CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor.

Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

Involvement of ET(A) and ET(B) receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes.

This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [32P]phosphatidylbutanol in [32P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ET(B)-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET(A) receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2+/-3.5% of the ET-1 response) was defined at low (up to 1 microM) concentrations of BQ-123, yielding an estimated Ki value for BQ-123 of 21.3+/-2.5 nM. In addition, the presence of 1 microM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD2 value. Increasing concentrations of the ET(B) selective antagonist BQ-788 inhibited the S6c response with a Ki of 17.8+/-0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing Ki values of 20.9+/-5.1 nM and 439+/-110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 microM BQ-788, also revealing two components. Only one of them, corresponding to 69.8+/-4.4% of the response, was sensitive to BQ-788 which showed a Ki value of 28.8+/-8.9 nM. Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET(A) and ET(B) receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET(A) and ET(B) receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET(B) receptors were not blocked, and 30-50% and 50-70%, respectively, when ET(B) receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

Group I metabotropic glutamate receptors mediate phospholipase D stimulation in rat cultured astrocytes.

We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD-catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi-prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR-mediated phosphoinositide phospholipase activation [quisqualate > (S)-3,5-dihydroxyphenylglycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid]. The response to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was inhibited by the mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and, less potently, by 1-aminoindan-1,5-dicarboxylic acid and 4-carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.

Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices.

Although reactive oxygen species (ROS) are conventionally viewed as toxic by-products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H(2)O(2))-induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H(2)O(2) stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)-catalyzed transphosphatidylation reaction. The effect of exogenous H(2)O(2) on the PLD response was mimicked by menadione-induced production of endogenous H(2)O(2). Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H(2)O(2) was totally suppressed by chelation of both extracellular and cytosolic Ca(2+) with EGTA and BAPTA/AM, respectively. Furthermore, H(2)O(2)-induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down-regulation. Activation of PLD by H(2)O(2) was also inhibited by the protein-tyrosine kinase inhibitor genistein. Finally, H(2)O(2) also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.

Pharmacological characterization of the putative cADP-ribose receptor.

cADP-ribose (cADPR), a naturally occurring metabolite of NAD(+), has been shown to be an important regulator of intracellular Ca(2+) release. Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). Indeed, cADPR-mediated Ca(2+) release is subject to functional regulation by other modulators of CICR, including Ca(2+), caffeine and calmodulin. However, the underlying basis behind the effect of such agents on cADPR activity (in particular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of (32)P-radiolabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR binds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of either an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [(32)P]cADPR binding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relatively insensitive to both endogenous and pharmacological modulators of RyR-mediated CICR. In turn, this suggests that such agents modulate cADPR-mediated Ca(2+) release primarily by tuning the 'gain' of the CICR system, upon which cADPR acts, rather than influencing the interaction of cADPR with its target receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.

Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells.

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (approximately 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.

Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: calcium-dependence of the phospholipase C response.

In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca(2+) mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca(2+) modulation of the PLC response. Both PLC activation and Ca(2+) signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1 alpha and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca(2+) for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re-exposure of cells to Ca(2+)-containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca(2+) concentration. On the other hand, loading of the intracellular Ca(2+) stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca(2+) in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca(2+) during stimulation and the chelation of cytosolic Ca(2+) with BAPTA/AM inhibited both glutamate-elicited Ca(2+) response and PLC activation. Overall, these results indicate that the mGluR-mediated activation of PLC depends on the presence of extracellular Ca(2+) and can be modulated by moderate changes of cytosolic Ca(2+). Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR-mediated activation of PLC by Ca(2+) release in predepolarized cells.