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

Abstract

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 microM. 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-competitive' behaviour. At concentrations of up to 10 micro M, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP(8-37).

Figure 1

Effects of BIBN4096BS on the stimulation of cAMP production by human αCGRP in the presence of the indicated concentrations of BIBN4096BS in (a) L6 cells, (b) SK-N-MC cells and (c) Col 29 cells. Data represent means±s.e.mean of two to five experiments. Points were measured in duplicate in each experiment. Data are expressed as percentage of maximum cAMP production, estimated by fitting each line to a logistic Hill equation as described in the Methods. Maximum cAMP values were as follows: L6 cells, 270±30 pmol per 10⁶ cells; SK-N-MC cells, 240±20 pmol per 10⁶ cells; Col 29 cells, 170±19 pmol per 10⁶ cells. Basal values were all below 10 pmol per 10⁶ cells.

Figure 2

Schild transform of concentration–response curves shown in Figure 1; (a) fitted to a straight line with unconstrained slope and (b) fitted to a straight line with slope constrained to 1.

Figure 3

Effects of CGRP_8–37 on the stimulation of cAMP production by human αCGRP in the presence of the indicated concentrations of antagonist in (a) L6 cells, (b) SK-N-MC cells and (c) Col 29 cells. Data represent means±s.e.mean of four to seven experiments. Points were measured in duplicate in each experiment. Data are expressed as percentage of maximum cAMP production, estimated by fitting each line to a logistic Hill equation as described in the Methods. Maximum cAMP values were as follows: L6 cells, 250±27 pmol per 10⁶ cells; SK-N-MC cells, 230±18 pmol per 10⁶ cells; Col 29 cells, 180±17 pmol per 10⁶ cells. Basal values were all below 10 pmol per 10⁶ cells.

Figure 4

Effects of CGRP_8–37 and BIBN4096BS on the stimulation of cAMP production by rat adrenomedullin in Rat 2 cells. Data represent means±s.e.mean of three to four experiments. Points were measured in triplicate in each experiment. Data are expressed as percentage of maximum cAMP production, estimated by fitting each line to a logistic Hill equation as described in the Methods. Maximum cAMP values were 25±2.7 pmol per 10⁶ cells. Basal values were all below 1 pmol per 10⁶ cells.