Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells

doi:10.1016/0196-9781(94)00195-C

Peptides

Volume 16, Issue 3, 1995, Pages 421-424

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Abstract

Inhibition of human [¹²⁵I]calcitonin gene-related peptide-I ([¹²⁵I]hCGRP-I) binding by human adrenomedullin (hADM), its N-terminal truncated fragments, CGRP and amylin, and cyclic AMP accumulation were examined in the human neuroblastoma cell line SK-N-MC. Binding of [¹²⁵I]hCGRP-I (125 pM) was inhibited by hCGRP-I, hADM(1–52), hADM(13–52), and human amylin with IC₅₀ of 0.32 ± 0.06, 2.11 ± 0.26, 3.45 ± 0.54, and 68.8 ± 6.6 nM, respectively. hCGRP-I(8–37) and hADM(22–52), which lack the N-terminal ring structure, inhibited [¹²⁵I]hCGRP-I binding with IC₅₀ of 2.35 ± 0.45 and > 1000 nM. hCGRP-I, hADM(1–52), hADM(13–52) and human amylin stimulated cAMP accumulation with EC₅₀ of 0.40 ± 0.05, 18.1 ± 2.6, 51.3 ± 9.0 and 925 ± 159 nM, respectively, hCGRP-I(8–37) (100 nM) antagonized hCGRP-I and hADM(1–52) stimulated cAMP production with the same K_i of 16.6 ± 1.2 and 16.8 ± 1.1 nM. In conclusion, human ADM, which is more distantly related to CGRP than amylin, interacts more potently with the CGRP receptor in SK-N-MC cells than amylin. The N-terminal ring structure of hADM, unlike that of hCGRP, is essential for binding to the CGRP receptor. Coupling of hADM binding to cAMP stimulation is less efficient than for hCGRP-I and is reduced by deletion of the unique 12 amino acid sequence of hADM N-terminal to the ring structure.

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Calcitonin gene-related peptide (CGRP) is a highly potent vasodilator known to be involved in many physiological functions within the cardiovascular, gastrointestinal, immune, and nervous systems. This study assessed the desensitization of CGRP receptors by measuring agonist-mediated activation of adenylate cyclase in a model system employing human neuroblastoma-derived SK-N-MC cells. In these cells, we demonstrated that pre-incubation with CGRP (20 nM) induces a rapid desensitization of CGRP signaling (t_1/2 ≤ 3 min) by causing a decrease in potency and efficacy. CGRP's desensitization potency (DC₅₀ = 0.29 nM) is similar to its activation potency on non-desensitized cells (EC₅₀ = 0.20 nM). The desensitized receptors exhibited slow and incomplete re-sensitization upon removal of the pre-incubated ligand, resulting in 52–65% functional recovery after 3–5 h while CGRP binding sites were completely restored. Additional agonists within the calcitonin/CGRP family of peptides (calcitonin, amylin, adrenomedullin, and adrenomedullin 2) were compared to CGRP with regard to their ability to activate and desensitize CGRP receptors. Calcitonin and amylin did not cause receptor activation nor did they produce desensitization. Adrenomedullin and adrenomedullin 2 activated the receptors and produced desensitization, but at a slower rate and with a weaker desensitization potency than CGRP-induced desensitization. Adrenomedullin exhibited similar potency for receptor activation and desensitization, whereas adrenomedullin 2 has a 4-fold higher preference for receptor desensitization than for receptor activation. Activation and desensitization induced by CGRP, adrenomedullin and adrenomedullin 2 were blocked by the CGRP receptor antagonist CGRP8-37. These data indicate that CGRP receptors are desensitized by select peptides in the calcitonin/CGRP family. Slow recovery from the desensitized state may provide a strategy for timed modulation of the CGRP signaling pathway.
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The effects of intravenous (iv) adrenomedullin (AM) on gastric emptying were investigated in conscious rats. AM induced a maximal 50% inhibition of gastric emptying at a dose of 1.2 nmol/kg. AM was about two-fold less potent than α-calcitonin gene-related peptide (α-CGRP), which induced a similar 50% maximal inhibition of gastric emptying at 0.6 nmol/kg. Delayed gastric emptying induced by i.v. AM and α-CGRP was prevented by peripheral injection of the selective CGRP₁ antagonist, CGRP_8–37, and by pretreatment with indomethacin, while not altered by blockade of the sympathetic nervous system with propranolol. These data indicate that peripheral AM inhibits gastric emptying through the interaction with CGRP_8–37-sensitive receptors, likely CGRP₁ receptors, and the recruitment of prostaglandin-dependent mechanisms.
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Only limited pharmacological characterization of the CGRP₁ receptor, a heterodimer of the calcitonin (CT) receptor-like receptor (CL) and receptor activity-modifying protein 1 has been performed in cells that do not endogenously express RAMP2. We characterized the receptor in RAMP-deficient Cos 7 cells by measuring cAMP responses following agonist treatment in the absence or presence of antagonists. Potent cAMP responses to human α-and β-CGRP (Cys(Et)^2,7)hαCGRP and human adrenomedullin (AM) were observed. Adrenomedullin_15–52 was also an effective agonist of the CGRP₁ receptor but human and salmon calcitonin and rat amylin were only weak agonists. As expected, BIBN4096BS and CGRP_8–37 were effective antagonists of the CGRP₁ receptor. (Cys(Acm)^2,7)hαCGRP also antagonized CGRP responses. Antagonists of related receptors were only weakly able to inhibit CGRP responses.
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Adrenomedullin (AM) is a peptide that possesses potentially beneficial properties. Since the initial discovery of the peptide by Kitamura et al. in 1993, the literature has been awash with reports describing its novel mechanisms of action and huge potential as a therapeutic target. Strong evidence now exists that AM is able to act as an autocrine, paracrine, or endocrine mediator in a number of biologically significant functions, including the endothelial regulation of blood pressure, protection against organ damage in sepsis or hypoxia, and the control of blood volume through the regulation of thirst. Its early promise as a potential mediator/modulator of disease was not, however, entirely as a result of the discovery of physiological functions but due more to the observation of increasing levels measured in plasma in direct correlation with disease progression. In health, AM circulates at low picomolar concentrations in plasma in 2 forms, a mature 52-amino acid peptide and an immature 53-amino acid peptide. Plasma levels of AM have now been shown to be increased in a number of pathological states, including congestive heart failure, sepsis, essential hypertension, acute myocardial infarction, and renal impairment. These earliest associations have been further supplemented with evidence of a role for AM in other pathologies including, most intriguingly, cancer. In this review, we offer a timely review of our current knowledge on AM and give a detailed account of the putative role of AM in those clinical areas in which the best therapeutic opportunities might exist.

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ArticleAdrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells

Abstract

Eur. J. Pharmacol.

FEBS Lett.

Life Sci.

FEBS Lett.

Eur. J. Pharmacol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Article
Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells