Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes.
Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.
Department of Surgery (Transplant Surgery), Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
Irina A
IA
Ionova
Jeannette
J
Vásquez-Vivar
Jennifer
J
Whitsett
Anja
A
Herrnreiter
Meetha
M
Medhora
Brian C
BC
Cooley
Galen M
GM
Pieper
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D001119:Arginase / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D006136:GTP Cyclohydrolase / Q000378:metabolism; D016027:Heart Transplantation; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011622:Pterins / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D017207:Rats, Sprague-Dawley; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013997:Time Factors
D016207:Cytokines; C491065:Gchfr protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011622:Pterins; D012333:RNA, Messenger; D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; C016727:sepiapterin; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D013762:Tetrahydrofolate Dehydrogenase; C113162:Arg2 protein, rat; D001119:Arginase; D006136:GTP Cyclohydrolase; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C003402:sapropterin