- Email: [email protected]
Fluoride-induced depletion of polyphosphoinositides in rat brain cortical slices: a rationale for the inhibitory effects on phospholipase C
Int. J. Devl Neuroscience, Vol. 17, No. 4, pp. 357±367, 1999 # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0736-5748/99 $20.00 + 0.00
www.elsevier.com/locate/ijdevneu
PII: S0736-5748(99)00034-9
FLUORIDE-INDUCED DEPLETION OF POLYPHOSPHOINOSITIDES IN RAT BRAIN CORTICAL SLICES: A RATIONALE FOR THE INHIBITORY EFFECTS ON PHOSPHOLIPASE C ELISABET SARRI$ and ENRIQUE CLARO%* $Departament de Bioquimica i de Biologia Molecular, Facultat de Medicina, Universitat AutoÁnoma de Barcelona, Barcelona, Spain; %Departamento de BioquõÂ mica y BiologõÂ a Molecular, Facultad de Veterinaria, Universidad de Extremadura, Avda. Universidad, s/n, 10071 CaÂceres, Spain (Received 17 November 1998; received in revised form 9 April 1999; accepted 12 April 1999) AbstractÐFluoride, which is used commonly as a pharmacological tool to activate phosphoinositidephospholipase C coupled to the heterotrymeric Gq/11 proteins, inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polyphosphoinositides (PtdIns4P and PtdIns4,5P2) in membranes from rat brain cortex. Fluoride enhanced basal production of 3H-inositol phosphates in membranes prepared from brain cortical slices that had been prelabeled with [3H]inositol, but inhibited the stimulation elicited by carbachol in the presence of GTPgS. However in both cases ¯uoride depleted [3H]PtdIns4P content by 95%. The inhibitory eects of ¯uoride on the release of 3H-inositol phosphates in slices were not apparent in a pulse [3H]inositol-labeling strategy, but became dramatic in a continuous labeling protocol, particularly at long incubation times. Prelabeling slices with [3H]inositol in the presence of ¯uoride precluded polyphosphoinositide labeling, and eliminated phospholipase C responsiveness to carbachol under normal or depolarizing conditions, and to the calcium ionophore ionomycin. The lack of response of 3H-polyphosphoinositide-depleted slices to phospholipase C stimuli was not due to ¯uoride poisoning, unaccesibility of the [3H]inositol label to phospholipase C or desensitization of Gq/11, as the eect of carbachol and GTPgS was restored, in the presence of ATP, in membranes prepared from slices that had been labeled in the presence of ¯uoride. In conclusion, our data show that ¯uoride, at a concentration similar to that used to stimulate directly Gq/11-coupled phospholipase C, eectively blocks the synthesis of phospholipase C substrates from PtdIns. # 1999 ISDN. Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION It is generally accepted that coupling of many neurotransmitter and hormone receptors with a seven transmembrane motif to the stimulation of phosphoinositide phospholipase C takes place through a Gq/11 protein.32,34 Receptor activation of heterotrymeric G proteins, which in the unstimulated state have GDP bound to the a subunit, can be bypassed with ¯uoroaluminate ions ÿ 3+ or even (AlFÿ 4 ). AlF4 is formed with ¯uoride in millimolar concentrations but micromolar Al 39 trace amounts of the ion and can directly dissociate G proteins by interacting with GDP-a subunit, as it mimicks the g-phosphate group of GTP.4 The ability of ¯uoroaluminate to stimulate phospholipase C has been shown in membrane preparations from brain tissue.6,27,28 However, ¯uoroaluminate also binds in phosphate sites of various ATPases and phosphatases.5 These actions may potentially complicate the interpretation of results regarding modulation of phospholipase C by ¯uoroaluminate, particularly when dealing with intact tissue or cell preparations. On the other hand, it has been demonstrated that ¯uoride ions (but not ¯uoroaluminate) inhibit phosphatidylinositol 4-kinase in liver and brain membranes.3,8 It is conceivable, therefore, that exposure of intact tissue to ¯uoride would lead to some depletion of polyphosphoinositide phospholipase C substrates. In fact, some reports have shown that agoniststimulated accumulation of 3H-inositol phosphates in brain slices is inhibited in the presence of ¯uoride20,41 and a polyphosphoinositide-depletion mechanism has been discussed to explain them.8,19 Interestingly, it has been shown that the inhibitory eect of ¯uoride is potentiated by lithium25, which also causes polyphosphoinositide depletion.1 In most instances, however, ¯uoride eects on the phosphoinositide system have been reported as stimulatory.17,21,36 In the present *To whom all correspondence should be addressed; E-mail: [email protected] 357
358
E. Sarri and E. Claro
paper, we show that ¯uoride induces an eective block of polyphosphoinositide formation in membranes and in slices from rat brain cortex, which is directly related to the inhibition of agonist-stimulated production of inositol phosphates. However, depending on the methodological strategy to label phospholipase C substrate with [3H]inositol, i.e. pulse or continuous labeling, ¯uoride inhibitory eects may not be apparent, or become as drastic as to completely eliminate phospholipase C responsiveness to all stimuli tested. MATERIALS AND METHODS Materials [g-32P]ATP (3,000 Ci/mmol) and myo-[2-3H]inositol (20 Ci/mmol) were purchased from NEN (Itisa, Madrid), GTPgS was from Boehringer Mannheim (Barcelona), and ionomycin from Calbiochem (BioNova, Madrid). Carbachol, sodium ¯uoride, sodium deoxycholate, phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphophate (PtdIns4,5P2) were from Sigma (Sigma-Aldrich QuõÂ mica, Madrid) and Dowex resin (AG 1X8, 100±200 mesh, formate form) from BioRad (Madrid). Silicagel-60 thin layer chromatography (TLC) plates, organic solvents, and other reagents were from Merck (Barcelona). Animals In the present work, Wistar rats (100±150 g) of either sex were used. These were breeded in the facilities of the Caceres campus of the University of Extremadura, and maintained under a 12-h light/dark cycle with free access to food and water. Preparation of membranes Unlabeled brain cortical membranes or membranes from [3H]inositol-prelabeled slices, used for experiments on [g-32P]ATP phosphorylation of phosphatidylinositol (PtdIns) or phospholipase C activation, respectively, were prepared as described.10,13 Phosphorylation of PtdIns in membranes Membranes were resuspended at 1 mg protein/ml in 25 mM TRIS-maleate buer, pH 6.8 with KOH, containing 3 mM EGTA, 10 mM MgCl2 and, when present, 10 mM sodium ¯uoride or 10 mM GTPgS and incubated 10 min at 48C. Afterwards, 100-ml samples of membrane suspension were transferred to tubes containing 2 ml each of 52 mM sodium deoxycholate and 104 mM [g-32P]ATP (106 cpm) and incubated 1 min at 378C. Reactions were stopped with 1.2 ml chloroform/methanol (1:2, v/v). After addition of 0.5 ml each of chloroform and 0.25 M HCl, the tubes were capped, shaken, and centrifuged to separate two phases. The upper (aqueous) phases were discarded and 1.2 ml methanol/water (1:1, v/v) were added to re-extract watersoluble products from the organic phases. After a new phase separation, 0.6-ml aliquots of the organic phases were evaporated and resuspended in 15 ml of chloroform/methanol (1:2, v/v) containing 10 mg each of PtdIns4P and PtdIns4,5P2 as carriers, then applied on silicagel-60 TLC plates, which were developed using chloroform/methanol/ammonia/water (90:90:10:19, v/v/v/v) as described.7 Lipids were detected by staining with iodine vapor and by autoradiography, then scrapped and counted for radioactivity. Preparation of cortical slices Rats were killed by decapitation, the brains were quickly removed and cortices were dissected free of meninges and white matter. Cross-chopped (350350 mm) slices were made with a McIlwain tissue chopper and dispersed in Krebs-Henseleit buer (KH buer) (in mM: 116 NaCl; 4.7 KCl; 1.2 MgSO4; 1.2 KH2PO4; 1 CaCl2; 25 NaHCO3; 11 glucose) pH 7.4, equilibrated with O2/CO2 (95:5, v/v). Production of 3H-inositol phosphates in [3H]inositol-labeled slices Slices were labeled with [3H]inositol according to two dierent strategies, namely pulse labeling protocol and continuous labeling protocol. In the former, slices were prelabeled in bulk
Fluoride-induced depletion of polyphosphoinositides
359
with [3H]inositol in KH buer during 2±4 h. After this period, the tracer was washed and 50 ml samples of gravity-packed slices were transferred to assay tubes and challenged with agonists in KH buer and in the presence of 10 mM LiCl in a ®nal volume of 250 ml. In the continuous labeling protocol, 50 ml samples of slices were incubated for various periods of time in assay tubes with KH buer containing 10 mM LiCl, agonists and [3H]inositol, in a ®nal volume of 250 ml. In both cases, reactions were stopped with 1.2 ml chloroform/methanol (1:2, v/v) as above. 1 ml-samples from the aqueous phases containing 3H-inositol phosphates were neutralized with 1.5 M NH4OH and diluted to 4 ml with water, then passed through columns with Dowex 18 resin (0.5 ml bed volume). After washing with 10 ml water and 10 ml 60 mM sodium formate/5 mM borax, 3H-inositol monophosphates were eluted with 8 ml of 0.2 M ammonium formate/0.1 M formic acid, and 3H-inositol polyphosphates with 8 ml of 1 M ammonium formate/0.1 M formic acid. To correct for inter- and intra-experimental variations due to dierent [3H]inositol incorporation and sample size, production of 3H-inositol phosphates was routinely expressed as percentage of total radioactivity present in the lipids. To do so, 100 ml aliquots of the organic phase from each individual incubation were evaporated and counted for radioactivity. Assay of phospholipase C in membranes from [3H]inositol-prelabeled slices This was done exactly as described.10,13 Deacylation of phosphoinositides In some cases, 3H-polyphosphoinositides present in the lipid fraction were separated from [ H]PtdIns. This was done by anion exchange chromatography of the glycerophosphoryl derivatives after alkaline deacylation as described.29 0.2 ml each of methanol and 1 M KOH in methanol/water (19:1, v/v) and 0.4 ml chloroform were added to 0.6 ml aliquots of the organic phases and left 20 min at RT. Then, 1 ml chloroform and 0.6 ml each of methanol and water were added and the tubes were shaken and centrifuged to separate phases. After this treatment, more than 98% of the original lipid radioactivity appeared in the aqueous phase. Deacylated 3 H-phosphoinositides were separated either by Dowex chromatography or HPLC (see below). In the ®rst case, after washing the columns with water, [3H]glycerophosphoryl-inositol ([3H]GroPIns) was eluted with 10 ml of 60 mM sodium formate/5 mM borax, then the higher deacylated phosphoinositides ([3H]GroPIns4P and [3H]GroPIns4,5P2) were eluted together with 8 ml of 1 M ammonium formate/0.1 M formic acid. 3
HPLC separation of deacylated 3H-phosphoinositides and 3H-inositol phosphates 1 ml samples were ®ltered through 0.2 mm ®lters and injected to a Waters HPLC system equipped with a Whatman Partisil 10 SAX column and a Whatman CSK I guard column. Separation was acomplished using ammonium phosphate (pH 3.7) gradients at a ¯ow rate of 1 ml/min.35 Fractions were colected every 30 s and counted for radioactivity. To calibrate the system, mixtures of [3H]Ins1P, [3H]Ins1,3P2 and [3H]Ins3,4P2 and mixtures of [3H]Ins4P and [3H]Ins1,4P2 were obtained after incubating [3H]Ins1,3,4P3 and [3H]Ins1,4,5P3, respectively, with membranes from rat brain cortex.35 Deacylated 3H-phosphoinositide standards were made as described above from [3H]PtdIns, [3H]PtdIns4P, and [3H]PtdIns4,5P2. RESULTS Fig. 1 shows that 10 mM ¯uoride inhibited phosphorylation of PtdIns to PtdIns4P in membranes of brain cortex. Over a 1-min incubation of membranes with [g-32P]ATP, which yielded a [32P]PtdIns4P spot on TLC of 815233 cpm under basal conditions, the presence of 10 mM NaF inhibited formation of [32P]PtdIns4P by 68% (259212 cpm), as well as that of [32P]PtdIns4,5P2 (16526 cpm vs 379242 cpm). Inclusion of 30 mM AlCl3 in the assay to allow formation of ¯uoroaluminate complex did not modify ¯uoride inhibitory eects (not shown). On the other hand, 10 mM GTPgS stimulated synthesis of [32P]PtdIns4,5P2 some 36% (517220 cpm), whereas no eect was detected regarding [32P]PtdIns4P synthesis (839221 cpm).
360
E. Sarri and E. Claro
Fig. 1. Fluoride inhibits PtdIns phosphorylation in cerebral cortical membranes. Membranes (100 mg protein) were incubated 1 min at 378C in 100 ml ®nal volume of 24 mM TRIS-maleate, pH 6.8, containing 2.9 mM EGTA, 9.5 mM MgCl2, 1 mM sodium deoxycholate, 1.9 mM [g-32P]ATP and no further addition (control), 9.5 mM NaF, 9.5 mM GTPgS or 9.5 mM NaF in combination with 9.5 mM GTPgS. Highly polar phospholipids were separated by TLC using chloroform/methanol/NH4OH/water (90:90:10:19, v/v/v/v) as mobile phase and autoradiographed, then scraped o the plate and counted for radioactivity. The image shown is representative of two independent experiments carried out with triplicate determinations.
Finally, GTPgS did not counteract the inhibitory eect of ¯uoride on the synthesis of [32P]PtdIns4P (230228 cpm), but was eective to a certain extent to revert the inhibition of [32P]PtdIns4,5P2 synthesis (22727 cpm). In brain membranes, phospholipase C as activated by carbachol in the presence of GTPgS does not hydrolyze endogenous PtdIns12, in contrast to what has been found when [3H]PtdIns is supplied as an exogenous substrate.7,33 Hence, as ¯uoride appeared to block PtdIns phosphorylation to PtdIns4P (Fig. 1), stimulation of Gq/11-coupled phospholipase C with ¯uoride should induce a depletion of polyphosphoinositide substrates. In order to test this, membranes were prepared from cortical slices that had been previously incubated with [3H]inositol to label endogenous phosphoinositides. Then, the membranes were exposed to the muscarinic agonist carbachol in the presence of GTPgS. This treatment stimulated the production of 3H-inositol phosphates, re¯ecting activation of phospholipase C (Fig. 2, left panel). Fluoride also stimulated phospholipase C (left panel) but, remarkably, inhibited the eect of carbachol plus GTPgS (left panel). On the other hand, the presence of ¯uoride alone or in combination with carbachol and GTPgS induced a 95% depletion of [3H]PtdIns4P content of membranes, as compared with the respective controls (right panel). Fig. 3 shows a purely stimulatory eect of ¯uoride on the release of 3H-inositol phosphates from slices that had been preincubated 4 h with [3H]inositol. After this pulse-labeling protocol, stimulation of phospholipase C with carbachol and ¯uoride gave a fairly linear and additive response. This suggests either that, unlike the situation in membranes, substrate depletion induced by ¯uoride did not take place in slices, or that a long [3H]inositol prelabeling period allowed a build up of labeled polyphosphoinositides that did not become limiting for the generation of 3H-inositol phosphates. This last possibility seems more likely, as shown by the
Fluoride-induced depletion of polyphosphoinositides
361
Fig. 2. Fluoride stimulates phospholipase C but inhibits [3H]PtdIns4P formation in membranes from [3H]inositol-prelabeled cortical slices. Membranes were prepared from cortical slices that had been previously labeled with [3H]inositol. 250 mg membrane protein, containing a total radioactivity in the lipid fraction of 8,500 dpm, were incubated 10 min at 378C in 250 ml ®nal volume of 25 mM TRIS-maleate, pH 6.8, containing 1 mM sodium deoxycholate, 6 mM MgCl2, 2 mM ATP, 100 nM free calcium buffered with 3 mM EGTA and, when present, 1 mM GTPgS, 1 mM carbachol (Cch) or 10 mM NaF as indicated. Left panel shows total production of 3H-inositol phosphates. Right panel shows [3H]PtdIns4P present at the end of the assay, after substracting [3H]PtdIns4P in zero time controls (463239 dpm). Results are means2S.E.M. of three independent experiments which were carried out with triplicate determinations.
next series of experiments which were designed using a continuous labeling protocol and gave clearly dierent ¯uoride eects. In this case, cortical slices were incubated with [3H]inositol together with carbachol, a depolarizing potassium concentration (20 mM), ¯uoride or combinations of these stimuli, so that [3H]inositol labeling of lipids and generation of 3Hinositol phosphates by phospholipase C took place in a concerted fashion. Then, after 30, 60 or
Fig. 3. Stimulatory eect of ¯uoride on the release of 3H-inositol phosphates from cortical slices in an [3H]inositol-prelabeling protocol. Cortical slices were labeled 4 h at 378C in KH buer containing 5 mCi/ml [3H]inositol, then washed with fresh KH buer. After labeling, 50 ml aliquots of gravitypacked sliced were transferred to assay tubes containing 250 ml ®nal volume of KH buer with 10 mM LiCl and, when present, 1 mM carbachol (Cch) and/or 10 mM NaF, and incubated at 378C as indicated. Results are means2range of two independent experiments carried out with triplicate determinations and represent total 3H-inositol phosphates, expressed as percent of 3H-inositol lipids. Zero-time controls averaged 5.0620.34.
362
E. Sarri and E. Claro
Fig. 4. Mixed stimulatory and inhibitory eects of ¯uoride in an [3H]inositol-continuous labeling protocol. Cortical slices (50 ml) were incubated at 378C during 30 min (open columns), 60 min (dashed columns) or 90 min (®lled columns) in 250 ml ®nal volume of KH buer containing 10 mCi/ml [3H]inositol, 10 mM LiCl and no further addition (control, CNT), 1 mM carbachol (Cch) or/and 10 mM NaF (F), both at normal (4.7 mM) or depolarizing (20 mM) potassium concentrations (K). Results are means2S.E.M. of three independent experiments carried out with triplicate determinations and represent 3H-inositol monophosphates (panel A), 3H-inositol polyphosphates (panel B), total 3H-inositol lipids (panel C) and 3H-polyphosphoinositides (panel D).
90 min, 3H-inositol phosphates and 3H-inositol lipids were determined (Fig. 4). Using this experimental design, carbachol stimulated the accumulation of 3H-inositol monophosphates (panel A) and 3H-inositol polyphosphates (panel B) time-dependently. Fluoride, on the other hand, also stimulated the accumulation of 3H-inositol monophosphates (panel A), but the eect was maximal at 30 min and decreased thereafter. A similar eect of ¯uoride was found when added together with carbachol at normal or depolarizing potassium concentrations. Noteworthy, the time-dependent inhibitory eects of ¯uoride were more apparent under all conditions tested regarding the accumulation of 3H-inositol polyphosphates (panel B). Bearing in mind that results on the accumulation of 3H-inositol phosphates, shown in panels A and B, are expressed as percent of lipid labeling with [3H]inositol, the time-dependent inhibition by ¯uoride is consistent with some degree of substrate depletion, as suggested by Tiger et al.41, and agrees with results obtained in membranes (Figs. 1 and 2). This was con®rmed after measuring total 3H-inositol lipids, consisting mainly of [3H]PtdIns (panel C) and 3H-polyphosphoinositides (panel D). Fluoride reduced total lipid labeling with [3H]inositol by 50% when present alone or in combination with high potassium and/or carbachol, as compared with the respective controls, either at 30, 60 or 90 min (panel C). Interestingly, a similar reduction of total lipid labeling was found after carbachol stimulation under depolarizing potassium conditions (panel C). Regarding polyphosphoinositide labeling ([3H]PtdIns4P+[3H]PtdIns4,5P2), which for the sake of clarity is expressed in panel D as percent of total 3H-inositol lipids, ¯uoride inhibitory eects were more drastic and showed a dierent time-dependency (panel D). Under basal or carbachol-stimulated conditions, 3H-polyphosphoinositide content in the slices increased from 4.5±6% of total lipid labeling at 30 min to 10±14% at 90 min, but remained 1±3% at 30, 60 and 90 min in the presence of ¯uoride. Unlike total lipid labeling, 3H-polyphosphoinositide content did not decrease after carbachol stimulation at high potassium concentration (panel D). These results show that, in slices, ¯uoride inhibited total [3H]inositol incorporation into lipids, an eect that
Fluoride-induced depletion of polyphosphoinositides
363
was reproduced after carbachol stimulation at high potassium concentration. However ¯uoride also promoted a speci®c depletion of 3H-polyphosphoinositides that was not mimicked after stimulation with carbachol under depolarizing conditions. Fig. 5 shows an experiment designed to obtain [3H]inositol-labeled slices depleted of 3Hpolyphosphoinositides. After a 2-h prelabeling incubation with [3H]inositol in the presence or absence of ¯uoride, lipids were extracted, deacylated and the resulting 3H-glycerophosphoryl derivatives were separated by HPLC (lower panel). Fluoride decreased total lipid labeling by 48%, in agreement with results in Fig. 4. However, relative to [3H]PtdIns, the label in PtdIns4P was 10.8% under control conditions and 0.8% with ¯uoride. Regarding [3H]PtdIns4,5P2, these values were 1.6 and 0.14%, respectively (lower panel). Thus, inhibition of polyphosphoinositide labeling was 93 and 91% eective for PtdIns4P and PtdIns4,5P2, respectively. These 3Hpolyphosphoinositide-depleted slices were unresponsive to carbachol at normal or depolarizing potassium concentrations or to the calcium ionophore ionomycin (upper panel), in terms of of
Fig. 5. Total block of the release of 3H-inositol phosphates after an [3H]inositol prelabeling protocol in the presence of ¯uoride. Cortical slices were incubated 2 h at 378C in KH buer containing 10 mCi/ml [3H]inositol and in the presence or absence of 10 mM NaF, then washed with fresh KH buer with or without 10 mM NaF. 50 ml-samples of slices were then transferred to tubes containing 250 ml ®nal volume of KH buer with 10 mM LiCl, in the presence or absence of 10 mM NaF, and no further additions (control, CNT), 1 mM carbachol (Cch), 20 mM KCl (K+), 1 mM carbachol plus 20 mM KCl (Cch+K+) or 10 mM ionomycin (ION) and incubated 30 min at 378C. Results in the upper panel are means2range of two independent experiments carried out with triplicate determinations and represent total 3H-inositol phosphates, expressed as percent of 3H-inositol lipids. Lower panel shows a typical HPLC separation of deacylated 3H-phosphoinositides present after the 2-h prelabeling period in the absence (open symbols) or presence (®lled symbols) of 10 mM NaF. The Y-axis represents radioactivity in each 30-s fraction, expressed as percent of total radioactivity in the combined fractions corresponding to [3H]glycerophosphorylinositol (deacylated [3H]PtdIns), which averaged 359,000212,000 and 188,00029,000 dpm in the absence or presence of NaF, respectively (n=5).
www.elsevier.com/locate/ijdevneu
PII: S0736-5748(99)00034-9
FLUORIDE-INDUCED DEPLETION OF POLYPHOSPHOINOSITIDES IN RAT BRAIN CORTICAL SLICES: A RATIONALE FOR THE INHIBITORY EFFECTS ON PHOSPHOLIPASE C ELISABET SARRI$ and ENRIQUE CLARO%* $Departament de Bioquimica i de Biologia Molecular, Facultat de Medicina, Universitat AutoÁnoma de Barcelona, Barcelona, Spain; %Departamento de BioquõÂ mica y BiologõÂ a Molecular, Facultad de Veterinaria, Universidad de Extremadura, Avda. Universidad, s/n, 10071 CaÂceres, Spain (Received 17 November 1998; received in revised form 9 April 1999; accepted 12 April 1999) AbstractÐFluoride, which is used commonly as a pharmacological tool to activate phosphoinositidephospholipase C coupled to the heterotrymeric Gq/11 proteins, inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polyphosphoinositides (PtdIns4P and PtdIns4,5P2) in membranes from rat brain cortex. Fluoride enhanced basal production of 3H-inositol phosphates in membranes prepared from brain cortical slices that had been prelabeled with [3H]inositol, but inhibited the stimulation elicited by carbachol in the presence of GTPgS. However in both cases ¯uoride depleted [3H]PtdIns4P content by 95%. The inhibitory eects of ¯uoride on the release of 3H-inositol phosphates in slices were not apparent in a pulse [3H]inositol-labeling strategy, but became dramatic in a continuous labeling protocol, particularly at long incubation times. Prelabeling slices with [3H]inositol in the presence of ¯uoride precluded polyphosphoinositide labeling, and eliminated phospholipase C responsiveness to carbachol under normal or depolarizing conditions, and to the calcium ionophore ionomycin. The lack of response of 3H-polyphosphoinositide-depleted slices to phospholipase C stimuli was not due to ¯uoride poisoning, unaccesibility of the [3H]inositol label to phospholipase C or desensitization of Gq/11, as the eect of carbachol and GTPgS was restored, in the presence of ATP, in membranes prepared from slices that had been labeled in the presence of ¯uoride. In conclusion, our data show that ¯uoride, at a concentration similar to that used to stimulate directly Gq/11-coupled phospholipase C, eectively blocks the synthesis of phospholipase C substrates from PtdIns. # 1999 ISDN. Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION It is generally accepted that coupling of many neurotransmitter and hormone receptors with a seven transmembrane motif to the stimulation of phosphoinositide phospholipase C takes place through a Gq/11 protein.32,34 Receptor activation of heterotrymeric G proteins, which in the unstimulated state have GDP bound to the a subunit, can be bypassed with ¯uoroaluminate ions ÿ 3+ or even (AlFÿ 4 ). AlF4 is formed with ¯uoride in millimolar concentrations but micromolar Al 39 trace amounts of the ion and can directly dissociate G proteins by interacting with GDP-a subunit, as it mimicks the g-phosphate group of GTP.4 The ability of ¯uoroaluminate to stimulate phospholipase C has been shown in membrane preparations from brain tissue.6,27,28 However, ¯uoroaluminate also binds in phosphate sites of various ATPases and phosphatases.5 These actions may potentially complicate the interpretation of results regarding modulation of phospholipase C by ¯uoroaluminate, particularly when dealing with intact tissue or cell preparations. On the other hand, it has been demonstrated that ¯uoride ions (but not ¯uoroaluminate) inhibit phosphatidylinositol 4-kinase in liver and brain membranes.3,8 It is conceivable, therefore, that exposure of intact tissue to ¯uoride would lead to some depletion of polyphosphoinositide phospholipase C substrates. In fact, some reports have shown that agoniststimulated accumulation of 3H-inositol phosphates in brain slices is inhibited in the presence of ¯uoride20,41 and a polyphosphoinositide-depletion mechanism has been discussed to explain them.8,19 Interestingly, it has been shown that the inhibitory eect of ¯uoride is potentiated by lithium25, which also causes polyphosphoinositide depletion.1 In most instances, however, ¯uoride eects on the phosphoinositide system have been reported as stimulatory.17,21,36 In the present *To whom all correspondence should be addressed; E-mail: [email protected] 357
358
E. Sarri and E. Claro
paper, we show that ¯uoride induces an eective block of polyphosphoinositide formation in membranes and in slices from rat brain cortex, which is directly related to the inhibition of agonist-stimulated production of inositol phosphates. However, depending on the methodological strategy to label phospholipase C substrate with [3H]inositol, i.e. pulse or continuous labeling, ¯uoride inhibitory eects may not be apparent, or become as drastic as to completely eliminate phospholipase C responsiveness to all stimuli tested. MATERIALS AND METHODS Materials [g-32P]ATP (3,000 Ci/mmol) and myo-[2-3H]inositol (20 Ci/mmol) were purchased from NEN (Itisa, Madrid), GTPgS was from Boehringer Mannheim (Barcelona), and ionomycin from Calbiochem (BioNova, Madrid). Carbachol, sodium ¯uoride, sodium deoxycholate, phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphophate (PtdIns4,5P2) were from Sigma (Sigma-Aldrich QuõÂ mica, Madrid) and Dowex resin (AG 1X8, 100±200 mesh, formate form) from BioRad (Madrid). Silicagel-60 thin layer chromatography (TLC) plates, organic solvents, and other reagents were from Merck (Barcelona). Animals In the present work, Wistar rats (100±150 g) of either sex were used. These were breeded in the facilities of the Caceres campus of the University of Extremadura, and maintained under a 12-h light/dark cycle with free access to food and water. Preparation of membranes Unlabeled brain cortical membranes or membranes from [3H]inositol-prelabeled slices, used for experiments on [g-32P]ATP phosphorylation of phosphatidylinositol (PtdIns) or phospholipase C activation, respectively, were prepared as described.10,13 Phosphorylation of PtdIns in membranes Membranes were resuspended at 1 mg protein/ml in 25 mM TRIS-maleate buer, pH 6.8 with KOH, containing 3 mM EGTA, 10 mM MgCl2 and, when present, 10 mM sodium ¯uoride or 10 mM GTPgS and incubated 10 min at 48C. Afterwards, 100-ml samples of membrane suspension were transferred to tubes containing 2 ml each of 52 mM sodium deoxycholate and 104 mM [g-32P]ATP (106 cpm) and incubated 1 min at 378C. Reactions were stopped with 1.2 ml chloroform/methanol (1:2, v/v). After addition of 0.5 ml each of chloroform and 0.25 M HCl, the tubes were capped, shaken, and centrifuged to separate two phases. The upper (aqueous) phases were discarded and 1.2 ml methanol/water (1:1, v/v) were added to re-extract watersoluble products from the organic phases. After a new phase separation, 0.6-ml aliquots of the organic phases were evaporated and resuspended in 15 ml of chloroform/methanol (1:2, v/v) containing 10 mg each of PtdIns4P and PtdIns4,5P2 as carriers, then applied on silicagel-60 TLC plates, which were developed using chloroform/methanol/ammonia/water (90:90:10:19, v/v/v/v) as described.7 Lipids were detected by staining with iodine vapor and by autoradiography, then scrapped and counted for radioactivity. Preparation of cortical slices Rats were killed by decapitation, the brains were quickly removed and cortices were dissected free of meninges and white matter. Cross-chopped (350350 mm) slices were made with a McIlwain tissue chopper and dispersed in Krebs-Henseleit buer (KH buer) (in mM: 116 NaCl; 4.7 KCl; 1.2 MgSO4; 1.2 KH2PO4; 1 CaCl2; 25 NaHCO3; 11 glucose) pH 7.4, equilibrated with O2/CO2 (95:5, v/v). Production of 3H-inositol phosphates in [3H]inositol-labeled slices Slices were labeled with [3H]inositol according to two dierent strategies, namely pulse labeling protocol and continuous labeling protocol. In the former, slices were prelabeled in bulk
Fluoride-induced depletion of polyphosphoinositides
359
with [3H]inositol in KH buer during 2±4 h. After this period, the tracer was washed and 50 ml samples of gravity-packed slices were transferred to assay tubes and challenged with agonists in KH buer and in the presence of 10 mM LiCl in a ®nal volume of 250 ml. In the continuous labeling protocol, 50 ml samples of slices were incubated for various periods of time in assay tubes with KH buer containing 10 mM LiCl, agonists and [3H]inositol, in a ®nal volume of 250 ml. In both cases, reactions were stopped with 1.2 ml chloroform/methanol (1:2, v/v) as above. 1 ml-samples from the aqueous phases containing 3H-inositol phosphates were neutralized with 1.5 M NH4OH and diluted to 4 ml with water, then passed through columns with Dowex 18 resin (0.5 ml bed volume). After washing with 10 ml water and 10 ml 60 mM sodium formate/5 mM borax, 3H-inositol monophosphates were eluted with 8 ml of 0.2 M ammonium formate/0.1 M formic acid, and 3H-inositol polyphosphates with 8 ml of 1 M ammonium formate/0.1 M formic acid. To correct for inter- and intra-experimental variations due to dierent [3H]inositol incorporation and sample size, production of 3H-inositol phosphates was routinely expressed as percentage of total radioactivity present in the lipids. To do so, 100 ml aliquots of the organic phase from each individual incubation were evaporated and counted for radioactivity. Assay of phospholipase C in membranes from [3H]inositol-prelabeled slices This was done exactly as described.10,13 Deacylation of phosphoinositides In some cases, 3H-polyphosphoinositides present in the lipid fraction were separated from [ H]PtdIns. This was done by anion exchange chromatography of the glycerophosphoryl derivatives after alkaline deacylation as described.29 0.2 ml each of methanol and 1 M KOH in methanol/water (19:1, v/v) and 0.4 ml chloroform were added to 0.6 ml aliquots of the organic phases and left 20 min at RT. Then, 1 ml chloroform and 0.6 ml each of methanol and water were added and the tubes were shaken and centrifuged to separate phases. After this treatment, more than 98% of the original lipid radioactivity appeared in the aqueous phase. Deacylated 3 H-phosphoinositides were separated either by Dowex chromatography or HPLC (see below). In the ®rst case, after washing the columns with water, [3H]glycerophosphoryl-inositol ([3H]GroPIns) was eluted with 10 ml of 60 mM sodium formate/5 mM borax, then the higher deacylated phosphoinositides ([3H]GroPIns4P and [3H]GroPIns4,5P2) were eluted together with 8 ml of 1 M ammonium formate/0.1 M formic acid. 3
HPLC separation of deacylated 3H-phosphoinositides and 3H-inositol phosphates 1 ml samples were ®ltered through 0.2 mm ®lters and injected to a Waters HPLC system equipped with a Whatman Partisil 10 SAX column and a Whatman CSK I guard column. Separation was acomplished using ammonium phosphate (pH 3.7) gradients at a ¯ow rate of 1 ml/min.35 Fractions were colected every 30 s and counted for radioactivity. To calibrate the system, mixtures of [3H]Ins1P, [3H]Ins1,3P2 and [3H]Ins3,4P2 and mixtures of [3H]Ins4P and [3H]Ins1,4P2 were obtained after incubating [3H]Ins1,3,4P3 and [3H]Ins1,4,5P3, respectively, with membranes from rat brain cortex.35 Deacylated 3H-phosphoinositide standards were made as described above from [3H]PtdIns, [3H]PtdIns4P, and [3H]PtdIns4,5P2. RESULTS Fig. 1 shows that 10 mM ¯uoride inhibited phosphorylation of PtdIns to PtdIns4P in membranes of brain cortex. Over a 1-min incubation of membranes with [g-32P]ATP, which yielded a [32P]PtdIns4P spot on TLC of 815233 cpm under basal conditions, the presence of 10 mM NaF inhibited formation of [32P]PtdIns4P by 68% (259212 cpm), as well as that of [32P]PtdIns4,5P2 (16526 cpm vs 379242 cpm). Inclusion of 30 mM AlCl3 in the assay to allow formation of ¯uoroaluminate complex did not modify ¯uoride inhibitory eects (not shown). On the other hand, 10 mM GTPgS stimulated synthesis of [32P]PtdIns4,5P2 some 36% (517220 cpm), whereas no eect was detected regarding [32P]PtdIns4P synthesis (839221 cpm).
360
E. Sarri and E. Claro
Fig. 1. Fluoride inhibits PtdIns phosphorylation in cerebral cortical membranes. Membranes (100 mg protein) were incubated 1 min at 378C in 100 ml ®nal volume of 24 mM TRIS-maleate, pH 6.8, containing 2.9 mM EGTA, 9.5 mM MgCl2, 1 mM sodium deoxycholate, 1.9 mM [g-32P]ATP and no further addition (control), 9.5 mM NaF, 9.5 mM GTPgS or 9.5 mM NaF in combination with 9.5 mM GTPgS. Highly polar phospholipids were separated by TLC using chloroform/methanol/NH4OH/water (90:90:10:19, v/v/v/v) as mobile phase and autoradiographed, then scraped o the plate and counted for radioactivity. The image shown is representative of two independent experiments carried out with triplicate determinations.
Finally, GTPgS did not counteract the inhibitory eect of ¯uoride on the synthesis of [32P]PtdIns4P (230228 cpm), but was eective to a certain extent to revert the inhibition of [32P]PtdIns4,5P2 synthesis (22727 cpm). In brain membranes, phospholipase C as activated by carbachol in the presence of GTPgS does not hydrolyze endogenous PtdIns12, in contrast to what has been found when [3H]PtdIns is supplied as an exogenous substrate.7,33 Hence, as ¯uoride appeared to block PtdIns phosphorylation to PtdIns4P (Fig. 1), stimulation of Gq/11-coupled phospholipase C with ¯uoride should induce a depletion of polyphosphoinositide substrates. In order to test this, membranes were prepared from cortical slices that had been previously incubated with [3H]inositol to label endogenous phosphoinositides. Then, the membranes were exposed to the muscarinic agonist carbachol in the presence of GTPgS. This treatment stimulated the production of 3H-inositol phosphates, re¯ecting activation of phospholipase C (Fig. 2, left panel). Fluoride also stimulated phospholipase C (left panel) but, remarkably, inhibited the eect of carbachol plus GTPgS (left panel). On the other hand, the presence of ¯uoride alone or in combination with carbachol and GTPgS induced a 95% depletion of [3H]PtdIns4P content of membranes, as compared with the respective controls (right panel). Fig. 3 shows a purely stimulatory eect of ¯uoride on the release of 3H-inositol phosphates from slices that had been preincubated 4 h with [3H]inositol. After this pulse-labeling protocol, stimulation of phospholipase C with carbachol and ¯uoride gave a fairly linear and additive response. This suggests either that, unlike the situation in membranes, substrate depletion induced by ¯uoride did not take place in slices, or that a long [3H]inositol prelabeling period allowed a build up of labeled polyphosphoinositides that did not become limiting for the generation of 3H-inositol phosphates. This last possibility seems more likely, as shown by the
Fluoride-induced depletion of polyphosphoinositides
361
Fig. 2. Fluoride stimulates phospholipase C but inhibits [3H]PtdIns4P formation in membranes from [3H]inositol-prelabeled cortical slices. Membranes were prepared from cortical slices that had been previously labeled with [3H]inositol. 250 mg membrane protein, containing a total radioactivity in the lipid fraction of 8,500 dpm, were incubated 10 min at 378C in 250 ml ®nal volume of 25 mM TRIS-maleate, pH 6.8, containing 1 mM sodium deoxycholate, 6 mM MgCl2, 2 mM ATP, 100 nM free calcium buffered with 3 mM EGTA and, when present, 1 mM GTPgS, 1 mM carbachol (Cch) or 10 mM NaF as indicated. Left panel shows total production of 3H-inositol phosphates. Right panel shows [3H]PtdIns4P present at the end of the assay, after substracting [3H]PtdIns4P in zero time controls (463239 dpm). Results are means2S.E.M. of three independent experiments which were carried out with triplicate determinations.
next series of experiments which were designed using a continuous labeling protocol and gave clearly dierent ¯uoride eects. In this case, cortical slices were incubated with [3H]inositol together with carbachol, a depolarizing potassium concentration (20 mM), ¯uoride or combinations of these stimuli, so that [3H]inositol labeling of lipids and generation of 3Hinositol phosphates by phospholipase C took place in a concerted fashion. Then, after 30, 60 or
Fig. 3. Stimulatory eect of ¯uoride on the release of 3H-inositol phosphates from cortical slices in an [3H]inositol-prelabeling protocol. Cortical slices were labeled 4 h at 378C in KH buer containing 5 mCi/ml [3H]inositol, then washed with fresh KH buer. After labeling, 50 ml aliquots of gravitypacked sliced were transferred to assay tubes containing 250 ml ®nal volume of KH buer with 10 mM LiCl and, when present, 1 mM carbachol (Cch) and/or 10 mM NaF, and incubated at 378C as indicated. Results are means2range of two independent experiments carried out with triplicate determinations and represent total 3H-inositol phosphates, expressed as percent of 3H-inositol lipids. Zero-time controls averaged 5.0620.34.
362
E. Sarri and E. Claro
Fig. 4. Mixed stimulatory and inhibitory eects of ¯uoride in an [3H]inositol-continuous labeling protocol. Cortical slices (50 ml) were incubated at 378C during 30 min (open columns), 60 min (dashed columns) or 90 min (®lled columns) in 250 ml ®nal volume of KH buer containing 10 mCi/ml [3H]inositol, 10 mM LiCl and no further addition (control, CNT), 1 mM carbachol (Cch) or/and 10 mM NaF (F), both at normal (4.7 mM) or depolarizing (20 mM) potassium concentrations (K). Results are means2S.E.M. of three independent experiments carried out with triplicate determinations and represent 3H-inositol monophosphates (panel A), 3H-inositol polyphosphates (panel B), total 3H-inositol lipids (panel C) and 3H-polyphosphoinositides (panel D).
90 min, 3H-inositol phosphates and 3H-inositol lipids were determined (Fig. 4). Using this experimental design, carbachol stimulated the accumulation of 3H-inositol monophosphates (panel A) and 3H-inositol polyphosphates (panel B) time-dependently. Fluoride, on the other hand, also stimulated the accumulation of 3H-inositol monophosphates (panel A), but the eect was maximal at 30 min and decreased thereafter. A similar eect of ¯uoride was found when added together with carbachol at normal or depolarizing potassium concentrations. Noteworthy, the time-dependent inhibitory eects of ¯uoride were more apparent under all conditions tested regarding the accumulation of 3H-inositol polyphosphates (panel B). Bearing in mind that results on the accumulation of 3H-inositol phosphates, shown in panels A and B, are expressed as percent of lipid labeling with [3H]inositol, the time-dependent inhibition by ¯uoride is consistent with some degree of substrate depletion, as suggested by Tiger et al.41, and agrees with results obtained in membranes (Figs. 1 and 2). This was con®rmed after measuring total 3H-inositol lipids, consisting mainly of [3H]PtdIns (panel C) and 3H-polyphosphoinositides (panel D). Fluoride reduced total lipid labeling with [3H]inositol by 50% when present alone or in combination with high potassium and/or carbachol, as compared with the respective controls, either at 30, 60 or 90 min (panel C). Interestingly, a similar reduction of total lipid labeling was found after carbachol stimulation under depolarizing potassium conditions (panel C). Regarding polyphosphoinositide labeling ([3H]PtdIns4P+[3H]PtdIns4,5P2), which for the sake of clarity is expressed in panel D as percent of total 3H-inositol lipids, ¯uoride inhibitory eects were more drastic and showed a dierent time-dependency (panel D). Under basal or carbachol-stimulated conditions, 3H-polyphosphoinositide content in the slices increased from 4.5±6% of total lipid labeling at 30 min to 10±14% at 90 min, but remained 1±3% at 30, 60 and 90 min in the presence of ¯uoride. Unlike total lipid labeling, 3H-polyphosphoinositide content did not decrease after carbachol stimulation at high potassium concentration (panel D). These results show that, in slices, ¯uoride inhibited total [3H]inositol incorporation into lipids, an eect that
Fluoride-induced depletion of polyphosphoinositides
363
was reproduced after carbachol stimulation at high potassium concentration. However ¯uoride also promoted a speci®c depletion of 3H-polyphosphoinositides that was not mimicked after stimulation with carbachol under depolarizing conditions. Fig. 5 shows an experiment designed to obtain [3H]inositol-labeled slices depleted of 3Hpolyphosphoinositides. After a 2-h prelabeling incubation with [3H]inositol in the presence or absence of ¯uoride, lipids were extracted, deacylated and the resulting 3H-glycerophosphoryl derivatives were separated by HPLC (lower panel). Fluoride decreased total lipid labeling by 48%, in agreement with results in Fig. 4. However, relative to [3H]PtdIns, the label in PtdIns4P was 10.8% under control conditions and 0.8% with ¯uoride. Regarding [3H]PtdIns4,5P2, these values were 1.6 and 0.14%, respectively (lower panel). Thus, inhibition of polyphosphoinositide labeling was 93 and 91% eective for PtdIns4P and PtdIns4,5P2, respectively. These 3Hpolyphosphoinositide-depleted slices were unresponsive to carbachol at normal or depolarizing potassium concentrations or to the calcium ionophore ionomycin (upper panel), in terms of of
Fig. 5. Total block of the release of 3H-inositol phosphates after an [3H]inositol prelabeling protocol in the presence of ¯uoride. Cortical slices were incubated 2 h at 378C in KH buer containing 10 mCi/ml [3H]inositol and in the presence or absence of 10 mM NaF, then washed with fresh KH buer with or without 10 mM NaF. 50 ml-samples of slices were then transferred to tubes containing 250 ml ®nal volume of KH buer with 10 mM LiCl, in the presence or absence of 10 mM NaF, and no further additions (control, CNT), 1 mM carbachol (Cch), 20 mM KCl (K+), 1 mM carbachol plus 20 mM KCl (Cch+K+) or 10 mM ionomycin (ION) and incubated 30 min at 378C. Results in the upper panel are means2range of two independent experiments carried out with triplicate determinations and represent total 3H-inositol phosphates, expressed as percent of 3H-inositol lipids. Lower panel shows a typical HPLC separation of deacylated 3H-phosphoinositides present after the 2-h prelabeling period in the absence (open symbols) or presence (®lled symbols) of 10 mM NaF. The Y-axis represents radioactivity in each 30-s fraction, expressed as percent of total radioactivity in the combined fractions corresponding to [3H]glycerophosphorylinositol (deacylated [3H]PtdIns), which averaged 359,000212,000 and 188,00029,000 dpm in the absence or presence of NaF, respectively (n=5).