Elsevier

Cell Calcium

Volume 46, Issue 3, September 2009, Pages 197-208
Cell Calcium

Calcium signals activated by ghrelin and D-Lys3-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

https://doi.org/10.1016/j.ceca.2009.07.003Get rights and content

Abstract

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys3-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca2+]i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca2+ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys3-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca2+ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys3-GHRP-6 ghrelin antagonist features ghrelin independent activities.

Introduction

Ghrelin is a 28 amino acid peptide produced primarily in the stomach, and secreted into the systemic circulation [1]. Through activation of the GH secretagogue receptor (GHSR), a member of the G-protein-coupled receptor family mostly expressed in the pituitary and hypothalamus, ghrelin induces pituitary GH secretion, food intake, and adiposity [2]. In addition, ghrelin exerts a range of peripheral activities, including inhibition of apoptosis in several cell types, stimulation of differentiation of skeletal myoblasts, pre-adipocytes and pre-osteoblats, neurogenesis and protection from chronic heart failure [2], [3], [4], [5].

Upon binding and activation of GHSR-1a, ghrelin elicits increase in intracellular calcium concentration through multiple mechanisms according to the different cell types. In several cell types ghrelin induces calcium mobilization both through PLC/IP3-mediated release from internal stores [1], [6] and through activation of voltage independent calcium influx [7]. However, several evidences suggest that voltage dependent calcium channels also regulate several ghrelin biological activities. N-type calcium channels mediate ghrelin-induced NPY release in neurons of arcuate nucleus. Conversely L-type voltage-sensitive calcium channels mediate ghrelin-induced GH release in somatotrophes and ghrelin-induced neurogenesis in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus [8], [9], [10], [11], [12].

The aim of this study was to characterize calcium signalling upon ghrelin stimulation of DRG cells from chick embryo. The sequences of both ghrelin and GHSR-1a genes are highly conserved from avian to the mammalian genome. Consistently, human ghrelin stimulates GH secretion in chickens [13], [14], [15], [16]. Herein we demonstrate that DRG cells express GHSR-1a and that ghrelin triggers calcium signalling in developing DRG by regulating two different calcium entry pathways, a receptor activated calcium entry (RACE) and a store operated calcium entry (SOCE) in a coordinate manner. Moreover, at our surprise, we observed that D-Lys3-GHRP-6 exerts a number of activities in the absence of exogenous ghrelin, including stimulation of calcium increase, inhibition of spontaneous calcium oscillations and stimulation of apoptosis.

Section snippets

Cell culture

DRG cells were obtained from seven-day-old chick embryos. Dissected ganglia were collected in Dulbecco's phosphate-buffered saline (PBS) with calcium and magnesium (Invitrogen) and then incubated in divalent cation-free PBS (Invitrogen) containing 0.0625% trypsin (Sigma) at 37 °C for 5 min. After gentle trituration, cells were re-suspended in a serum free N2 medium composed of a 1:1 mixture of DMEM/F-12 (Invitrogen) supplemented with N-2 supplement (human transferrin 100 μM, insulin recombinant

Immunocytochemical identification of DRG cells

DRG is a vascularised structure composed of neurons and satellite glial cells surrounded by a connective capsule. To unambiguously identify cultured DRG cells utilized in this study, we have characterized by immunocytochemistry in three different experiments cells derived from the neural crest, neurons and glial cells. Both DRG neurons and associated glia are indeed generated from trunk neural crest cells [17]. Neural crest-derived cells were identified by HNK-1 antibodies, which recognize an

Discussion

In this paper we show for the first time that ghrelin coordinately activates two distinct calcium pathways, RACE and SOCE, in developing DRG non-neuronal cells. These two routes of calcium entry can be easily distinguished on the basis of their sensitivity to the pharmacological blocker Gd3+. As previously described in other cell types [24], [25], [26], [27], [28], [29], we showed that upon ghrelin stimulation of glial cells, RACE and SOCE are not simultaneously activated, as SOCE develops only

Acknowledgements

We thank Dr. R. Billington for helpful discussion, Prof. D. Lovisolo and Prof. M. Grilli for their generous gift of antibodies and reagents for immunocytochemistry. This work was financed by Italian Ministry for University and Research (PRIN grant to AG), Telethon (grant N̊ GGP030386 to AG), and Regione Piemonte (to AG). S. Bernascone is a recipient of “Fondazione CRT-Progetto Lagrange” Ph.D. scholarship.

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