Calcitonin (CT) receptors dimerise with receptor activity modifying proteins (RAMPs) to create high affinity amylin (AMY) receptors. The CT _(a) receptor/RAMP1 complex is termed the AMY1 _(a) receptor (Poyner et al., 2002 for review). Although characterised by binding, the functional effects of antagonists at these receptors have not been established.

Here, we transiently transfected COS-7 cells with the human (h) CT _(a) receptor with, or without hRAMP1 using Lipofectamine 2000 and assessed the effects of antagonists via assay of cAMP responses. After ~36 hours, transfected cells were incubated with 1mM isobutylmethylxanthine/0.1 % bovine serum albumin in serum free Dulbecco's Modified Eagle Medium for 30 minutes before addition of drugs diluted in the same buffer. After 30 minutes, reactions were terminated with lysis buffer (0.3 % v/v Tween 20, 5 mM HEPES, 0.1 % w/v BSA in water, pH 7.4) and the cAMP content assayed by ALPHA screen (Perkin Elmer). Data were analysed using global Schild analysis in Graphpad Prism 4. Schild slopes were not significantly different from 1 so were constrained to 1 to calculate pK _B values. Statistical analysis was by Student’s unpaired t-test or by one-way ANOVA followed by Tukey’s test where multiple comparisons were made.

Agonist pEC₅₀ values were 8.99±0.1 (n=9) and 6.95±0.18 (n=8) for hCT and rat (r) AMY at CT _(a), respectively. At AMY _1(a) the rAMY pEC₅₀ was 9.12±0.16 (10), the hCT pEC₅₀ was 8.93±0.09 (7). As predicted and as shown in table 1, AC187 was most effective at inhibiting AMY responses at AMY _1(a) receptors. sCT _8-32 was not discriminatory between receptors. The results also indicate that RAMP1 is required for CGRP _8-37 to attenuate rAMY responses. The pK _B obtained for CGRP_8-37 at AMY 1_(a) was significantly lower (P<0.05) than that obtained against CGRP at CGRP₁ receptors (pK _B 8.67±0.37 n=3), transfected into COS-7 cells in parallel experiments.

The affinity of CGRP _8-37 that we report for AMY _1(a) is comparable with that for putative CGRP ₂ receptors (Poyner et al., 2002). This potential explanation for CGRP₂ receptors is consistent with previous reports that CGRP _8-37 is an antagonist of AMY receptors and early reports that AMY acted via CGRP receptors to produce its effects.

Table 1. pK _B values ± s.e.mean (n) for antagonists at CT _(a) and AMY1 _(a) receptors using hCT or rAMY as agonists. n.s., no significant shift by up to 10 µM antagonist. n.d., not done. **significantly different (P<0.01) to the same conditions at CT _(a) (Student’s t-test).