CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22-52, CGRP8-37 and BIBN4096BS

Abstract

Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3: we report the effects of the antagonist fragments of human AM and CGRP (AM22-52 and CGRP8-37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells. AM22-52 (10 microM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34+/-0.14 (hCL/hRAMP2), 7.28+/-0.06 (Rat 2), 7.00+/-0.05 (L6), 6.25+/-0.17 (rCL/hRAMP2)). CGRP8-37 (10 microM) resembled AM22-52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04+/-0.13 (hCL/hRAMP2), 6.72+/-0.06 (Rat2), 7.03+/-0.12 (L6)). On CL/RAMP3 receptors, 10 microM CGRP8-37 was an effective antagonist at all combinations (apparent pA2 values: 6.96+/-0.08 (hCL/hRAMP3), 6.18+/-0.18 (rCL/rRAMP3), 6.48+/-0.20 (rCL/hRAMP3)). However, 10 microM AM22-52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73+/-0.14). BIBN4096BS (10 microM) did not antagonise AM at any of the receptors. Where investigated (all-rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM approximately betaCGRP>alphaCGRP. rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. This study shows that on CL/RAMP complexes, AM22-52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8-37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

Figure 1

Characterisation of Cos 7 cells, (a) Responses of cells transfected with rCL/hRAMP3, cloning vector (pcDNA3), rCL and hRAMP3, and subsequently challenged with either 100 nM AM or serum-free medium (SFM). (b) Concentration–response curves to hαCGRP in cells transfected with hCL/hRAMP1, hCL and hRAMP1. Points are the mean±s.e.m. of triplicate determinations. These are representative data from experiments repeated 10 times.

Figure 2

Characterisation of the stimulation of cyclic AMP production by rat AM in Cos 7 cells transfected with CL/RAMP2 combinations. Points are the mean±s.e.m. of triplicate determinations. Concentration–response curves are representative of three to seven experiments. Data are expressed as the percentage of maximum cyclic AMP production, estimated by fitting each line to a logistic Hill equation, as described in Methods. Maximum cyclic AMP values were 250±20 pmol per 10⁶ cells for hCL/hRAMP2, and 450±60 pmol per 10⁶ cells for hCL/hRAMP3; basal values were all below 10 pmol per 10⁶ cells, (a) hCL/hRAMP2, AM_22–52; (b) hCL/hRAMP2, CGRP_8–37; (c) rCL/hRAMP2, AM_22–52; (d) rCL/hRAMP2, CGRP_8–37; (e) Schild plot, antagonism of AM by AM_22–52 on hCL/hRAMP2 and hCL/hRAMP3 receptors.

Figure 3

Characterisation of the stimulation of cyclic AMP production by rat AM in L6 and Rat 2 cells (endogenous rCL/rRAMP2). Points are the mean±s.e.m. of triplicate determinations. Concentration–response curves are representative of three or four experiments. Data are expressed as the percentage of maximum cyclic AMP production, estimated by fitting each line to a logistic Hill equation, as described in Methods. Maximum cyclic AMP values were 290±20 pmol per 10⁶ cells for Rat 2 cells, and 2000±300 pmol per 10⁶ cells for L6 cells; basal values were all below 10 pmol per 10⁶ cells, (a) L6 cells, AM_22–52 and BIBN4096BS against rAM; (b) L6 cells CGRP_8–37 against rAM; (c) L6 cells, AM_22–52 against αCGRP, (d) Rat 2 cells, AM_22–52 against rAM.

Figure 4

Characterisation of the stimulation of cyclic AMP production by rat AM in Cos 7 cells transfected with CL/RAMP3 combinations. Points are the mean±s.e.m. of triplicate determinations. Concentration–response curves are representative of three to six experiments. Data are expressed as the percentage of maximum cyclic AMP production, estimated by fitting each line to a logistic Hill equation, as described in Methods. Maximum cyclic AMP values were 450±60 pmol per 10⁶ cells for hCL/hRAMP3, 300±25 pmol per 10⁶ cells for hCL/rRAMP3 and 630±70 pmol per 10⁶ cells for rCL/rRAMP3; basal values were all below 10 pmol per 10⁶ cells, (a) hCL/hRAMP3, AM_22–52; (b) hCL/hRAMP3 CGRP_8–37; (c) rCL/hRAMP3, AM_22–52; (d) rCL/hRAMP3, CGRP_8–37; (e) rCL/rRAMP3, AM_22–52; (f) rCL/rRAMP3, CGRP_8–37.

Figure 5

Characterisation of the stimulation of cyclic AMP production by agonists in Cos 7 cells transfected with CL/RAMP3 combinations. Points are the mean±s.e.m. of triplicate determinations. Concentration–response curves are representative of three to 12 experiments. Data are expressed as the percentage of maximum cyclic AMP production to rAM, estimated by fitting each line to a logistic Hill equation, as described in Methods, (a) hCL/rRAMP3; (b) rCL/rRAMP3.

Figure 6

Summary of apparent pA₂ or pK_b values for CGRP_8–37 and AM_22–52 on CL/RAMP combinations. Values that are significantly different from each other according to Tukey's test are indicated. Where the affinity of the antagonist was too low to be calculated, the following comparisons have been highlighted: ⁺P<0.05; outside the 95% confidence limit for AM_22–52 on rCL/hRAMP2. ⁺⁺⁺P<0.001; outside the 99.9% confidence limits for AM_22–52 on rCL/rRAMP2. ^#P<0.05; outside the 95% confidence limit for CGRP_8–37 on rCL/hRAMP3.