Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFα production in cardiomyocytes by activating a glycine receptor
Abstract
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-α (TNFα) 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 TNFα 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 μg/mL significantly stimulated TNFα production. GLY did not inhibit TNFα production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFα release stimulated by 100 μg/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 α1 and β 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 TNFα production induced by LPS at high doses in neonatal rat cardiomyocytes.
Keywords:
Methods: Neonatal rat cardiomyocytes were isolated, and the [Ca2+]c and TNFα 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 μg/mL significantly stimulated TNFα production. GLY did not inhibit TNFα production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFα release stimulated by 100 μg/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 α1 and β 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 TNFα production induced by LPS at high doses in neonatal rat cardiomyocytes.