Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma × glioma hybrid cells (NG108-15)

doi:10.1016/0006-8993(96)00337-X

Brain Research

Volume 724, Issue 2, 17 June 1996, Pages 238-245

https://doi.org/10.1016/0006-8993(96)00337-X Get rights and content

Abstract

Adrenomedullin (ADM) is a hypotensive peptide with structural homology, including a ring structure linked by a disulfide bridge, to calcitonin gene-related peptide (CGRP), calcitonin and amylin. ADM is predominantly synthesized in the adrenal medulla, but immunoreactive ADM has also been detected in the human brain. Here we have characterized ADM binding sites in cultured rat astrocytes using human [¹²⁵I]ADM(1–52) as radioligand. Half-maximal inhibition of [¹²⁵I]ADM(1–52) binding by intact rat ADM(1–50) amounted to 0.27 ± 0.03 nM (n = 15). The related peptides rat α-CGRP, rat amylin and salmon calcitonin displaced [¹²⁵I]ADM(1–52) at 85-, 148-, and > 4000-fold higher concentrations. Half-maximal stimulation of CAMP accumulation by rat ADM(1–50) was obtained with 1.00 ± 0.12 nM (n = 16). Rat α-CGRP was 214-fold, and rat amylin and salmon calcitonin were > 1000-fold less potent. Concerning cAMP accumulation the results were indistinguishable in mouse neuroblastoma × rat glioma hybrid cells (NG108-15), but here rat α-CGRP was > 1000-fold less potent than rat ADM(1–50). Human ADM(22–52) and human CGRP-I(8–37), which lack the ring structure, failed to stimulate cAMP accumulation, but they antagonized rat ADM(1–50) stimulated cAMP accumulation with inhibitory constants of 365 ± 93 nM and 92 ± 2 nM in astrocytes, and 45 ± 3 nM and 1300 ± 500 nM in NG108-15 cells. Rat ADM(1–50) did not raise cytosolic free calcium concentrations in astrocytes and NG108-15 cells. In conclusion, we have identified novel ADM receptors coupled to cAMP formation in cultured rat astrocytes and NG108-15 cells. Different interactions with the homologous peptide CGRP as well as the truncated receptor antagonists ADM(22–52) and CGRP(8–37) in rat astrocytes and neuroblastoma × glioma hybrid cells are consistent with ADM receptor isotypes in the brain.

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The transcriptional profile of adrenomedullin (AM), a new metastasis-related factor involved in hepatocellular carcinoma (HCC), and its specific receptors (CLR, RAMP1, RAMP3) were evaluated in liver tissues of HCV-positive HCC subjects undergoing liver transplantation (LR) and in donors (LD). AM and its specific receptor expression were also assessed in extracellular vesicles (EVs) secreted by tumorigenic (HepG2) and non-tumorigenic (WRL68) cells by Real-Time PCR. AM expression resulted significantly elevated in LR concerning LD (p = 0.0038) and, for the first time, significantly higher levels in HCC patients as a function of clinical severity (MELD score), were observed. RAMP3 and CLR expression increased in LR as a function of clinical severity while RAMP1 decreased. Positive correlations were found among AM, its receptors, and apoptotic markers. No AM mRNA expression difference was observed between HepG2 and WRL68 EVs. RAMP1 and RAMP3 resulted lower in HepG2 concerning WRL68 while significantly higher levels were observed for CLR. While results at tissue level characterize AM as a regulator of carcinogenesis-tumor progression, those obtained in EVs do not indicate AM as a target candidate, neither as a pathological biomarker nor as a marker involved in cancer therapy.
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AM exerts multiples biological activities through the activation of two specific receptors, the AM1 and AM2 receptors, formed from the obligate co-expression of a class-B, G protein coupled receptor (GPCR), the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) 2 or 3, respectively. The calcitonin gene-related peptide 1 (CGRP1) receptor is formed of a complex between CRLR and RAMP1 (Zimmerman et al., 1996; Belloni et al., 1999). AM and its receptor has been detected in a variety of organs, such as the kidney, heart, adrenal gland and spleen, and has also been found to be secreted from endothelial and vascular smooth muscle cells (Kitamura et al., 1993; Eguchi et al., 1994; Sakata et al., 1994).
Human adrenomedullin (AM) is a 52-amino acid peptide involved in cardiovascular control. AM has two specific receptors formed by the calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP) 2 or 3, known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the calcitonin gene-1 related peptide receptor (CGRP1), composed of CRLR/RAMP1. In brain, AM and their receptors are expressed in several localized areas, including the cerebellum. AM has been reported as an antioxidant. Little is known about the role of AM in the regulation of cerebellar reactive oxygen species (ROS) metabolism. We assessed the effect of AM on three antioxidant enzymes activity: catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) and on thiobarbituric acid reactive substances (TBARS) production in rat cerebellar vermis, as well the receptor subtypes involved in AM actions. Additionally, we evaluated the role of angiotensin II (ANG II), protein kinase A (PKA) activity, and protein kinase C/nicotinamide adenine dinucleotide phosphate oxidase (PKC/NAD(P)H) (oxidase) pathway. Sprague-Dawley rats were sacrificed by decapitation and cerebellar vermis was microdissected under stereomicroscopic control. CAT, GPx, SOD activity and TBARS production was determined spectrophotometrically. Our findings demonstrated that in cerebellar vermis, AM decreased and ANG II increased CAT, GPx and SOD activity and TBARS production. Likewise, AM antagonized ANG II-induced increase antioxidant enzyme activity. AM(22–50) and CGRP(8–37) blunted AM-induced decrease of antioxidant enzymes activity and TBARS production indicating that these actions are mediated through AM and CGRP₁ receptors. Further, PKA inhibitor (PKAi) blunted AM action and apocynin and chelerythrine reverted ANG II action, suggesting that AM antioxidant action is mediated through stimulation of PKA activity, while ANG II-induced stimulation through PKC/NAD(P)H oxidase pathway. Our results support the role of AM in the regulation of cerebellar antioxidant enzymes activity and suggest a physiological role for AM in cerebellum.
Adrenomedullin: A potential therapeutic target for retinochoroidal disease
2016, Progress in Retinal and Eye Research
Citation Excerpt :
AM22-52 is the only AM receptor antagonist that has been widely used and validated to date. It reportedly antagonizes AM in cell lines such as NG108-15 neuroblastoma cells (Zimmermann et al., 1996), pancreatic cancer cells (Ishikawa et al., 2003), and renal carcinoma cells in a selective manner (Tsuchiya et al., 2010). In renal carcinoma cell lines, the effects of AM22-52 are specific to tumor endothelial cells and have no effects on normal endothelial cells.
Adrenomedullin (AM) is a 52-amino acid peptide with anti-inflammatory, anti-apoptotic, and anti-oxidative properties discovered in a human pheochromocytoma. It is a member of the calcitonin peptide superfamily, and its signal is mediated by calcitonin receptor-like receptor (CLR). CLR interacts with receptor activity-modifying proteins (RAMPs), among which RAMP-2 and RAMP-3 carry CLR from the endoplasmic reticulum to the cellular membrane to confer high affinity for AM.
In addition to being implicated in a variety of systemic diseases, AM is a critical contributor to the pathogenesis of retinochoroidal disease. It is robustly upregulated in retinochoroidal disease models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularisation (CNV) as well as in human patients with retinochoroidal diseases.
In this review, we discuss the most salient recent findings that strongly illustrate the role of AM in retinochoroidal disease. In the OIR model, AM was identified as a key angiogenic mediator of retinal vascularisation, and AM inhibition suppressed only pathological angiogenesis, not physiological angiogenesis. On the contrary, lesion size was larger in AM^+/− CNV model mice, presumably due to the anti-inflammatory function of AM.
Despite the success of anti-vascular endothelial growth factor agents for the treatment of retinochoroidal disease, therapeutic shortcomings remain. Finding ways to modulate AM activity will provide new treatment avenues. Potential treatment strategies modulating the action of AM and its signaling pathway have been studied extensively. AM and its signaling molecules are intriguing future treatment targets for retinochoroidal disease.
Hypoxia-inducible adrenomedullin accelerates hepatocellular carcinoma cell growth
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In addition, our clinico-pathological analysis demonstrates that adrenomedullin up-regulation is related to the vascular invasion of HCCs and that the extent of adrenomedullin is correlated with N-cadherin intensity, which suggests that adrenomedullin has prognostic implications in HCC patients. Adrenomedullin expression has been demonstrated in several cancer cell lines, e.g., brain tumor [30], neuroblastoma [31], and in a number of lung cancer cell lines [32]. In addition, the expressions of adrenomedullin and its receptor have been demonstrated in many human epithelial cancer cell lines of diverse origins (e.g., lung, colon, ovary, breast, bone marrow, prostate, and cartilage cell lines [33].
Adrenomedullin is implicated in tumor progression and induced by hypoxia. We evaluated if adrenomedullin signaling is active in hepatocellular carcinoma (HCC), especially under hypoxic conditions, and to analyze its prognostic implication in HCC patients. HCC cells expressed adrenomedullin and its receptor, and hypoxia induced adrenomedullin expression. Adrenomedullin stimulated HCC cell growth via Akt activation, which was prevented by adrenomedullin peptide inhibitor. Clinico-pathological analysis revealed adrenomedullin extent was related to vascular invasion and N-cadherin intensity, which were reported to indicate a poor prognosis. In conclusion, adrenomedullin signaling is hypoxia-inducible and functionally active in HCCs, and its expression may be a prognostic factor.
Adrenomedullin In Gastrointestinal Function
2006, Handbook of Biologically Active Peptides
Adrenomedullin, first isolated in 1993 from human pheochromocytoma, is a member of the calcitonin family of peptides that includes calcitonin, calcitonin-gene-related peptide (CGRP), and amylin. The adrenomedullin gene encodes a 185-amino-acid precursor, preproadrenomedullin, which is processed to form the biologically active peptides adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP). Adrenomedullin circulates in the plasma and is expressed in a variety of gastrointestinal tissues, including the liver, pancreas, stomach, duodenum, small intestine, and colon. The biologic activities of adrenomedullin are mediated via CGRP receptors and specific adrenomedullin receptors, termed AM₁ and AM₂, primarily through activation of adenylate cyclase. In the stomach, adrenomedullin (1) stimulates somatostatin secretion and thus inhibits histamine and acid secretion; (2) protects the stomach from injury by increasing mucosal blood flow, stimulating epithelial cell proliferation and restitution, and promoting angiogenesis; and (3) inhibits gastric emptying. Adrenomedullin inhibits sodium absorption in the colon and amylase secretion in the pancreas. It may also contribute to the hemodynamic alterations observed in cirrhotic portal hypertension. More potent and selective adrenomedullin receptor antagonists are required to define the physiological and pathophysiological roles of adrenomedullin in the regulation of gastrointestinal function.
Lipopolysaccharide-mediated cell activation without rapid mobilization of cytosolic free calcium
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The involvement of cytosolic free calcium (Ca²⁺) as a second messenger in lipopolysaccharide (LPS)-activated cellular pathways remains controversial. Using human monocyte-like THP-1 cells stably transfected with glycosylphosphatidylinositol-anchored human CD14, we have monitored the concentration of intracellular Ca²⁺ after sequential addition of LPS, ATP, and ionomycin. Whereas ATP and ionomycin induced rapid elevations of cytosolic Ca²⁺, no such changes were observed after LPS addition. These data argue against the participation of cytosolic Ca²⁺ in the CD14-dependent LPS activation pathway.

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Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma × glioma hybrid cells (NG108-15)

Abstract

Neurone

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J. Biol. Chem.

Biochem. Biophys. Res. Commun.

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