the measurement of NSE in the CSF had significance as a monitor of therapeutic efficacy and prognosis for the brain tumors.