Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFα production in cardiomyocytes by activating a glycine receptor

Aim: Previous studies have demonstrated that glycine (GLY) markedly reduces lipopolysaccharide (LPS)-induced myocardial injury. However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration ([Ca2+](c)) and tumor necrosis factor-alpha (TNF alpha) production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process. Methods: Neonatal rat cardiomyocytes were isolated, and the [Ca2+](c) and TNF alpha levels were determined by using Fura-2 and a Quantikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively. Results: LPS at concentrations ranging from 10 ng/mL to 100 mu g/mL significantly stimulated TNF alpha production. GLY did not inhibit TNF alpha production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNF alpha release stimulated by 100 mu g/mL LPS and prevented an LPS-induced increase in [Ca2+](c), which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca2+](c), but did prevent the potassium chloride-induced increase in [Ca2+](c) in cardiomyocytes. Strychnine reversed the inhibition of the KCl-stimulated elevation in [Ca2+](c) by GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca2+](c). Furthermore, GLY receptor alpha(1) and beta subunit-immunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine. Conclusion: Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca2+](c) and inhibits the TNF alpha production induced by LPS at high doses in neonatal rat cardiomyocytes.