A comparison of the actions of BIBN4096BS and CGRP 8-37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells

1. The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. 2. On human SK-N-MC cells (expressing human calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA 2 of 9.95 although the slope of the Schild plot (1.37±0.16) was significantly greater than 1. 3. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA 2 of 9.25 and a Schild slope of 0.89±0.05, significantly less than 1. 4. On human Colony (Col) 29 cells, CGRP 8-37 had a significantly lower pA 2 than on SK-N-MC cells (7.34±0.19 (n=7) compared to 8.35±0.18, (n=6)). BIBN4096BS had a pA 2 of 9.98 and a Schild plot slope of 0.86±0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK-N-MC cells. 5. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. CGRP 8-37 had a pA 2 of 6.72 against adrenomedullin. 6. BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK-N-MC cells and the CGRP-responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent 'non-competive' behaviour. At concentrations of up to 10 μM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP 8-37.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells:Studies with chimeric and other peptides

1. Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8-37 (hαCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase). 2. On SK-N-MC cells the potency order was hαCGRP8-37 > hαCGRP19-37 = AC187 > rat amylin8-37 > hα[Tyr0]-CGRP28-37 (apparent pKBS of 7.49 ± 0.25, 5.89 ± 0.20, 6.18 ± 0.19, 5.85 ± 0.19 and 5.25 ± 0.07). The SK-N-MC receptor appeared CGRP1-like. 3. On Col 29 cells, only hαCGRP8-37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB = 6.48 ± 0.28). Its receptor appeared CGRP2-like. 4. hα[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP8-37. 5. On SK-N-MC cells, hαCGRP(8-18, 28-37) (M433) and mastoparan-hαCGRP28-37 (M432) had apparent pKBS of 6.64 ± 0.16 and 6.42 ± 0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. 6. M433 was almost as potent as hαCGRP8-37 on Col 29 cells (apparent pKB = 6.17 ± 0.20). Other antagonists were inactive.