Elisabet Sarri

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 (H2O2)‐induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H2O2 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 H2O2 on the PLD response was mimicked by menadione‐induced production of endogenous H2O2. Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H2O2 was totally suppressed by chelation of both extracellular and cytosolic Ca2+ with EGTA and BAPTA/AM, respectively. Furthermore, H2O2‐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 H2O2 was also inhibited by the protein‐tyrosine kinase inhibitor genistein. Finally, H2O2 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.

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

Abstract: 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‐α‐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.

Vascular Smooth Muscle Cell Phenotypic Changes in Patients With Marfan Syndrome

Objective— Marfan’s syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-&bgr; signaling. TGF-&bgr; is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-&bgr; signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan’s syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Approach and Results— Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (&agr;-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-&bgr; pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. Conclusions— In Marfan VSMC, both in tissue and in culture, there are variable TGF-&bgr;-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan’s syndrome aneurysm formation.

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 Ca2+ mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca2+ modulation of the PLC response. Both PLC activation and Ca2+ signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1α and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca2+ for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re‐exposure of cells to Ca2+‐containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca2+ concentration. On the other hand, loading of the intracellular Ca2+ stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca2+ in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca2+ during stimulation and the chelation of cytosolic Ca2+ with BAPTA/AM inhibited both glutamate‐elicited Ca2+ response and PLC activation. Overall, these results indicate that the mGluR‐mediated activation of PLC depends on the presence of extracellular Ca2+ and can be modulated by moderate changes of cytosolic Ca2+. 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 Ca2+ release in predepolarized cells.

Phospholipid Synthesis Participates in the Regulation of Diacylglycerol Required for Membrane Trafficking at the Golgi Complex

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin‐1, and trans‐(1S,3R)‐aminocyclopentyl‐1,3‐dicarboxylic acid (trans‐ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi‐prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D‐catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin‐1, and trans‐ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 µM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin‐1‐stimulated phospholipase D (EC50 44 nM) was not blocked by BQ‐123, a specific antagonist of the ETA receptor. Endothelin‐3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin‐1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin‐1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.

Involvement of ETA and ETB 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 ETB‐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 ETA 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 μM) concentrations of BQ‐123, yielding an estimated Ki value for BQ‐123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ‐123 significantly reduced the maximal response to ET‐1 but did not change the pD2 value. Increasing concentrations of the ETB 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 μM 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 ETA and ETB receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ETA and ETB receptors to the ET‐1 response was found to be approximately 20% and 80%, respectively, when ETB receptors were not blocked, and 30–50% and 50–70%, respectively, when ETB 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.

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 Ca2+ mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca2+ modulation of the PLC response. Both PLC activation and Ca2+ signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1α and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca2+ for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re‐exposure of cells to Ca2+‐containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca2+ concentration. On the other hand, loading of the intracellular Ca2+ stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca2+ in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca2+ during stimulation and the chelation of cytosolic Ca2+ with BAPTA/AM inhibited both glutamate‐elicited Ca2+ response and PLC activation. Overall, these results indicate that the mGluR‐mediated activation of PLC depends on the presence of extracellular Ca2+ and can be modulated by moderate changes of cytosolic Ca2+. 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 Ca2+ release in predepolarized cells.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

Summary The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER–Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER–Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

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

Abstract: 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 (∼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.