Ruth Lezama

Potentiation of the osmosensitive taurine release and cell volume regulation by cytosolic Ca2+ rise in cultured cerebellar astrocytes

Hyposmolarity (−30%) in cultured cerebellar astrocytes raised cytosolic Ca2+ concentration ([Ca2+]i) from 160 to 400 nM and activated the osmosensitive taurine release (OTR) pathway. Although OTR is essentially [Ca2+]i‐independent, further increase in [Ca2+]i by ionomycin strongly enhanced OTR, with a more robust effect at low and mild osmolarity reductions. Ionomycin did not affect isosmotic taurine efflux. OTR was decreased by tyrphostin A25 and increased by ortho‐vanadate, suggesting a modulation by tyrosine kinase or phosphorylation state. Inhibition of phosphatidylinositol‐3‐kinase activity by wortmannin markedly decreased OTR and the ionomycin increase. Conversely, OTR and the ionomycin effect were independent of ERK1/ERK2 activation. OTR and its potentiation by ionomycin differed in their sensitivity to CaM and CaMK blockers and in the requirement of an intact cytoskeleton for the ionomycin effect, but not for normal OTR. Changes in the actin cytoskeleton organization elicited by hyposmolarity were not observed in ionomycin‐treated cells, which may permit the operation of CaM/CaMK pathways involved in the OTR potentiation by [Ca2+]i rise. OTR potentiation by [Ca2+]i requires the previous or simultaneous activation/operation of the taurine release mechanism and is not modifying its set point, but rather increasing the effectiveness of the pathway, resulting in a more efficient volume regulation. This may have a beneficial effect in pathological situations with concurrent swelling and [Ca2+]i elevation in astrocytes.

Epidermal Growth Factor Receptor is a Common Element in the Signaling Pathways Activated by Cell Volume Changes in Isosmotic, Hyposmotic or Hyperosmotic Conditions

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume.

Osmolytes and Mechanisms Involved in Regulatory Volume Decrease Under Conditions of Sudden or Gradual Osmolarity Decrease

A decrease in external osmolarity results in cell swelling and the immediate activation of a mechanism to restore cell volume, known as regulatory volume decrease (RVD). When exposed to a gradual osmolarity decrease (GODE), some cells do not swell. This reflects the operation of an active regulatory process known as isovolumetric regulation (IVR). The mechanisms underlying IVR appear similar to those activated during RVD, namely the extrusion of K+, Cl−, amino acids, and other organic molecules. A previous study has documented IVR in cerebellar granule neurons, parallel to an early efflux of taurine and Cl−, whereas K+ efflux is delayed. In this work we briefly review the importance of amino acids in the mechanisms of cell volume control in the brain, with emphasis on IVR. We also present experiments showing the response to GODE in cerebellar astrocytes. The currents activated during GODE, recorded in the whole-cell configuration of the patch clamp technique, indicate the early activation of an anion current, followed by a more delayed cation current. A correlation between the time course of amino acid efflux during GODE and the occurrence or not of IVR in various cell types, suggest the importance of these osmolytes in the volume regulatory process in this model.

Tyrosine kinases and osmolyte fluxes during hyposmotic swelling

Recent evidence documents the involvement of protein tyrosine kinases (TK) in the signalling network activated by hyposmotic swelling and regulatory volume decrease. Both receptor type and cytosolic TK participate as signalling elements in the variety of cell adaptive responses to volume changes, which include adhesion reactions, reorganization of the cytoskeleton, temporal deformation/remodelling of the membrane and stress‐detecting mechanisms. The present review refers to the influence of TK on the activation/operation of the osmolyte efflux pathways, ultimately leading to cell volume recovery, i.e. the osmosensitive Cl− channel (Cl), the K+ channels activated by swelling in the different cell types and the taurine efflux pathway as representative of the organic osmolyte pathway.

Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons.

Exposure of cultured cerebellar granule neurons (24 h serum‐starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non‐receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal‐regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl‐inositol‐3‐kinase (PI3K). The hyposmotic‐induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85–95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co‐immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84–91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4‐Src‐PI3K signaling cascade is indicated by the 48–66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Are Membrane Tyrosine Kinase Receptors Involved in Osmotransduction

The activation of EGFR and possibly of other TKR by hyposmolarity suggests a role for these receptors in the mechanisms of cell volume sensing or as early signals in osmotransduction. The plethora of interplaying connections with both external and internal messengers, molecules and signaling cascades, a characteristic feature of these receptors, make them exceptionally well-positioned as transducers of the changes in cell volume and the adaptive mechanisms to re-establish normal volume. Moreover, TKR receptors may also offer an interesting link to be considered in cell functions in which both changes in cell volume and TKR activation concur such as proliferation, adhesion and survival.

Tyrosine Kinases and Taurine Release

Amino acids are part of the pool of organic osmolytes contributing to volume regulation after hyposmotic swelling in most animal cells, including brain and retina1–5. Swelling occurs under ischemic conditions consequent to energy failure, which impairs the Na/K ATPase and leads to an increase in intracellular Na+followed by CF influx and osmotically obligated water. The subsequent depolarization results in excessive extracellular K+ which is removed by uptake, and is also followed by ci and water6. While an active response to volume correction is known to occur following hyposmotic swelling, it is unclear whether ischemic swelling activates a similar volume regulatory mechanism.