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Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary ( calcitonin / cerebellum / spinal cord / thyroid )
| Content Provider | Semantic Scholar |
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| Author | Tschopp, Fritz A. Henke, Hermann Petermann, Josef B. Tobler, P. H. Lundberg, Jan M. Cuello, Claudio A. Fischer, Jan A. |
| Abstract | Binding sites for synthetic human 1251-labeled calcitonin gene-related peptide (125I-CGRP) have been demonstrated in membranes of the human nervous system. Binding was high in the cerebellar cortex (1.35 ± 0.27 fmol/mg of tissue; mean ± SEM), spinal cord (1.06 ± 0.27 to 1.27 ± 0.23 fmol/mg), and nucleus dentatus (1.02 ± 0.15 fmol/mg), intermediate in the inferior colliculus (0.80 ± 0.14 fmol/mg) and substantia nigra (0.75 ± 0.14 fmol/mg), low in the neocortex, globus pallidus, nucleus caudatus, hippocampus, amygdala, superior colliculus, thalamus, and hypothalamus (0.15-0.32 fmol/mg), and negligible in spinal and sympathetic ganglia and pituitary (<0.04 fmol/mg). Autoradiography showed distinct 1251-CGRP binding over the molecular and Purkinje cell layers of the cerebellar cortex and over the substantia gelatinosa posterior of the spinal cord. The highest levels of CGRP-like components were recognized in the dorsal part of the spinal cord and the pituitary gland. In the ventral part of the spinal cord as well as in the pituitary and thyroid glands, CGRP values were higher when measured by radioreceptorassay as compared to RIA, indicating that at least two CGRP-like components are present. The predominant CGRPlike peak on HPLC had the retention time of synthetic human CGRP. Immunohistochemistry revealed the presence of a dense plexus of CGRP immunoreactive nerve fibers in the dorsal horn of the spinal cord. The existence of rat and human calcitonin gene-related peptide (CGRP) has been predicted by analysis of the nucleotide sequence of the calcitonin gene (1, 2). CGRP is a unique 37 amino acid peptide that shares greater structural homology with salmon calcitonin-(1-32) than with rat and human calcitonin-(1-32) (1-3). Expression ofCGRP was shown immunohistochemically and by specific RIA in combination with gelpermeation chromatography and HPLC in the nervous system and in the pituitary and thyroid glands (4-6). The amino acid sequence was derived from human medullary carcinoma of the thyroid (7). Here we report distinct regional distribution of CGRP and calcitonin (CT) binding sites in the human central nervous system. The presence of endogenous CGRP-like components in the nervous system, pituitary, and thyroid was revealed by a radioreceptorassay (RRA) using human cerebellar membranes, by RIA, and by immunohistochemistry. MATERIALS AND METHODS Tissues and Peptides. Tissue obtained at autopsy within 20 hr postmortem was frozen on dry ice and stored at -80°C for a maximum of 3 wk. Patients with malignant tumors, chronic renal insufficiency, or metabolic bone disease were excluded from the study. Synthetic salmon CT-(1-32) and salmon [3H]CT-(1-32) (28 Ci/mmol; 1 Ci = 37 GBq) were donated by S. Guttmann (Sandoz AG, Basel, Switzerland); human [3H]CT-(1-32)(27 Ci/mmol), by R. Wade (Ciba-Geigy, Horsham, U.K.); and synthetic human CT-(1-32), a-melanotropin, corticotropin(1-39), and fragments thereof, by W. Rittel (Ciba-Geigy AG, Basel, Switzerland). Synthetic human and rat CGRP(1-37) and PDN-21 were purchased from Peninsula Laboratories (Belmont, CA); synthetic angiotensin I, ,B-endorphin, vasopressin, oxytocin, somatostatin, somatoliberin, and substance P, from Beckman Instruments (Geneva, Switzerland); [Leu5]and [Met5lenkephalin, luliberin, and thyroliberin, from Bachem Fine Chemicals (Torrance, CA); and morphine sulfate, from Pentax (Lyndhurst, NJ). Fluorescein isothiocyanate-conjugated second antibodies were obtained from Dakopatts (Copenhagen, Denmark). Receptor Binding Studies. Membranes were prepared and incubated as described (8, 9) with 17,000 dpm (1 dpm = 16.7 mBq) of human 125I-labeled CGRP (125I-CGRP) (330-380 Ci/mmol) or salmon 125I-labeled CT (125I-CT) (570-630 Ci/mmol). Specific binding was defined as total binding minus binding in the presence of 0.7 ,uM unlabeled human CGRP or salmon CT, respectively. A RRA for CGRP was developed using membranes from the human cerebellar cortex; tissue extracts or peptides were incubated with membranes obtained from 6 mg of wet tissue for 3 hr at 6°C in 0.4 ml of 50 mM Hepes/Tris containing 1% (wt/vol) bovine serum albumin and 0.02% (wt/vol) sodium azide. Results are expressed in ng equivalents of synthetic human CGRP. Autoradiography. Autoradiography was performed with sections (25 ,m) of cerebellum and spinal cord that were incubated with human 125I-CGRP as described elsewhere (10). The exposure time was 4 wk. Nonspecific binding was determined in the presence of 2 AM unlabeled CGRP. The sections were stained with cresyl violet for histological examination. RIA. Immunoreactive CGRP was estimated in a heterologous system using antibodies raised to synthetic rat CGRP (6). Fifty percent inhibition of specific binding was achieved with 0.08 nM rat CGRP and 0.3 nM human CGRP, respectively. Human and salmon CT and the peptides listed above were not recognized in amounts of up to 5 ,uM. Results are expressed in ng equivalents of synthetic human CGRP. Immunohistochemistry. Human postmortem spinal cord obtained 3-24 hr after death was cut into 1to 3-mm-thick Abbreviations: CGRP, calcitonin gene-related peptide; CT, calcitonin; RRA, radioreceptorassay. IlTo whom reprint requests should be addressed. 248 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. NatL Acad Sci. USA 82 (1985) 249 |
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| Alternate Webpage(s) | http://www.pnas.org/content/82/1/248.full.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
