Debbie L. Hay

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

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 μM) 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 μM) 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 μM 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 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14). BIBN4096BS (10 μM) 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∼βCGRP>αCGRP. 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.

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines.

The calcitonin receptor‐like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene‐related peptide (CGRP) and/or adrenomedullin in transfected cells. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. We confirmed CGRP subtype 1 receptor (CGRP1) pharmacology in SK‐N‐MC neuroblastoma cells. L6 myoblast cells expressed both CGRP1 and adrenomedullin receptors whereas Rat‐2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP2 receptor pharmacology for Col‐29 colonic epithelial cells, which, instead were CGRP1‐like in this study. L6, SK‐N‐MC and Col‐29 cells expressed mRNA for RAMP1 and RAMP2 but Rat‐2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. SK‐N‐MC, Col‐29 and Rat‐2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus, circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

A comparison of the actions of BIBN4096BS and CGRP8–37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells

The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. On human SK‐N‐MC cells (expressing human calcitonin receptor‐like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA2 of 9.95 although the slope of the Schild plot (1.37±0.16) was significantly greater than 1. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA2 of 9.25 and a Schild slope of 0.89±0.05, significantly less than 1. On human Colony (Col) 29 cells, CGRP8–37 had a significantly lower pA2 than on SK‐N‐MC cells (7.34±0.19 (n=7) compared to 8.35±0.18, (n=6)). BIBN4096BS had a pA2 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. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. CGRP8–37 had a pA2 of 6.72 against adrenomedullin. 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 CGRP8–37.

Knockouts and transgenics confirm the importance of adrenomedullin in the vasculature

Adrenomedullin might be a much more important player in vascular function than previously thought. The Adm knockout models presented at this conference confirm the role of adrenomedullin in control of blood pressure and also support the argument for more research on adrenomedullin as an angiogenic factor 8xPCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non-estrogenic mechanism in the human endometrium. Zhao, Y et al. Oncogene. 1998; 16: 409–415Crossref | PubMedSee all References8. The adrenomedullin binding proteins also challenge our preconceptions of effective circulating adrenomedullin levels.Key conference outcomes Adrenomedullin receptors are combinations of a calcitonin receptor-like receptor (CRLR) with receptor activity modifying proteins (RAMPs). Two germ-line deletions of the gene encoding adrenomedullin (Adm) were demonstrated, one deleting only adrenomedullin and the other deleting adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP). Knockout of the Admgene is lethal in the homozygous form. The phenotype of the Admknockout mouse might be associated with a failure of normal vessel development. Knockout of the Adm –PAMP gene increases blood pressure whereas overexpression of adrenomedullin lowers blood pressure. The effects of injury on blood vessels are enhanced in both Admknockout mice but are reduced in mice overexpressing adrenomedullin. Complement factor H is an adrenomedullin binding protein that enhances adrenomedullin-receptor-mediated effects.

Desensitisation of adrenomedullin and CGRP receptors

Adrenomedullin (AM), a potent vasoactive peptide, is elevated in certain disease states such as sepsis. Its role as a physiologically relevant peptide has been confirmed with the advent of the homozygous lethal AM peptide knockout mouse. So far, there have been few and conflicting studies which examine the regulatory role of AM at the receptor level. In this article, we discuss the few studies that have been presented on the desensitisation of AM receptors and also present novel data on the desensitisation of endogenous AM receptors in Rat-2 fibroblasts.