Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them.

The aim of this study was to determine whether volume-activated taurine and Cl- effluxes occur via the same system in skate (Raja erinacea) red blood cells (RBC). The effluxes were measured in isotonic and hypotonic elasmobranch Ringer solutions, in which NaCl was replaced by mannitol and the remaining exchangeable anions with gluconate. Methazolamide (0.1 mM) was added to minimize HCO3- formation. RBC Cl- content fell approximately 50%/h in both isotonic and hypotonic media, with no detectable K- loss in either medium. The observed Cl- loss was accompanied by an increase in pH. Both the Cl- loss and pH rise were inhibited by 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM), suggesting that Cl- efflux was due to H(+)-Cl- cotransport. 36Cl- effluxes in isotonic and hypotonic media were (means +/- SE, n = 11) 2.8 +/- 0.6 and 3.5 +/- 0.9 mumol.g dry wt RBC-1.min-1, respectively, whereas [3H]taurine effluxes in the same media were 0.045 +/- 0.02 and 2.1 +/- 0.05 mumol.g dry wt RBC-1.min-1, respectively (n = 6). These results indicate that taurine and Cl- effluxes occur via different pathways in skate RBC. In addition, the swelling-activated Cl- channel reported in epithelial cells does not appear to be present in skate RBC. This conclusion was confirmed by Western blots with an antibody to swelling-activated Cl- channels. Taurine and Cl- fluxes are apparently under different pathway influences in these RBC: taurine diffuses via a channel, whereas Cl- is transported by cotransporters.

Erythrocytes of the skate (Raja erinacea) exposed to hypotonic stress swell and then undergo a volume regulatory decrease by releasing taurine and other osmolytes. Previous studies showed that taurine release occurs via a volume-activated, Na(+)-independent, bi-directional transporter that has the properties of a size-limited channel. We now report on the transport characteristics of this channel and its regulation. Kinetic, competition and inhibitor studies indicate that polyols (myo-inositol) and trimethylamines (betaine) are transported by the same channel as taurine. Although the identity of the channel is still unknown a variety of evidence suggests that band 3 is involved in either channel formation or regulation. Hypotonicity causes phosphorylation and structural changes in band 3. Under isotonic conditions band 3 is predominantly in the dimeric form. Hypotonicity causes a shift to tetrameric band 3. We hypothesize that the band 3 tetramer either forms or regulates an osmolyte channel. The finding that expression of band 3 protein increases osmolyte channel activity in Xenopus oocytes supports this hypothesis.

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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.

Taurine, a β amino acid, is a primary osmolyte in nucleated skate erythrocytes and is involved in the regulation of cell volume. Growth factors may be involved in the regulation of cell volume which occurs during cell division. Erythropoietin (EPO) is the primary growth factor controlling erythropoiesis. To investigate its mechanism of action, we used nucleated skate erythrocytes. EPO stimulates Na+‐independent uptake of taurine in a concentration‐dependent manner. The uptake was inhibited by the tyrosine kinase inhibitor genistein. Concomitantly, EPO stimulates tyrosine phosphorylation of a number of proteins, particularly ones of molecular masses 145, 120, 100, 80, 65, and 35 kDa. Using specific antibodies, the 145 kDa protein is identified as phospholipase C γ‐1 (PLC γ‐1) and the 100 kDa protein as the skate homolog of the anion exchanger band 3. Since PLC γ‐1 is activated, turnover of membrane lipids was determined. EPO increased 1,2‐diacylglycerol formation from phosphatidylinositols (phosphatidylinositol‐4‐monophosphate and 4,5‐biphosphate) during an early phase and later preferentially from phosphatidylcholine. The early hydrolysis of phosphoinositides was confirmed measuring generation of inositol‐1,4,5‐trisphosphate, demonstrating an activation of PLC γ‐1 activity. To determine if phospholipase D (PLD) stimulation also occurred, ethanol was included in the reactions. Phosphatidylethanol, synthesized by PLD‐mediated transphosphatidylation, appeared at times longer than 5 min, suggesting delayed activation of PLD. These results demonstrate that EPO, via simulation of tyrosine phosphorylation, stimulates taurine transport in skate erythrocytes. © 1996 Wiley‐Liss, Inc.

Volume expansion of erythrocytes of little skate, Raja erinacea, triggers the opening of an osmolyte channel. We review this transport mechanism and further investigate the channel's physicochemical nature by probing the channel with a series of pyridoxine derivatives in skate RBC as well as in epithelial cells: MDCK and C6 glioma cells and in skate hepatocytes. The identity of the transport mechanism (band 3 vs. An anion channel) which mediates the swelling‐activated efflux of osmolytes in fish RBC is controversial. Therefore, we compared taurine and Cl– effluxes in similar conditions. We found that there is significant Cl– loss from volume‐expanded skate RBC. However, there was no effect of either hypotonicity or a number of taurine transport inhibitors on this loss. Utilizing changes in intracellular pH as a means of indirectly measuring H+/Cl– cotransport, we found that a rise in cell pH accompanied the loss of Cl–. This suggests that Cl– efflux could occur via a H+/Cl– cotransporter. To probe and compare the osmolyte channel (taurine efflux) of the skate RBC and three other cell types we used a family of pyridoxine inhibitors. The inhibitory patterns for the skate erythrocytes and hepatocytes differed from those for MDCK and C6 glioma cells and the two former cell types differed from each other. Therefore, the results show that the osmolyte channel in the skate differs from that in other epithelial cells with regard to pyridoxine derivative binding properties. J. Exp. Zool. 279:456–461, 1997. © 1997 Wiley‐Liss, Inc.

Taurine, a major osmolyte of vertebrate hearts, is released from the skate heart at increased rates during hypotonic stress. We tested the hypothesis that this taurine release is mediated by chloride channels activated by swelling. Two inhibitors of the channels, NPPB and DIDS, inhibited the volume-activated release of taurine from embryonic skate hearts. These results support the hypothesis that swelling-activated chloride channels mediate the release of cardiac taurine.