Acta Pharmacologica Sinica (2009) 30:1107-1114; doi: 10.1038/aps.2009.106; published online 20 July 2009

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: cardiomyocytes; lipopolysaccharides; intracellular calcium; tumor necrosis factor; glycine-gated chloride channel