High glucose suppresses expression of equilibrative nucleoside transporter 1 (ENT1) in rat cardiac fibroblasts through a mechanism dependent on PKC-ζ and MAP kinases

Recently it was demonstrated that the elevated concentration of glucose but not lack of insulin is responsible for suppression of equilibrative nucleoside transporter (ENT 1) in diabetic rat cardiac fibroblasts (CFs). The present study was undertaken to determine the signaling pathway utilized by glucose to regulate the expression of ENT I in the primary culture of rat CFs. Pretreatment of CFs with Go 6983, an isozyme non-selective PKC inhibitor, prevented the high glucose (25 mM) effect on ENT I mRNA level and nitrobenzylthioinosine (NBTI)-sensitive adenosine uptake. Similar effect was observed with a cell-permeable PKC-zeta pseudosubstrate, whereas Go 6976 a selective inhibitor of Ca2+-dependent PKC-alpha and PKC-beta isozymes had little effect on high glucose-induced suppression of ENT I mRNA level. Incubation of Us with nitric oxide (NO) donors (SNAPE, SNP) or NO synthase inhibitors (L-NAME, L-NMMA) prior to exposition of CFs to high glucose did not change the glucose effect on ENT I mRNA level. The high glucose-induced suppression of ENT I expression was blocked by PD9859 (an inhibitor of MEK), whereas neither wortmannin (an inhibitor of PI3K) nor rapamycin (an inhibitor of mTOR) affected the glucose action on ENT I transcript level. Highly effective in preventing the high glucose effect on ENT I mRNA level were GW 5074 (an inhibitor of Raf kinase) and SB 203580 (selective p38 MAPK inhibitor). These findings indicate that high glucose suppresses the expression of ENT I in CFs by NO independent manner involving the signaling through PKC-zeta, Raf-1, MEK, and p38 MAPK pathways.