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Characterization of CGRP receptor sites in rat airways
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11 FUNCTIONAL AND BIOCHEMICAL CHARACTERIZATION OF RAT LYMPHOCYTE CGRP RECEPTORS
J.P. MCGILLIS, S. HUMPHREYS, and S. REID, Department of Microbiology and Immunology, MS415, University of Kentucky College of Medicine, Lexington KY 40536-0084 CGRP receptors were studied in the rat immune system by radioligand binding, affinity labelling, and 2nd messenger responses. CGRP binding studies on purified rat T and B cells revealed the presence of high affinity binding sites for 1251-[HisI0]-CGRP, Binding at 4 ° and 22 ° was rapid, reaching equilibrium by 10 minutes. Saturation binding determined that T and B cell CGRP receptors have Kds of 0.807 + 0.168 nM and 0.387 + 0.072 nM and densities of 774 + 387 and 747 + 244 binding sites/cell, respectively. Rat CGRP was the most potent inhibitor of CGRP binding, Ki = 0.192 + 0.073 nM, followed by human CGRP and the CGRP antagonist CGRP 8-37" 1251-[HisI0]-CGRP binding to lymphocytes was not inhibited by calcitonin, neuropeptide Y, or substance P. The lymphocyte CGRP receptor was further characterized by SDSPAGE analysis of CGRP receptors which were affinity labelled with 1251[HisI0]-CGRP and disuccinimidyl suberate. The predominant affinity labelled receptor protein had a mw of 74,500. Minor protein bands of mw 220,000 and one of higher mw which barely penetrated the gel were also observed. The effect of CGRP on cAMP production was also studied. CGRP stimulated cAMP in lymphocytes in a dose dependent manner with an EDs0 of---8 pM, and could be inhibited by the CGRP antagonist CGRP8_37. The cAMP response to CGRP was rapid and sustained, with CGRP still elevated at i hour. In contrast, cAMP elevation following isoproterinol treatment returned to baseline by 30 minutes. Further studies are being done to determine the immunoregulatory and inflammatory effects of CGRP on lymphocyte function.
12 CHARACTERIZATION OF CGRP RECEPTOR SITES IN RAT AIRWAYS C. LANOUE 1, A. FOURNIER2, S. ST-PIERRE2 and A. CADIEUX1, IDepartment of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke (Quebec), Canada J1H 5N4, and 2INRS-Santt, Universit6 du Qutbec, Pointe-Claire (Quebec), Canada H9R 1G6 We have found CGRP to exert a potent and dose-related antibronchoconstrictor action in several mammalian species including man. This inhibitory effect of the peptide is non-specific and most powerfully expressed in distal airways. The purpose of this study was to characterize the receptors involved in this CGRP action by using synthetic peptide derivatives, hCGRPtx(8-37) or hCGRP~x(12-37) or [Cys(ACM)27]hCGRPtz, known to present different affinities between two putative CGRP receptor subtypes. All experiments were carded out in vitro and each CGRPrelated peptide was tested for its potency and efficiency as agonist and/or antagonist in lung parenchymal strips. Our results revealed that [Cys(ACM)2"7]hCGRPct maintained the inhibitory action of the native molecule in carbamylcholine-and 5-HT-indueed contractions. On an equimolar basis the linear analogue was 100 fold less potent than CGRP. In contrast, C-terminal hCGRP fragments (8-37) and (12-37) retained any CGRP like activity in rat isolated airways. However, when present in the incubation medium, both hCGRP fragments, in concentrations ranging from 10.7 to 10.2 M, displayed competitive and dose-related antagonist properties toward the inhibitory effect of CGRP. These results indicate that the antibronchoconstrictor action of CGRP is mediated via specific receptor interactions and that the receptors involved are of the CGRP 1 subtype. (Supported by the MRCC).
11 FUNCTIONAL AND BIOCHEMICAL CHARACTERIZATION OF RAT LYMPHOCYTE CGRP RECEPTORS
J.P. MCGILLIS, S. HUMPHREYS, and S. REID, Department of Microbiology and Immunology, MS415, University of Kentucky College of Medicine, Lexington KY 40536-0084 CGRP receptors were studied in the rat immune system by radioligand binding, affinity labelling, and 2nd messenger responses. CGRP binding studies on purified rat T and B cells revealed the presence of high affinity binding sites for 1251-[HisI0]-CGRP, Binding at 4 ° and 22 ° was rapid, reaching equilibrium by 10 minutes. Saturation binding determined that T and B cell CGRP receptors have Kds of 0.807 + 0.168 nM and 0.387 + 0.072 nM and densities of 774 + 387 and 747 + 244 binding sites/cell, respectively. Rat CGRP was the most potent inhibitor of CGRP binding, Ki = 0.192 + 0.073 nM, followed by human CGRP and the CGRP antagonist CGRP 8-37" 1251-[HisI0]-CGRP binding to lymphocytes was not inhibited by calcitonin, neuropeptide Y, or substance P. The lymphocyte CGRP receptor was further characterized by SDSPAGE analysis of CGRP receptors which were affinity labelled with 1251[HisI0]-CGRP and disuccinimidyl suberate. The predominant affinity labelled receptor protein had a mw of 74,500. Minor protein bands of mw 220,000 and one of higher mw which barely penetrated the gel were also observed. The effect of CGRP on cAMP production was also studied. CGRP stimulated cAMP in lymphocytes in a dose dependent manner with an EDs0 of---8 pM, and could be inhibited by the CGRP antagonist CGRP8_37. The cAMP response to CGRP was rapid and sustained, with CGRP still elevated at i hour. In contrast, cAMP elevation following isoproterinol treatment returned to baseline by 30 minutes. Further studies are being done to determine the immunoregulatory and inflammatory effects of CGRP on lymphocyte function.
12 CHARACTERIZATION OF CGRP RECEPTOR SITES IN RAT AIRWAYS C. LANOUE 1, A. FOURNIER2, S. ST-PIERRE2 and A. CADIEUX1, IDepartment of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke (Quebec), Canada J1H 5N4, and 2INRS-Santt, Universit6 du Qutbec, Pointe-Claire (Quebec), Canada H9R 1G6 We have found CGRP to exert a potent and dose-related antibronchoconstrictor action in several mammalian species including man. This inhibitory effect of the peptide is non-specific and most powerfully expressed in distal airways. The purpose of this study was to characterize the receptors involved in this CGRP action by using synthetic peptide derivatives, hCGRPtx(8-37) or hCGRP~x(12-37) or [Cys(ACM)27]hCGRPtz, known to present different affinities between two putative CGRP receptor subtypes. All experiments were carded out in vitro and each CGRPrelated peptide was tested for its potency and efficiency as agonist and/or antagonist in lung parenchymal strips. Our results revealed that [Cys(ACM)2"7]hCGRPct maintained the inhibitory action of the native molecule in carbamylcholine-and 5-HT-indueed contractions. On an equimolar basis the linear analogue was 100 fold less potent than CGRP. In contrast, C-terminal hCGRP fragments (8-37) and (12-37) retained any CGRP like activity in rat isolated airways. However, when present in the incubation medium, both hCGRP fragments, in concentrations ranging from 10.7 to 10.2 M, displayed competitive and dose-related antagonist properties toward the inhibitory effect of CGRP. These results indicate that the antibronchoconstrictor action of CGRP is mediated via specific receptor interactions and that the receptors involved are of the CGRP 1 subtype. (Supported by the MRCC).