CONCURRENT PRODUCTION OF REACTIVE OXYGEN AND NITROGEN SPECIES BY AIRWAY EPITHELIAL-CELLS IN-VITRO

Intracellularly generated reactive species of both oxygen (ROS) and ni trogen (RNS) have been implicated in signaling responses in airway epi thelial cells, but these radicals have not been measured directly in s uch cells. In this study, intracellular production of both ROS and RNS were measured in the same cell lysates of guinea pig tracheal epithel ial (GPTE) cells maintained in primary culture. ROS and RNS were quant ified under basal (constitutive) conditions and in response to differe nt stimuli: LPS and TNF alpha [activators of inducible nitric oxide sy nthase (iNOS)]; several activators of calcium-dependent cNOS (ATP, bra dykinin, ionophore A23187, and thapsigargin); and exogenous oxidant st ress generated by addition of xanthine oxidase to purine (p + XO). Stu dies with LPS and TNF alpha also were performed using the murine macro phage cell line, RAW 264.7, as a positive control. Intracellular oxida nt production was detected from oxidation of dihydrorhodamine to rhoda mine. NOx was quantified by either chemiluminescent or fluorescent det ection. NOS activity was measured as citrulline production from argini ne. Basal production of oxidants by GPTE cells (0.08 + 0.00 nmol rhoda mine) was less than 10% that of RAW.267 cells (0.91 + 0.03 nmol rhodam ine). TNF alpha and LPS significantly increased intracellular oxidant production in GPTE cells, as did p + XO, but none of the cNOS activato rs affected production of oxidants in these cells. Concentrations of N O2 after 4 h in unstimulated RAW 264.7 and GPTE cells were similar and comprised 63% of total NOx in GPTE and 62% in RAW cells. TNF alpha an d LPS both increased NO2 in GPTE cells, but none of the Ca++-mobilizin g agents nor p + XO significantly affected intracellular RNS. The resu lts suggest both ROS and RNS can be measured in the same lysates from airway epithelial cells, and that both ROS and RNS are produced in the se cells in response to different stimuli. (C) 1998 Elsevier Science I nc.