Thrombin increases hyposmotic taurine efflux and accelerates ICI-swell and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation.

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

Hypobaric hypoxia-reoxygenation diminishes band 3 protein functions in human erythrocytes.

We have previously shown that subjects exposed to acute hypobaric hypoxia display an erythrocyte membrane protein band 3 with an increased susceptibility to proteolytic degradation. We suggested it was due to an oxidative damage of band 3. We now report that exposure to hypobaric hypoxia followed by reoxygenation affects protein band 3 functions such as anion transport and binding of glyceraldehyde-3P-dehydrogenase. Transport capacity was assessed with the fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] ethanesulfonate (NBD-taurine). Binding capacity was evaluated from the activity of the membrane-associated enzyme. Healthy young men were exposed for 20 min to hypobaric hypoxia, simulating an altitude of 4,500 m above sea level and after recompression band 3 function was assessed. An inhibition of band 3 anion transport function and a decrease in the binding of glyceraldehyde-3P-dehydrogenase to band 3 were observed. Evidence is given supporting the hypothesis that functional alteration of band 3 is due to its oxidative modification originated as a consequence of the exposure to hypobaric hypoxia and further reoxygenation.

Differential taurine uptake in central and peripheral regions of goldfish retina.

The transport system of taurine was investigated in fragments of goldfish retina prepared from the total tissue and from concentric regions: center and periphery. A high-affinity, saturable, sodium-dependent system was demonstrated in the three types of fragments. The Km for one-site analysis was similar in the two regions and the total retina. The analysis for two sites revealed a significant higher Km for the high-affinity site in fragments from the central region. The maximal uptake rate was higher in the central zone than in the total retina or the periphery. The Hill slopes obtained from saturation experiments of fragments of total retina, center, and periphery were similar to one other and near to 1. The slope of the time course uptake was intermediate for total retina and higher in the center than in the periphery. Hypotaurine and beta-alanine were found to inhibit taurine uptake, but GABA was a weak inhibitor. The values of Ki for hypotaurine by one- and two-site analysis were lower in the central region. The disruption of photoreceptors by shaking did not modify significantly the uptake of the amino acid. Remotion of endogenous taurine by dialysis of central and peripheral fragments increased the uptake in the periphery, but not in the center. The differences observed among the three samples revealed less affinity and high capacity for taurine uptake in the center, plus a higher sensitivity of inhibition. In addition, the peripheral zone had a greater affinity for taurine, and the maximal velocity of the entrance seems to be inhibited by the higher concentration of the amino acid in this zone. These observations may reflect differences between proliferating and non-proliferating regions of the retina (i.e., periphery and center).