Abstract
Neuronal activity in the retina generates osmotic gradients that lead to Muller cell swelling, followed by a regulatory volume decrease (RVD) response, partially due to the isoosmotic efflux of KCl and water. However, our previous studies in a human Muller cell line (MIO-M1) demonstrated that an important fraction of RVD may also involve the efflux of organic solutes. We also showed that RVD depends on the swelling-induced Ca2+ release from intra-cellular stores. Here we investigate the contribution of taurine (Tau) and glutamate (Glu), the most relevant amino acids in Muller cells, to RVD through the volume-regulated anion channel (VRAC), as well as their Ca2+ dependency in MIO-M1 cells. Swelling-induced [H-3]Tau/[H-3]Glu release was assessed by radiotracer assays and cell volume by fluorescence videomicroscopy. Results showed that cells exhibited an osmosensitive efflux of [H-3]Tau and [H-3]Glu (Tau > Glu) blunted by VRAC inhibitors 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)-oxybutyric acid and carbenoxolone reducing RVD. Only [H-3]Tau efflux was mainly dependent on Ca2+ release from intracellular stores. RVD was unaffected in a Ca2+-free medium, probably due to Ca2+-independent Tau and Glu release, but was reduced by chelating intracellular Ca2+. The inhibition of phosphatidylinositol-3-kinase reduced [H-3]Glu efflux but also the Ca2+-insensitive [H-3]Tau fraction and decreased RVD, providing evidence of the relevance of this Ca2+-independent pathway. We propose that VRAC mediated Tau and Glu release has a relevant role in RVD in Muller cells. The observed disparities in Ca2+ influence on amino acid release suggest the presence of VRAC isoforms that may differ in substrate selectivity and regulatory mechanisms, with important implications for retinal physiology.
NEW & NOTEWORTHY The mechanisms for cell volume regulation in retinal Muller cells are still unknown. We show that swelling induced taurine and glutamate release mediated by the volume regulated anion channel (VRAC) largely contributes the to the regulatory volume decrease response in a human Muller cell line. Interestingly, the hypotonic-induced efflux of these amino acids exhibits disparities in Ca2+-dependent and -independent regulatory mechanisms, which strongly suggests that Muller cells may express different VRAC heteromers formed by the recently discovered leucine-rich repeat containing 8 (LRRC8) proteins.