Oxidative Stress Causes a Protein Kinase C-independent Increase of Paracellular Permeability in an In Vitro Epithelial Model

Abstract

To evaluate the response of an epithelial barrier to a moderate but sustained oxidative stress, we cultured monolayers of Madin Darby canine kidney cells on microporous filters and exposed them to the hypoxanthine-xanthine oxidase (HX-XO) reaction. The transepithelial permeability coefficient for mannitol (Pm) was assessed as a marker of paracellular permeability. When the oxidative stress was limited in intensity and duration (production of 10 nmol/ml/min O2- with generation of 467 +/- 30 nmol/ml H2O2 over 1 h), we observed an increase of Pm with a delay of several hours (324 +/- 65% of baseline by 6 h, P < 0.005). There was complete return to control values by 24 h. The increase of Pm did not appear to be related to a depletion of cellular ATP. Protein kinase C (PKC) activity did not increase, and the rise in Pm was not prevented by CGP 41,251, a specific inhibitor of PKC. By contrast, CGP 41,251 inhibited the Pm increase that was elicited by PDBU, a phorbol ester that activates PKC. In our model, we conclude that a reversible increase of paracellular permeability occurs after oxidative stress independently of ATP depletion or PKC activation. Other, as yet unknown mechanisms have to be involved in this process.

Continuous exposure of airway epithelial cells to hydrogen peroxide: Protection by KGF

“…The current study was the first to investigate the recovery of barrier function after oxidant injury in AEC. Other recovery studies after oxidant injury (Welsh et al, 1985;Rochat et al, 1993;Meyer et al, 2001) and after surfactant treatment (Pasternak and Miller, 1996) have been performed on MDCK cell monolayers. The severity of the insult resulting from increased exposure time (Welsh et al, 1985;Rochat et al, 1993;Pasternak and Miller, 1996;Meyer et al, 2001) and the agent of injury (Pasternak and Miller, 1996) were found to be important factors in the ability of the BF to recover.…”

“…Other recovery studies after oxidant injury (Welsh et al, 1985;Rochat et al, 1993;Meyer et al, 2001) and after surfactant treatment (Pasternak and Miller, 1996) have been performed on MDCK cell monolayers. The severity of the insult resulting from increased exposure time (Welsh et al, 1985;Rochat et al, 1993;Pasternak and Miller, 1996;Meyer et al, 2001) and the agent of injury (Pasternak and Miller, 1996) were found to be important factors in the ability of the BF to recover. Although the ability of KGF to protect against oxidant-induced permeability has been well documented (Mason et al, 1996;Yi et al, 1996;Waters et al, 1997;Savla and Waters, 1998;Gillis et al, 1999), no previous attempts have been made to determine whether KGF's influence extends to repair of the barrier after it had been compromised.…”

“…However, higher surfactant concentrations and/or longer exposure times led to inhibition or elimination of TER recovery (Pasternak and Miller, 1996). Others have demonstrated that ROS exposure affects MDCK cells in a similar dose-and time-dependent fashion (Welsh et al, 1985;Rochat et al, 1993;Meyer et al, 2001). To our knowledge, recovery of AEC barrier function after oxidant injury has yet to be investigated.…”

“…Reactive oxygen species (ROS) have been shown in vivo (Royston et al, 1990;Mason et al, 1996;Wells et al, 1997) and in vitro (Welsh et al, 1985;McBride et al, 1993;Rochat et al, 1993;Yamaya et al, 1995;Rao et al, 1997Rao et al, , 2000Waters et al, 1997;Savla and Waters, 1998) to compromise the epithelial barrier, leading to increases in epithelial permeability in the airways, kidneys, and intestine. The changes in permeability associated with oxidant injury due to bolus doses of ROS (Rao et al, 1997(Rao et al, , 2000Waters et al, 1997) or short exposure to enzymatically-generated ROS (Welsh et al, 1985;McBride et al, 1993;Rochat et al, 1993;Yamaya et al, 1995) have been well documented. However, longerterm exposure to ROS has not been explored.…”

Actin re‐distribution in response to hydrogen peroxide in airway epithelial cells

“…The relationship between the tight junction proteins and the actin cytoskeleton appears to be important for the maintenance of barrier function during exposure to ROS. ROS-induced increases in albumin and manitol permeability (Rochat et al, 1993;Waters et al, 1997) and conductance (Welsh et al, 1985;Yamaya et al, 1995) and decreases in TER (Chapman et al, 2002) were accompanied by altered actin cytoskeleton structure (Welsh et al, 1985;Waters et al, 1997;Chapman et al, 2002). In intestinal epithelial cells, ROS-induced barrier dysfunction was correlated with a decrease in the amount of actin associated with the cytoskeleton (Banan et al, 2000).…”