The oxidative-stress-induced alteration in paracellular junctional complexes was analysed in Caco-2 cell monolayer. Oxidative stress induced a rapid increase in tyrosine phosphorylation of occludin, zonula occludens (ZO)-1, E-cadherin and beta-catenin. An oxidative-stress-induced decrease in transepithelial electrical resistance was associated with a redistribution of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the intercellular junctions. Genistein, a tyrosine kinase inhibitor, prevented the oxidative-stress-induced decrease in resistance and redistribution of protein complexes. Occludin, ZO-1, E-cadherin and beta-catenin in the Triton-insoluble cytoskeletal fraction were reduced by oxidative stress, which was prevented by genistein. Oxidative stress also reduced the co-immunoprecipitation of ZO-1 with occludin, which was prevented by genistein. Co-immunoprecipitation of beta-catenin with E-cadherin was unaffected by oxidative stress or genistein. ZO-1, E-cadherin and beta-catenin in the plasma membrane or membrane-cytoskeleton were either slightly reduced or unaffected by oxidative stress or genistein. These results show that oxidative stress induces tyrosine phosphorylation and cellular redistribution of occludin-ZO-1 and E-cadherin-beta-catenin complexes by a tyrosine-kinase-dependent mechanism.

The specific binding and inhibitory action of [3H]ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to [3H]ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 /xM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid.[3H]Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cells. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaC1 transport.Of various biochemical mechanisms thought to play important roles in salt-transporting epithelia, none has received more attention than the ouabain-sensitive, sodium-and potassium-dependent adenosine triphosphatase (Na,K-ATPase). The main role of this membrane-bound enzyme, associated with most animal cells, is thought to be the Na pump involved in maintenance of ion gradients

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K. J. DEGNAN AND OTHERS net Cl-blood side to seawater side flux of 162-8 isA/cm2 which was not statistically different (P > 0.70) from the mean short-circuit current of 158-6 + 163 ,uA/cm2 for these flux studies. The mean Na+ blood side to seawater side flux was 32-2 + 3-3 ,uA/cm2 and the mean Na+ seawater side to blood side flux was 34-8 + 4-1 isA/cm2, resulting in no significant (P > 0*20) net flux of this cation. Similar results were obtained with short-circuited epithelia of seawater-adapted fish when bathed on both sides with Ringer and gassed with 95 % 02/5 % CO2.5. Ouabain (105 M), furosemide (10-3 M), thiocyanate (10-2 M), adrenaline (1I0 M), and anoxia (100 % N2) decreased the short-circuit current 92*7, 85-0, 45-3, 62-6, and 83'3 % respectively. Theophylline (104 M) stimulated the short-circuit current 54-9 %. Increasing the HC03-concentration in the bathing solutions had a stimulatory effect on the short-circuit current and the potential difference across epithelia from seawater-adapted fish.6. The opercular epithelia of freshwater-adapted F. heteroclitU8, when bathed on both sides with Ringer, displayed a mean short-circuit current of 94-1 + 10 4 #sA/cm2, a mean transepithelial potential difference of 14-8 + 1*9 mV (blood side positive), and a mean d.c. resistance of 169-0 + 14-0 Q. cm2 (mean + S.E. of mean; n = 20). Isotope flux studies across these short-circuited epithelia revealed a net Cl-blood side to freshwater side flux of 95-2 + 16-1 ,uA/cm2 and no significant net flux of Na+.7. The opercular epithelia of 200 % seawater-adapted F. heteroclitus, when bathed on both sides with Ringer, displayed a mean short-circuit current of 33-5 + 8-5 IzA/cm2, a mean transepithelial potential difference of 10*5 + 2*5 mV (blood side positive), and a mean transepithelial d.c. resistance of 440 7 + 62*6 Q.cm2 (mean + S.E. of mean n = 18). Isotope flux studies across these short-circuited epithelia revealed a net Cl-blood side to seawater side flux of 96-2 + 51*5 ,uA/cm2 and a net Na+ blood side to seawater side flux of 65'3 + 28-6 ,uA/cm2.

The opercular epithelium of Fundulus heteroclitus contains typical gill chloride-secreting cells at the high density of 4 X 10(5) cells per square centimeter. When isolated, mounted as a membrane, and short-circuited, it actively transports chloride ions from the blood side to the seawater side of the preparation. This preparation offers a useful approach to the study of osmoregulation in bony fishes.