title: Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells.
abstract: Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE(2), which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in hepatic parenchymal cells by agonists released during stress, such as with PGE(2) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca(2+)](i) in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE(2) caused a dose-dependent increase in [Ca(2+)](i) from basal values of 130 +/- 10 to maximal levels of 434 +/- 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca(2+) dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca(2+) from intracellular stores, also partially blocked the increase in [Ca(2+)](i) caused by PGE(2), suggesting that intracellular Ca(2+) pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca(2+) pools are involved in the increase in [Ca(2+)](i) caused by PGE(2). Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca(2+)](i) due to PGE(2) in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl(-) was omitted, glycine was much less effective in preventing the increase in [Ca(2+)](i) due to PGE(2). Phenylephrine, an alpha(1)-type adrenergic receptor agonist, also increased [Ca(2+)](i), as expected, from 159 +/- 20 to 432 +/- 43 nM. Glycine also blocked the increase in [Ca(2+)](i) due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca(2+)](i) in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.
authors.affiliation: Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill 27599-7365, USA. qu@niehs.nih.gov
authors.forename: Wei
authors.initials: W
authors.lastname: Qu
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ikejima
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zhong
authors.forename: Michael P
authors.initials: MP
authors.lastname: Waalkes
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Thurman
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002317:Cardiovascular Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology; D015232:Dinoprostone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000316:Adrenergic alpha-Agonists; D002121:Calcium Channel Blockers; D002317:Cardiovascular Agents; D018118:Chloride Channels; D010656:Phenylephrine; D004533:Egtazic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D006493:Heparin; D015232:Dinoprostone; D002118:Calcium; D005998:Glycine