Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

Sarri, Elisabet; Servitja, Joan Marc; Picatoste, Fernando; Claro, Enrique

doi:10.1016/0014-5793(96)00906-4

Cited by 9 publications

(3 citation statements)

References 27 publications

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“…Phospholipids were labelled by incubating astrocytes with [ 32 P]orthophosphoric acid ([ 32 P]P i ; 1.5 μCi/well) in 300 μl of Krebs‐Henseleit buffer (KHB) without added P i (in mM: NaCl 116.0, KCl 4.7, MgSO 4 1.2, NaHCO 3 25.0, CaCl 2 1.3, and glucose 11.0) for 4 h at 37°C in an atmosphere of 5% CO 2 /95% air. This procedure was selected to avoid the heterogeneous labelling of different molecular species of phosphatidylcholine found when using preincubation with precursor 3 H‐fatty acids (Sarri et al , 1996). After the labelling period, cells were rinsed twice with KHB to remove non‐incorporated [ 32 P]P i and cells were incubated for the indicated times with agonists in the presence of butanol (final concentration 25 mM).…”

Section: Methodsmentioning

confidence: 99%

Involvement of ET_A and ET_B receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Servitja

Masgrau

Sarri

et al. 1998

British J Pharmacology

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1 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 [ 2 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 eects evoked by the ET B -preferring agonists, ET-3, S6c and IRL 1620, were signi®cantly lower than the maximal response to the non-selective agonist ET-1. 3 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 de®ned at low (up to 1 mM) concentrations of BQ-123, yielding an estimated K i value for BQ-123 of 21.3+2.5 nM. In addition, the presence of 1 mM BQ-123 signi®cantly reduced the maximal response to ET-1 but did not change the pD 2 value. 4 Increasing concentrations of the ET B selective antagonist BQ-788 inhibited the S6c response with a K i of 17.8+0.8 nM. BQ-788 also inhibited the eect of ET-1, although, in this case, two components were de®ned, accounting for approximately 50% of the response, and showing K i 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 mM 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 K i value of 28.8+8.9 nM. 5 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. 6 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 eects shown by endothelins on cultured and reactive astrocytes.

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Section: Methodsmentioning

confidence: 99%

Involvement of ET_A and ET_B receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Servitja

Masgrau

Sarri

et al. 1998

British J Pharmacology

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“…Brain cortical slices were obtained from Sprague‐Dawley rats (weighing 100‐120 g) and prelabeled for PLD and PLC determinations as described previously by Sarri et al (1996) and Claro et al (1993), respectively. In brief, cortices were dissected and cross‐chopped in 350 × 350 μm slices.…”

Section: Determination Of Pld and Plc In Brain Slicesmentioning

confidence: 99%

Effects of Oxidative Stress on Phospholipid Signaling in Rat Cultured Astrocytes and Brain Slices

Servitja

Masgrau

Pardo

et al. 2000

Journal of Neurochemistry

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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 menadioneinduced 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 2ϩ 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.

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“…There are various reports in the literature describing slight but significant activations of PLD by CCK A agonists [12,13], the protein kinase C activator phorbol myristate acetate (PMA) [14], and several growth factors acting through tyrosine kinase activation [15]. In most of these works, the experimental design consisted of labeling pancreatic acini with [ 3 H]myristic acid, which incorporates preferentially to phosphatidylcholine [16,17]. The fact that phosphatidylcholine is the main substrate for PLD makes [ 3 H]myristic acid a suitable phospholipid marker in this kind of study.…”

Section: Introductionmentioning

confidence: 99%

Colecystokinin octapeptide CCK‐8 and carbachol reduce [³²P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini

et al. 2000

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We have studied phospholipase D activation in [ 32 P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of 32 P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [ 32 P]phosphatidylethanol and [ 32 P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32 P-phosphatidylalcohol, yet they decreased [ 32 P]phosphatidylcholine content and stimulated the generation of [ 32 P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity. ß

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Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

Cited by 9 publications

References 27 publications

Involvement of ET_A and ET_B receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Involvement of ET_A and ET_B receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Effects of Oxidative Stress on Phospholipid Signaling in Rat Cultured Astrocytes and Brain Slices

Colecystokinin octapeptide CCK‐8 and carbachol reduce [³²P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini

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Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

Cited by 9 publications

References 27 publications

Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Effects of Oxidative Stress on Phospholipid Signaling in Rat Cultured Astrocytes and Brain Slices

Colecystokinin octapeptide CCK‐8 and carbachol reduce [32P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini

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Involvement of ET_A and ET_B receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Involvement of ET_A and ET_B receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

Colecystokinin octapeptide CCK‐8 and carbachol reduce [³²P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini