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

Abstract

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.

Hydrogen Peroxide Potentiates Volume-sensitive Excitatory Amino Acid Release via a Mechanism Involving Ca2+/Calmodulin-dependent Protein Kinase II

“…Similar to our findings, Cardin et al have demonstrated that the Ca 2ϩ ionophore ionomycin enhances volume-sensitive taurine release in cerebellar astrocyte cultures through a CaMKII-dependent mechanism (27). In the same study, CaMKII inhibition was ineffective in blocking control swelling-induced release in the absence of ionomycin (27). Using a phospho-Thr286 antibody recognizing the autophosphorylated active form of CaMKII (55), we confirmed that H 2 O 2 stimulates CaMKII activity in swollen astrocytes.…”

“…These data suggest that CaMKII is a critical component of the H 2 O 2 -dependent potentiation of swelling-sensitive EAA release. Similar to our findings, Cardin et al have demonstrated that the Ca 2ϩ ionophore ionomycin enhances volume-sensitive taurine release in cerebellar astrocyte cultures through a CaMKII-dependent mechanism (27). In the same study, CaMKII inhibition was ineffective in blocking control swelling-induced release in the absence of ionomycin (27).…”

“…Two Ca 2ϩ /calmodulin-dependent enzymes known to be activated by H 2 O 2 , the myosin light chain kinase (MLCK), and CaMKII, have also been found to regulate VRACs (20,27,49,50). We therefore explored whether changes in intracellular Ca 2ϩ and the activation of Ca 2ϩ /calmodulin-dependent enzymes are involved in the H 2 O 2 effect.…”

“…In line with such a suggestion, reactive oxygen species positively modulate the activity of a number of protein kinases, such as tyrosine kinases, mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) (20 -22). All of these kinases have been found to contribute to VRAC activation and/or modulation in a variety of cell types (9,(23)(24)(25)(26)(27)(28). Robust changes in tyrosine phosphorylation and MAPK activity, as well as the translocation of PKC and CaMKII from the cytosol to the membrane fraction, have been found in ischemia (29 -32).…”

Physiology of Cell Volume Regulation in Vertebrates

“…VB). Addition of Ca 2ϩ -mobilizing agonists does not elicit taurine release under isotonic conditions but potentiates the swelling-induced taurine efflux from a variety of cells presumably via pathways involving CaM and CaMKII, PI3K, and PKC (112,222,451,551,661). In contrast, a concentration (3 nM) of LTD 4 too low to elicit detectable Ca 2ϩ mobilization induces taurine release under isotonic conditions (see Fig.…”