Osmolyte channel regulation by ionic strength in skate RBC.

The aim of this study was to determine whether the opening of the osmolyte channel in skate red blood cells (RBC) is regulated by intracellular electrolyte concentration and conductivity. Consistent with previous studies, experiments with hyperosmotic preincubation before cell swelling or swelling with an isosmotic electrolyte (e.g., ammonium chloride) showed that an increase in ionic strength inhibits the opening of the taurine channel. However, a decrease in intracellular ionic strength did not always stimulate taurine efflux to the same degree. Whereas hyposmotic swelling caused a large increase in taurine efflux, swelling induced by treatment with isosmotic nonelectrolytes produced much smaller stimulation. Results with assays for band 3 phosphorylating enzymes were consistent with those from the taurine efflux studies; stimulation of enzyme activity was lower in cells that were swollen with isosmotic nonelectrolyte media than in cells swollen in hyposmotic media. These results indicate that a decrease in ionic strength is not the only signal for the opening of the taurine channel in skate RBC. Ionic strength does affect channel activity, but there must also be some other regulator.

Inhibition of volume-stimulated taurine efflux and tyrosine kinase activity in the skate red blood cell.

Phosphorylation of the band 3 anion exchange protein by the tyrosine kinases p72syk and p56lyn is thought to play a role in the pathway that regulates swelling-activated taurine efflux from the skate red blood cell. In this study, the protein tyrosine kinase (PTK) inhibitors piceatannol and tyrphostin A23 both inhibited taurine efflux and the activities of the tyrosine kinases p72syk and p56lyn in the skate erythrocyte. However, the PTK inhibitors genistein and tyrphostin A46 had only small effects on taurine efflux and PTK activities. In general, a strong correlation between the extent of inhibition of taurine efflux and of tyrosine kinase activity was observed. PTK inhibitors showed a similar pattern of inhibition of band 3 phosphorylation, with the greatest inhibition observed in cells treated with piceatannol. The protein kinase C inhibitors staurosporine and bisindolylmaleimide tested alone or in combination with piceatannol had little or no significant effect on swelling-activated taurine efflux. Overall the results support the hypothesis that phosphorylation of the skate band 3 protein by p72syk and p56lyn contributes to the regulation of volume-activated taurine efflux in skate red cells, and suggest that protein kinase C may not be involved in this regulation.

Volume expansion stimulates p72(syk) and p56(lyn) in skate erythrocytes.

Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60(src), and p72(syk), demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with 32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72(syk) appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety.