Hypotonic-stimulated taurine efflux in skate erythrocytes: regulation by tyrosine phosphatase activity

Abstract

Treatment of skate erythrocytes with FCCP, dinitrophenol, or sodium azide lowers ATP levels and inhibits Na+-independent taurine uptake after hypotonic volume expansion. Inside-out vesicles isolated from hypotonic volume-expanded cells demonstrate greater Na+-independent taurine uptake, and pretreatment of cells with FCCP abolishes this stimulation. Addition of ATP to the vesicles does not restore stimulated taurine uptake, suggesting that ATP does not act as a ligand modulator on the transporter. Therefore the role of protein phosphorylation was investigated. Because known protein kinase inhibitors have previously been found to have little effect on taurine fluxes in skate erythrocytes, we focused on the effects of protein phosphatase inhibition. When volume-expanded cells were returned to isotonic medium, taurine flux returned to basal values more slowly after treatment with the tyrosine phosphatase inhibitor pervanadate, suggesting that dephosphorylation may regulate inactivation. A similar effect of phosphatase inhibitors was observed in the inside-out vesicles from volume-expanded cells: the reversal of stimulated taurine uptake takes place more slowly in vesicles prepared from cells that had been incubated with pervanadate. Band 3, a major protein involved in the taurine transport pathway, shows increased tyrosine phosphorylation after hypotonic volume expansion. Pervanadate treatment of the cells potentiates and prolongs the increased phosphorylation. Therefore tyrosine phosphorylation of band 3 may play an important role in the activation of taurine fluxes after volume expansion.

Volume Expansion Stimulates p72 and p56 in Skate Erythrocytes

“…7, pervanadate enhanced the level of protein tyrosine phosphorylation produced by p72 syk both in the presence and absence of cdb3 at all time points examined under both isotonic and hypotonic conditions. The enhancement of the level of protein tyrosine phosphorylation by pervanadate is quite similar to the stimulation of taurine efflux by this agent previously observed in intact erythrocytes and membrane vesicles from skate red blood cells (13).…”

“…In the present study we have shown that the time courses for activation of the taurine flux measured in skate red blood cells and activation of p72 syk activity measured in red blood cell extracts using the cytoplasmic domain of band 3 as a substrate are quite similar. Furthermore, pervanadate, which increases the level of phosphorylation of cdb3 brought about by volume-activated p72 syk activity, also enhances the volume-activated efflux of taurine in both intact skate erythrocytes and membrane vesicles (13). This evidence raises the possibility that activation of p72 syk may be a crucial step in the volume recovery process.…”

“…Pervanadate and p72 syk Activity-We previously found that pervanadate stimulated taurine efflux in both intact skate erythrocytes and in inside-out membrane vesicles prepared from these erythrocytes (13). Stimulation was observed in both control and volume-expanded cells.…”

“…Activation of Lyn and Syk may both be required to stimulate taurine efflux maximally. Inclusion of the phosphatase inhibitor pervanadate, which has the physiologic effect in skate erythrocytes of potentiating a volume-induced taurine efflux during the regulatory volume decrease (13), also increases the tyrosine phosphorylation of band 3 by p72 syk . Because many of the tyrosine kinases are activated by tyrosine phosphorylation, inhibition of the phosphatases that inactivate these kinases may leave them in an active state.…”

Signalling pathways involved in the control of sperm cell volume

“…Similar swelling-dependent activation of these channels has been demonstrated for spermatozoa of a variety of species, such as cattle, swine, human and dog (Kulkarni et al 1997, Yeung et al 2003, Petrunkina et al 2001a. In somatic cells, RVD mediated by chloride and potassium currents is known to involve signalling cascades mediated by protein phosphorylation and dephosphorylation (Klein et al 1993, Bize & Dunham 1994, Robson & Hunter 1994, Crepel et al 1998, Musch et al 1998, Voets et al 1998, Thoroed et al 1999, Hubert et al 2000. In contrast, very little is known about whether phosphorylation processes are linked to the activation of osmo-sensing regulatory mechanisms in spermatozoa.…”