title: Biphasic regulation of P-glycoprotein function and expression by NO donors in Caco-2 cells.
abstract: OBJECTIVE
  To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.
  
  
  METHODS
  Caco-2 cells were exposed to NO donors for designated times. P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting, respectively. Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits, respectively.
  
  
  RESULTS
  Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners. A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS. In contrast, a long-term (24 h) exposure stimulated the P-gp function and expression. The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP. The other NO donors SIN-1 and SNAP showed similar effects. Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function. On the other hand, free radical scavengers ascorbate, glutathione and uric acid (2 mmol/L for each), PKC inhibitor chelerythrine (5 μmol/L), PI3K/Akt inhibitor wortmannin (1 μmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP, but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.
  
  
  CONCLUSIONS
  NO donors time-dependently regulate P-gp function and expression in Caco-2 cells: short-term exposure impairs P-gp function and expression, whereas long-term exposure stimulates P-gp function and expression. The regulation occurs via a NO-independent mechanism.
authors.affiliation: Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China.
authors.forename: Ru
authors.initials: R
authors.lastname: Duan
authors.forename: Nan
authors.initials: N
authors.lastname: Hu
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: Liu
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.forename: Hai-fang
authors.initials: HF
authors.lastname: Guo
authors.forename: Can
authors.initials: C
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Xiao-dong
authors.initials: XD
authors.lastname: Liu
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D018938:Caco-2 Cells; D016166:Free Radical Scavengers / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016166:Free Radical Scavengers; D020030:Nitric Oxide Donors; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009599:Nitroprusside