Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor-like receptor (CLR)/receptor-activity-modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells.Both neuronal and non-neuronal CLR immunopositive cells were present in our model.High affinity, specific [125I]-AM binding sites (Kd 79±9 pM and Bmax 571±34 fmol mg−1 protein) were more abundant than specific [125I]-CGRP binding sites (Kd 12±0.7 pM and Bmax 32±2 fmol mg−1 protein) in embryonic spinal cord cells.Specific [125I]-AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM22–52>rCGRPα>CGRP8–37≫[r-(r*,s*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide (BIBN4096BS).Specific [125I]-CGRP binding was competed by rCGRPα>rAMCGRP8–37BIBN4096BS>hAM22–52.Cellular levels of cAMP were increased by AM (pEC50 10.2±0.2) and less potently by rCGRPα (pEC50 8.9±0.4). rCGRPα-induced cAMP accumulation was effectively inhibited by CGRP8–37 (pA2 7.63±0.44) and hAM22–52 (pA2 6.18±0.21) while AM-stimulation of cAMP levels was inhibited by CGRP8–37 (pA2 7.41±0.15) and AM22–52 (pA2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA2 8.40±0.30) on cAMP accumulation.These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM1 (CLR/RAMP2) receptor subtype.British Journal of Pharmacology (2006) 148, 459–468. doi:10.1038/sj.bjp.0706750; published online 15 May 2006 [ABSTRACT FROM AUTHOR]