Biomedical subjects

Ruth Lezama

Publications and source records attributed to Ruth Lezama.

5 recordsLinked to original sources

PubMed2005

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.

Animals↗

PubMed2004

Epidermal growth factor receptor is activated by hyposmolarity and is an early signal modulating osmolyte efflux pathways in Swiss 3T3 fibroblasts.

Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically induced taurine release, as suggested by the latter's reduction when EGFR phosphorylation was blocked by 50 microM AG213 or AG112 and, conversely, its potentiation by EGF (200 ng/ml). The relationship between hyposmotically induced taurine efflux and reduced osmolarity showed saturable kinetics, following a sigmoidal function. EGF shifted the relationship to the left, implying an increase in sensitivity to hyposmolarity. EGF increased taurine efflux only marginally under isosmotic conditions. EGF and agglutinins also potentiated the hyposmotically induced release of 86Rb but, in contrast to taurine, the efflux was unaffected by EGFR inhibition. EGF and agglutinins markedly increased 86Rb release under isosmotic conditions. The EGF-evoked isosmotic 86Rb release, together with the hyposmotic efflux, accounted fully for the observed potentiation by EGF, raising the possibility of an overlapping of these two effects, rather than a true potentiation. A link between EGFR, phosphatidylinositide-3-kinase (PI3K) and hyposmotically induced taurine (but not 86Rb) release is suggested by the increase in PI3K activity elicited by hyposmolarity, which was fully prevented by EGFR inhibition, and by a marked reduction of hyposmotically induced taurine (but not 86Rb) release, by wortmannin. The present findings, together with results showing EGF activation of osmosensitive Cl- fluxes implicate EGFR as an important modulator of osmolyte efflux pathways.

Animals↗

PubMed2004

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.

Cell Size↗

PubMed2003

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.

Animals↗

PubMed2002

Tyrosine kinases and amino acid efflux under hyposmotic and ischaemic conditions in the chicken retina.

The chicken retina was exposed to 20% hyposmotic or ischaemia-like (54 mM KCl and 1 mM ouabain) conditions and changes in cell volume, amino acid release and activation of protein tyrosine kinases measured. To investigate possible connection between these cellular events, the effect of tyrosine kinase blockers on (3)H-taurine, (3)H-GABA and (3)H- D-aspartate (as a tracer for glutamate) efflux was examined. Both hyposmotic and ischaemic conditions increased phosphorylation of the tyrosine kinase p125 focal adhesion kinase (p125(FAK)) and the mitogen-activated protein kinase-p38 (MAPK-p38), but not of the extracellular-signal-related kinases-1/2 (ERK1/ERK2), and markedly activated the tyrosine kinase target enzyme phosphatidylinositide 3-kinase (PI3K). Hyposmolarity and ischaemia both led to rapid retinal swelling followed by active volume recovery of 84% (hyposmolarity) and 40% (ischaemia), together with rapid release of taurine, GABA and D-aspartate. Taurine and GABA efflux under both conditions was reduced markedly by tyrosine kinase and PI3K blockers (50 microM tyrphostin A23, 50 microM genistein, 100 nM wortmannin, 25 microM LY294002) and was decreased by 85% when ischaemia-induced swelling was prevented. About 65% of D-aspartate efflux occurred irrespective of swelling in ischaemia and was either less sensitive (hyposmotic) or largely resistant (ischaemia) to the blockers. These results suggest that in ischaemia, GABA and taurine react primarily to swelling with a typical osmolyte response, while glutamate differs in its release mechanisms under both hyposmotic and ischaemic conditions. These findings suggest new strategies for evaluating the contribution of swelling to excitotoxicity in ischaemia.

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