A novel peroxide-induced calcium transient regulates interleukin-6 expression in cardiac-derived fibroblasts.
Reperfusion of ischemic myocardium leads to a local burst of free radicals, increased [Ca(2+)](i), and the release of proinflammatory cytokines. The purpose of this study was to determine whether brief exposure of cardiac fibroblasts to H(2)O(2) is associated with transient changes in [Ca(2+)](i) levels and whether this stimulus is sufficient to induce interleukin-6 (IL-6) expression. Cardiac derived fibroblasts were isolated from adult male rats and cultured under standard conditions. Individual coverslip-attached fibroblasts were loaded with the calcium probe Fura-2/AM and exposed to a single 3-min pulse of 100 microm H(2)O(2). In addition, low passage cultures were exposed to a pulse of H(2)O(2) and assayed for IL-6 expression. A brief exposure of H(2)O(2) led to a large intracellular Ca(2+) transient with a mean transient magnitude of 318 +/- 28 nm (mean +/- S.D., n = 12). Stimulation in the absence of [Ca(2+)](o) led to a 59% reduction in mean transient magnitude (129 +/- 23 nm, n = 10, p < 0.001), whereas pretreatment with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C resulted in a 37% reduction (199 +/- 25 nm, n = 10, p < 0.01). Cells treated with xestospongin C and stimulated in the absence of [Ca(2+)](o) did not exhibit a Ca(2+) transient. Time-dependent IL-6 release was significantly elevated by 4 h (368 +/- 64 pg/mg protein, p < 0.01) and increased further by 24 h (1030 +/- 76 pg/mg protein). The depletion of cellular Ca(2+) by pretreatment with thapsigargin in the absence of [Ca(2+)](o) attenuated H(2)O(2)-induced IL-6 mRNA expression while blocking protein release. These data show that the exposure of cardiac fibroblasts to a brief pulse of physiological levels of H(2)O(2) resulted in a large Ca(2+) transient with intracellular and extracellular Ca(2+) contributions. Furthermore, brief H(2)O(2) exposure led to calcium-dependent IL-6 expression.
Colston
James T
JT
Division of Cardiology, University of Texas Health Science Center, South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, Texas 78284-7872, USA.
Chandrasekar
Bysani
B
Freeman
Gregory L
GL
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D047028:Macrocyclic Compounds; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011921:Rats, Inbred WKY
D015850:Interleukin-6; D047028:Macrocyclic Compounds; D010080:Oxazoles; D012333:RNA, Messenger; C108644:xestospongin A; D006861:Hydrogen Peroxide; D002118:Calcium