Pharmacological characterization of rat amylin receptors: implications for the identification of amylin receptor subtypes

Abstract

Background and purpose: Amylin (Amy) is an important glucoregulatory peptide and AMY receptors are clinical targets for diabetes and obesity. Human (h) AMY receptor subtypes are complexes of the calcitonin (CT) receptor with receptor activity-modifying proteins (RAMPs); their rodent counterparts have not been characterized. To allow identification of the most clinically relevant receptor subtype, the elucidation of rat (r) AMY receptor pharmacology is necessary.

Experimental approach: Receptors were transiently transfected into COS-7 cells and cAMP responses measured in response to different agonists, with or without antagonists. Competition binding experiments were performed to determine rAmy affinity.

Key results: rCT was the most potent agonist of rCT((a)) receptors, whereas rAmy was most potent at rAMY(1(a)) and rAMY(3(a)) receptors. rAmy bound to these receptors with high affinity. Rat α-calcitonin gene-related peptide (CGRP) was equipotent to rAmy at both AMY receptors. Rat adrenomedullin (AM) and rAM2/intermedin activated all three receptors but were most effective at rAMY(3(a)) . AC187, AC413 and sCT(8-32) were potent antagonists at all three receptors. rαCGRP(8-37) displayed selectivity for rAMY receptors over rCT((a)) receptors. rAMY(8-37) was a weak antagonist but was more effective at rAMY(1(a)) than rAMY(3(a)) .

Conclusions and implications: AMY receptors were generated by co-expression of rCT((a)) with rRAMP1 or 3, forming rAMY(1(a)) and rAMY(3(a)) receptors, respectively. CGRP was more potent at rAMY than at hAMY receptors. No antagonist tested was able to differentiate the rAMY receptor subtypes. The data emphasize the need for and provide a useful resource for developing new CT or AMY receptor ligands as pharmacological tools or potential clinical candidates.

Figure 1

Amino acid sequences of rAmy_8-37, sCT_8-32, AC413 and AC187. Alignments were performed with ClustalW. Identical residues are underlined.

Figure 2

Stimulation of cAMP production by (A) rat (r) CT, (B) rAmy, (C) rαCGRP, (D) rβCGRP, (E) rAM and (F) rAM2 in COS-7 cells transfected to express either rCT_(a) receptors, rAMY_1(a) receptors or rAMY_3(a) receptors as indicated. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin and are mean ± SEM of four to 23 combined experiments, performed in duplicate or triplicate.

Figure 3

Distribution of pEC₅₀ values for the agonists rCT, rAmy, rαCGRP, rβCGRP, (Cys(Et)^2,7)hαCGRP, (Cys(ACM)^2,7)hαCGRP, rAM and rAM2 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are mean ± SEM of four to 23 combined experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way anova followed by Tukey's test. For simplification, only comparisons against the cognate agonist for each receptor are shown (rCT for CT_(a), rAmy for rAMY_1(a) and rAMY_3(a)); other comparisons are shown in supplementary Tables S1–3. (Cys(Et)^2,7)hαCGRP and (Cys(ACM)^2,7)hαCGRP were excluded from the analysis except in the rAMY_3(a) receptor, where (Cys(Et)^2,7)hαCGRP produced a sufficient response to be included.

Figure 4

Antagonism of rAmy-induced cAMP production by the indicated concentrations of sCT_8-32 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin. Data are mean ± SEM of four combined experiments, performed in triplicate or quadruplicate.

Figure 5

Antagonism of rAmy-induced cAMP production by the indicated concentrations of AC187 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin. Data are mean ± SEM of three to four combined experiments, performed in triplicate or quadruplicate.

Figure 6

Antagonism of rAmy-induced cAMP production by the indicated concentrations of AC413 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin. Data are mean ± SEM of four combined experiments, performed in triplicate or quadruplicate.

Figure 7

Antagonism of rCT or rAmy-induced cAMP production by 10 µM rAmy_8-37 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin. Data are mean ± SEM of four to five combined experiments, performed in triplicate or quadruplicate.

Figure 8

Antagonism of rAmy-induced cAMP production by the indicated concentrations of rαCGRP_8-37 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin. Data are mean ± SEM of three to four combined experiments, performed in triplicate or quadruplicate.

Figure 9

Distribution of pK_B values for the antagonists sCT_8-32, AC187, rAmy_8-37, rαCGRP_8-37 and hαCGRP_8-37 in COS-7 cells transfected to express either (A) rCT_(a) receptors, (B) rAMY_1(a) receptors or (C) rAMY_3(a) receptors. Data are mean ± SEM of three to five combined experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way anova followed by Tukey's tests. Data points in boxes are significantly different, as indicated, from the comparator.

Figure 10

Specific binding of 100 pM [¹²⁵I]-rAmy in (A) Cos-7 cells transfected to express either rCT_(a) receptors, rAMY_1(a) receptors or rAMY_3(a) receptors and competitive displacement of 100 pM [¹²⁵I]-rAmy by (B) rAmy or (C) rαCGRP in COS-7 cells transfected to express either rat AMY_1(a) receptors or rat AMY_3(a) receptors expressed as a percentage maximum of 100 pM [¹²⁵I]-rAmy specific binding. Data are mean ± SEM of three to five (A) or two to three (B and C) combined experiments, performed in duplicate or triplicate.

Figure 11

Antagonism of rαCGRP-induced cAMP production by the indicated concentrations of rαCGRP_8-37 or hαCGRP_8-37 in COS-7 cells transfected to express either (A) and (D) rAMY_1(a) receptors or (B) and (E) rCGRP receptors or (C) rAM₂ receptors. Data are expressed as a percentage of the cAMP response generated by 50 µM forskolin. Data are mean ± SEM of three to four combined experiments, performed in triplicate or quadruplicate.

Figure 12

Distribution of pK_B values for (A) rαCGRP_8-37 or (B) hαCGRP_8-37 in COS-7 cells transfected to express either rAMY_1(a) receptors, rCGRP receptors or rAM₂ receptors using rαCGRP as the agonist. Data are mean ± SEM of three to five combined experiments. Comparisons were performed by (A) one-way anova followed by Tukey's tests or (B) unpaired t-test (**P < 0.01).

Figure 13

Representative gel electrophoresis image showing the expression of mRNA encoding rCLR, rRAMP1 and rCT receptor in rat IAS and VD. Similar results were obtained from tissue derived from three to four adult male Wistar rats.

Figure 14

Stimulation of cAMP production by rAM, rAM2 or rαCGRP in COS-7 cells transfected to express either (A) rCGRP receptors, (B) rAM₂ receptors or (C) mouse (m) AM₂ receptors. Data are expressed as the concentration of cAMP generated and are mean ± SEM of three to eight combined experiments, performed in triplicate.