Regulation of System A Transporters in Retinal Neurons by Hyperosmotic Stress and Substrate Deprivation.

Abstract

Abstract: : Purpose: SNAT1 and SNAT2 are two members of the SLC38 gene family that mediate the transport of system A substrates such as glutamine into retinal neurons. The goal of this study is to investigate the regulation of SNAT1 and SNAT2 gene expression in retinal neurons following hyperosmotic stress or substrate limitation, conditions which may be associated various retinal pathologies. Methods: Primary retinal cultures are prepared from rat pups (P1–P3) and cultured in Neurobasal medium. At DIV12–15, medium is supplemented with sucrose (260–450 mOsM_final) or replaced with Krebs–Ringers bicarbonate solution (270 mOsM) containing defined concentrations of substrates/non–substrates amino acids for varying lengths of time (1–8hr). System A activity is assessed by ¹⁴C–MeAIB transport, a selective system A substrate. Molecular analysis is performed using SNAT1 and SNAT2 specific probes and antibodies. Results: SNAT1 and SNAT2 are expressed in neuronal populations in retinal neurons in culture as they are in vivo. System A transport activity is up–regulated in response to both osmotic and substrate–deprivation stress. The adaptive response of system A to substrate–deprivation is completely suppressed by addition of glutamine (4mM). Both an acute and a chronic adaptive response are observed. The long term effects (>6hr) are sensitive to cycloheximide and actinomycin indicating de novo synthesis of transporter molecules. Conclusions: Transport of neutral amino acids such as glutamine in retinal neuronal cultures is highly regulated. The adaptive response of System A to hyperosmotic stress could support increased glutamate synthesis and release.

Keywords: neurotransmitters/neurotransmitter systems • ischemia • gene/expression