Elsevier

Peptides

Volume 22, Issue 11, November 2001, Pages 1795-1801
Peptides

Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells

https://doi.org/10.1016/S0196-9781(01)00520-4Get rights and content

Abstract

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex consisting of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2. To explore possible pathophysiological roles of adrenomedullin and its receptor component RAMP2 in hypoxic tissues, we studied effects of hypoxia on expression of adrenomedullin and RAMP2 in two human neuroblastoma cell lines, IMR-32 and NB69, by radioimmunoassay and Northern blot analysis. Expression levels of adrenomedullin were increased by hypoxia in both cell lines. Treatment with cobalt chloride or desferrioxamine mesylate also increased expression levels of adrenomedullin mRNA. On the other hand, expression levels of RAMP2 mRNA were decreased in IMR-32 cells and were not changed in NB69 cells by hypoxia. Treatment with cobalt chloride or desferrioxamine mesylate decreased expression levels of RAMP2 mRNA in both IMR-32 and NB69 cells. These findings indicate that adrenomedullin expression is induced during hypoxia in IMR-32 and NB69 neuroblastoma cells, but RAMP2 expression is rather suppressed under the same conditions. The decreased expression of RAMP2 and the ADM expression induction under hypoxia may constitute one mechanism of cellular adaptation to hypoxic stress.

Introduction

Adrenomedullin (ADM) is a 52 amino-acid peptide that was initially identified in human pheochromocytoma tissue [11]. ADM is a potent vasodilator peptide with stimulating action on platelet cyclic AMP production [11]. The C-terminal portion-(16–52) of ADM shares about 27% homology with calcitonin gene-related peptide (CGRP).

ADM and ADM-binding sites are expressed in almost all organs [12], [26], including the brain [28], [29], [35]. ADM has been shown to have various biological functions such as the regulation of cardiovascular tone, and the stimulating action on cAMP accumulation and Ca2+ mobilization in bovine aortic endothelial cells [13], [27]. It also has shown the natriuretic and diuretic actions, the bronchodilator action, and the inhibition of endothelin production in vascular smooth muscle cells [13], [27]. ADM modulated the proliferation of cells. ADM acts as a growth stimulator in some cell types, such as Swiss 3T3 cells [37] and MCF-7 human breast cancer cells [20], while ADM acts as a growth inhibitor in other cell types, such as rat mesangial cells [5] and vascular smooth muscle cells [10]. ADM may act as a neurotransmitter or neuromodulator in the brain. For example, intracerebroventricular administration of ADM increased blood pressure [30], and inhibited water drinking [21], feeding [32] and gastric emptying [18].

McLatchie et al. reported novel receptor complexes consisting of calcitonin-receptor-like receptor (CRLR) and receptor-activity modifying proteins (RAMPs) [19]. Three isoforms of RAMPs have been identified. CRLR functions as a CGRP receptor when coexpressed with RAMP1, and as a ADM receptor when coexpressed with RAMP2 or RAMP3. RAMP3 expression level is very low, and ADM binding tends to vary with RAMP2 mRNA levels in rat tissues [4]. It is therefore likely that RAMP2 is the major component for the ADM receptor. The expression of RAMP2 was regulated under pathophysiological states such as congestive heart failure [33] or sepsis [25] in vivo.

Low cellular oxygen tension is the physiological feature of embryogenesis, wound repair, carcinogenesis, and ischemic disease [2], [8], [17]. The decrease in oxygen tension is one of the strongest stimuli for the induction of ADM [6], [7], [15], [16], [22], [23], [24], [34]. Ladoux et al. showed that the three RAMPs and CRLR mRNAs expression was insensitive to hypoxia in cultured rat astrocytes by the RT-PCR method [16]. The regulation of RAMP2 expression under hypoxic conditions, however, has not been extensively studied in cultured tumor cells. In our preliminary studies, Northern blot analysis could clearly detect RAMP2 mRNA only in two human neuroblastoma cell lines, IMR-32 and NB69, among various human tumor cell lines examined. We therefore studied expression of ADM and RAMP2 by hypoxia and hypoxia-mimetic reagents such as cobalt chloride (CoCl2) or desferrioxamine mesylate (DFX) [9], [36] in cultured human neuroblastoma cells.

Section snippets

Materials

A human neuroblastoma cell line, IMR-32, was obtained from the Cancer Cell Repository, Institute of Development, Aging and Cancer Tohoku University (Sendai, Japan). A human neuroblastoma cell line, NB69, was obtained from the Riken Cell Bank (Tsukuba, Japan). CoCl2, DFX, and actinomycin D were obtained from Wako (Osaka, Japan); [α-32 P] dCTP from Amersham Pharmacia Biotech (Tokyo, Japan); Cell Counting Kit-8 from Dojindo (Kumamoto, Japan); restriction endonucleases from Takara Shuzo (Otsu,

Results

Northern blot analysis showed that hypoxia induced ADM mRNA expression in two human neuroblastoma cell lines, IMR-32 and NB69 (Fig. 1A and 1B). The expression levels of ADM mRNA were increased after 6-h exposure to hypoxia (about 1.8-fold in IMR-32 cells, and about 2.4-fold in NB69 cells), and remained to be increased for 24 h compared with the control in both the cells. In IMR-32 cells, the expression levels of RAMP2 mRNA started to decrease after 12-h hypoxia and continuously decreased for

Discussion

In this study, we have demonstrated for the first time that hypoxia and hypoxia-mimetic reagents decreased expression of RAMP2 mRNA in IMR-32 human neuroblastoma cells. Expression levels of RAMP2 mRNA in NB69 cells were also decreased by hypoxia-mimetic reagents, but were not noticeably changed by hypoxia. This different response to hypoxia may reflect the difference in the metabolic properties between these two cell lines. In addition, we have confirmed that the production and secretion of ADM

Acknowledgements

We thank Prof. T. Yamamoto (Tohoku University) for β-actin cDNA. This study has been supported by a Grant-in-aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports and Culture of Japan, and by the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

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