Review Secretagogue induction of GH release in QNR/D cells: Prevention of cell death C.G. Martínez-Moreno, D. Giterman, D. Henderson, S. Harvey ⇑ Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada article info Article history: Available online 19 April 2014 Keywords: Growth hormone Retinal ganglion cells QNR/D cells GHRH TRH Cell survival abstract Retinal ganglion cells (RGCs) in the chick embryonic neural retina are extrapituitary sites of growth hor- mone (GH) synthesis and release. The regulation of GH secretion by these cells is largely unknown, although we recently discovered several of the hypothalamic releasing factors involved in pituitary GH regulation (including GH-releasing hormone (GHRH) and thyrotropin releasing hormone, TRH) to be present in the cytoplasm of immortalized quail RGCs (QNR/D cells). QNR/D cells may therefore provide an experimental model for studies on GH regulation in the chick neural retina. The possibility that GHRH and TRH might stimulate GH secretion in QNR/D cells was therefore investigated. Both peptides acutely depleted the GH content of the QNR/D cells, as demonstrated by immunocytochemistry and ELISA, whilst increasing the GH content in incubation media. Both peptides also increased the immunochemical and ELISA content of the QNR/D cells and the content of GH in the incubation media after long-term incuba- tion. Cell survival, determined by metabolic activity of the QNR/D cells and by TUNEL-labeling, was reduced when the endogenous GH content was reduced by GH immunoneutralization, even in the pres- ence of exogenous GHRH or TRH. Cell survival was also reduced when endogenous GHRH was blocked by GHRH immunoneutralization, although the immunoneutralization of endogenous TRH did not affect QNR/D cell survival. In summary, these results demonstrate secretagogue actions of exogenous GHRH and TRH on the secretion of GH from QNR/D cells. They also suggest that endogenous GHRH, but not endogenous TRH, prevents cell death by increasing endogenous GH secretion in QNR/D cells. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Pituitary growth hormone (GH) secretion is known to be stim- ulated by numerous hypophysiotropic factors (Gahete et al., 2009), including GH-releasing hormone (GHRH) and thyrotrophin releasing hormone (TRH) (Harvey, 1990, 1999; Harvey et al., 1991). In marked contrast, the regulation of GH secretion in extrapituitary tissues is largely unknown. GHRH and TRH have, however been detected in quail QNR/D cells (a commercial cell line) (Harvey et al., 2012), that provide an experimental model to study retinal ganglion cell (RGC) function (Martínez-Moreno et al., 2014). Within these cells, preliminary data indicate a stimu- latory effect of exogenous GHRH on the expression of the GH gene, but the action of GHRH on the secretion of the GH protein has yet to be determined. The possibility that GHRH acts as a secretagogue for QNR/D GH has therefore been evaluated in the present study and compared with the action of TRH, a potent GH-releasing factor for avian pituitary glands (Harvey et al., 1991). In addition, as the immunoneutralization of endogenous GHRH in QNR/D cells results in cell death (Martínez-Moreno et al., 2014) the possibility that this reflects a reduction in QNR/D GH was assessed. 2. Materials and methods 2.1. Cell culture A quail neural retina cell line (QNR/D; American Type Culture Collection; No. CRL-2532) was obtained from Cedarlane Laboratories (Burlington, Ontario, Canada). Cells were incubated in a water-jacketed incubator (Forma Scientific) at 40 °C with 5.0% carbon dioxide in 25 cm 2 tissue culture flasks (Corning Incor- porated, NY, USA). The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen Corporation, Burlington, Ontario, Canada, No. 11995) supplemented with fetal bovine serum (FBS, 10% v/v) (Gibco, Invitrogen Corp.) and 1% antibiotic- antimitotic (containing 10,000 l/L of penicillin, 10,000 lg/L of streptomycin and 25 lg/L of amphotericin B) (Gibco, Invitrogen Corp.). They were subcultured at a ratio of 1:8 every 5–6 days, when they reached 80% of confluence. QNR/D cells for experiments were used between subculture passes 20 and 35 in order to pre- serve phenotypical characteristics. http://dx.doi.org/10.1016/j.ygcen.2014.04.004 0016-6480/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +1 780 492 3956. E-mail address: steve.harvey@ualberta.ca (S. Harvey). General and Comparative Endocrinology 203 (2014) 274–280 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen