Monoclonal Antibody Analysis of Phosphatidylserine and Protein Kinase C Localizations in Developing Rat Cerebellum

N-Methyl-D-aspartate (NMDA) stimulated the release of endogenous dopamine from striatal slices prepared from adult Sprague-Dawley rats. A mixture of sodium fluoride and aluminum chloride (AlF4-) added to the slices significantly potentiated the NMDA-stimulated release of dopamine in a concentration- and time-dependent manner. The AlF4- mixture had no effect on the nonstimulated basal efflux of dopamine, and no increases in NMDA-stimulated release were observed when NaF was replaced with NaCl. Similarly, AlCl3 or a mixture of NaCl and AlCl3 had no effect on NMDA-stimulated release. The AlF(4-)-induced increase in NMDA-stimulated dopamine release was totally blocked by magnesium or the selective NMDA glycine antagonist 7-chlorokynurenic acid. Striatal slices depolarized with KCl (15 mM) also released dopamine and this release was similarly potentiated by AlF4-. However, KCl-stimulated dopamine release from striatal synaptosomes was not potentiated by concentrations of AlF4- that greatly increased release from striatal slices. NMDA did not stimulate the release of dopamine from striatal synaptosomes in the absence or presence of aluminum fluoride. Modulators of adenylate cyclase (forskolin) and protein kinase C (phorbol esters) did not enhance NMDA-stimulated dopamine release. The protein kinase C inhibitor H-7 also did not reduce the potentiating effects of AlF4-. The mixed cholinergic agonist carbachol and the calcium ionophore A23187 mimicked the AlF4- effect although the increase in NMDA-stimulated dopamine release produced by these agents was less than that seen with AlF4-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoride plus aluminum: useful tools in laboratory investigations,but messengers of false information

Aluminofluoride complexes (AlF(x)) form spontaneously in aqueous solutions containing fluoride and traces of aluminum ions and appear to act as phosphate analogs. These complexes have become widely utilized in laboratory investigations of various guanine nucleotide-binding proteins. Reflecting on many laboratory studies, a new mechanism of fluoride and aluminum action on the cellular level is being suggested. The long-term synergistic effects of these ions in living environment and their hidden danger for human health are not yet fully recognized.     

The giant protein HERC1 is recruited to aluminum fluoride-induced actin-rich surface protrusions in HeLa cells

HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF(4)(-)) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF(4)(-), in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions.     

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.     

Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway.     

Interaction of aluminum ions with phosphoinositide metabolism in rat cerebral cortical membranes

Aluminum (Al) is believed to exert a primary role in the neurotoxicity associated with dialysis encephalopathy and has been suggested to be involved in a number of other neurological disorders, including Alzheimer's disease. Al, complexed with fluoride to form fluoroaluminate (AlF4-), can activate the GTP-binding (G) proteins of the adenylate cyclase and retinal cyclic GMP phosphodiesterase systems. Since an involvement of G-proteins with cerebral phosphoinositide (PtdIns) metabolism has also been suggested, in this study we investigated the interaction of the stable GTP analogue GTP(S), Al salts and NaF with this system. In rat cerebral cortical membranes, GTP(S) dose-dependently stimulated [3H]inositol phosphates ([3H]InsPs) accumulation. This effect was potentiated by carbachol and was partially prevented by the GTP-binding antagonist GDP(S), indicating that CNS muscarinic receptor activation is coupled to PtdIns hydrolysis via putative G-protein(s). GTP(S) stimulation was also inhibited by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, which is known to exert a negative feedback control on agonist-stimulated PtdIns metabolism. Both Al salts and NaF mimicked the action of GTP(S) in stimulating PtdIns turnover. Their actions were highly synergistic, suggesting that AlF4- could be the active stimulatory species. However, the stimulatory effects of AlCl3 and/or NaF were not potentiated by carbachol and were not inhibited by GDP(S) and PMA, suggesting that separate sites of action might exist for GTP(S) and AlF4-. In the nervous tissue, activation of PtdIns hydrolysis by Al (probably as AlF4-) may be mediated by activating a regulatory G-protein at a location distinct from the GTP-binding site or by a direct stimulation of phospholipase C.     

Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate.

Treatment of BC3H1 myocytes or 3T3-L1 fibroblasts with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42-kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from growth factor-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within 15 min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by growth factors in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase.     

Mechanism of fluoride activation of G protein-gated muscarinic atrial K+ channels.

Aluminum fluoride (AlF4-) activates the heterotrimeric G protein Gs (stimulatory G protein of adenylylcyclase) (Sternweis, P. C., and Gilman, A. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4888-4891) and GT (transducin), and for GT, Bigay et al. (Bigay, J., Deterre, P., Pfister, C., and Chabre, M. (1985) FEBS Lett. 191, 181-185) have made the intriguing proposal that AlF4- acts by mimicking the gamma-phosphate of GTP. The endogenous G protein (probably G alpha i-2 or G alpha i-3 (Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnbaumer, L. (1988) Nature 336, 680-682) that stimulates the muscarinic atrial K+ (K+[ACh]) channel is also thought to be activated by AlF4- (Kurachi, Y., Nakajima, T., and Ito, H. (1987) Circulation 76, 105P). To investigate the AlF4- mechanism, we applied potassium fluoride (KF) to the cytoplasmic face of inside-out membrane patches excised from guinea pig atria. We found that KF activated single K+[ACh] channel currents in both a concentration- and a Mg(2+)-dependent manner. Activation persisted following removal of KF, but unlike activation by guanosine 5'-(3-thiotriphosphate) (GTP gamma S), was fully reversed by removal of Mg2+. Evidence for Al3+ involvement was that the Al3+ chelator deferoxamine (500 microM) inhibited KF activation and that at low concentrations of KF (less than 1 mM), micromolar AlCl3 concentrations potentiated KF stimulation. The rate of activation produced by KF was far slower than the rate produced by GTP or GTP gamma S, and unlike these guanine nucleotides, the rate was unchanged in the presence of agonist. To test the gamma-phosphate-mimicking hypothesis, we evaluated the requirement for GDP; and to accomplish this, it was necessary to establish a condition that ensured exchange of guanine nucleotides. This condition was satisfied by using the muscarinic agonist carbachol because both the rate and the extent of activation of the K+[ACh] channels produced by GTP were much faster in carbachol, and both were greatly slowed when GDP was added along with GTP. By contrast, the effects of KF were unchanged by carbachol in the presence or absence of GDP. Further evidence that GDP is not essential for activation by AlF4- was provided by the observation that during carbachol activation and following extensive washing with GMP, guanosine 5'-O-(2-thiodiphosphate) at blocking concentrations had no effect on activation produced by KF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ inhibits guanine nucleotide-activated phospholipase D in neural-derived NG108-15 cells.

We have investigated the regulation of phospholipase D (PLD) activity by guanine nucleotides and Ca2+ in cells of the NG108-15 neuroblastoma X glioma line that were permeabilized with digitonin. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) caused a nearly sixfold increase (EC50 = 3 microM) in production of [3H]phosphatidylethanol (specific product of the PLD transphosphatidylation reaction). Other GTP analogues were less effective than GTP gamma S, and guanosine-5'-O-(2-thiodiphosphate) inhibited PLD activation by GTP gamma S. Both basal and GTP gamma S-stimulated PLD activities were potentiated by MgATP and Mg2+. Adenosine-5'-O-(3-thiotriphosphate) and ADP also potentiated the effect of GTP gamma S, but non-phosphorylating analogues of ATP had no such effect. The activation of PLD by GTP gamma S did not require Ca2+ and was independent of free Ca2+ ions up to a concentration of 100 nM (resting intracellular concentration). Higher Ca2+ concentrations (greater than or equal to 1 microM) completely inhibited PLD activation by GTP gamma S. It is concluded that elevated intracellular Ca2+ concentrations may negatively modulate PLD activation by a guanine nucleotide-binding protein, thus affecting receptor-PLD coupling in neural-derived cells.     

RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium

Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.     

Inhibition of phosphate uptake in corn roots by aluminum-fluoride complexes

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.     

Direct inhibition of in vitro PLD activity by 4-(2-aminoethyl)-benzenesulfonyl fluoride

While conducting a purification protocol of phospholipase D (PLD) from human granulocytes, we observed that PLD activity was inhibited by a commonly-used protease inhibitor cocktail. Of the six inhibitors present in the cocktail, the serine protease inhibitor, 4-(2-aminoethyl)-benezensulfonyl fluoride (AEBSF), was found to be the sole inhibitor of PLD. AEBSF caused a loss of neutrophil and purified plant PLD activities in vitro, but not in intact cells at the concentrations used, nor did it affect the related phospholipases A(2) and C, that were utilized as specificity controls. The compound AEBSNH(2), which has the fluoride replaced by an -NH(2) group, failed to affect PLD activity as did other compounds structurally related to AEBSF with known protease inhibitory capabilities. Finally, basal- and agonist-stimulated PLD activity was inhibited in phosphatidylcholine-specific anti-PLD immunoprecipitates (IC(50) = 75 microM). These results suggest that AEBSF, in an effect probably unrelated to its anti-proteolytic ability, directly interferes with PLD enzymatic activity, making it a significant compound to begin analyzing the role of PLD in mammalian cell signaling.     

Fluoride inhibits agonist-induced formation of inositol phosphates in rat cortex

Sodium fluoride inhibited carbachol, 5-hydroxytryptamine and noradrenaline stimulated formation of inositol phosphates in rat cerebral cortex. For example, carbachol (1 mM) induced a 337% increase of inositol phosphates above basal in 30 min which was reduced to 69% in the presence of NaF (10 mM). The IC50 for NaF was approximately 1.5 mM and inhibition was mediated by a decrease in maxima of the carbachol dose response curve rather than a shift to the right. This inhibitory action was not mimicked by NaBr or NaI, or by agents which increase cAMP. Inhibition did not appear to result from a toxic action of NaF since it had no effect on the formation of inositol phosphates by high K+; moreover, in higher concentrations NaF stimulated phospholipase C activity. Since fluoride ions are known to activate G-proteins in the concentrations used in this study, these results may indicate the existence of a novel G-protein linked to receptor inhibition of phospholipase C.     

Formation and characterization of kinesin.ADP.fluorometal complexes

Recent crystallographic studies of motor proteins showed that the structure of the motor domains of myosin and kinesin are highly conserved. Thus, these motor proteins, which are important for motility, may share a common mechanism for generating energy from ATP hydrolysis. We have previously demonstrated that, in the presence of ADP, myosin forms stable ternary complexes with new phosphate analogues of aluminum fluoride (AlF(4)(-)) and beryllium fluoride (BeF(n)), and these stable complexes mimic the transient state along the ATPase kinetic pathway [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. In this study, we examined the formation of kinesin.ADP.fluorometals ternary complexes and analyzed their characteristics using the fluorescent ATP analogue NBD-ATP (2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ADP). Our results suggest that these ternary complexes may mimic transient state intermediates in the kinesin ATPase cycle. Thus, the kinesin.ADP.AlF(4)(-) complex resembles the kinesin.ADP state, and the kinesin.ADP.BeF(n) complex mimics the kinesin.ADP.P(i) state.     

PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-g-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-g-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-g-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis.The preincubation of plasma membranes in the buffer without any other addition at 37øC for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-g-S but was insensitive to 5 mM AlF4-, a concentration that inhibited by 60% the enzyme assayed without preincubation.These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF4- was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.     

Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.

Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.     

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of neuronal phospholipase D activity under depolarizing conditions

Neuronal phospholipase D (PLD) activity was hypothesized to be involved in vesicle trafficking and endocytosis and, possibly, transmitter release. We here report that prolonged depolarization of rat hippocampal slices by potassium chloride (KCl) or 4-aminopyridine inhibited PLD activity. Similarly, PLD activity in rat cortical synaptosomes was significantly inhibited by depolarizing agents including veratridine and ouabain. Inhibition of calcium/calmodulin kinase II (CaMKII) which positively modulates synaptosomal PLD activity [Sarri et al. (1998) FEBS Lett. 440, 287-290] by KN-62 caused a further reduction of PLD activity in depolarized synaptosomes. Depolarization-induced inhibition of PLD activity was apparently not due to transmitter release or activation of other kinases. We observed, however, that KCl-induced depolarization caused an increase of inositol phosphates and a reduction of the synaptosomal pool of phosphatidylinositol-4, 5-bisphosphate (PIP(2)). Moreover, in synaptosomes permeabilized with Staphylococcus aureus alpha-toxin, PLD activation induced by calcium was abolished by neomycin, a PIP(2) chelator. We conclude that depolarizing conditions cause an inhibition of neuronal PLD activity which is likely due to breakdown of PIP(2), a required cofactor for PLD activity. Our findings suggest that neuronal PLD activity is regulated by synaptic activity.     

Involvement of guanine nucleotides in superoxide release by fluoride-treated neutrophils. Implications for a role of a guanine nucleotide regulatory protein

Previous studies demonstrating hydrolysis of phosphatidylinositol bisphosphate (PIP2) and generation of inositol phosphates in neutrophils exposed to 20.0 mM NaF provide indirect evidence that activation of phospholipase-associated guanine nucleotide regulatory protein, a guanine nucleotide binding protein which regulates the activation of a membrane inositol-specific phospholipase C, is an early event in the neutrophil stimulus-response pathway triggered by fluoride. Consistent with this hypothesis, exposure of a plasma membrane rich preparation isolated from 32P labeled neutrophils to 20.0 mM NaF resulted in hydrolysis of labeled PIP2. Levels of other phospholipids were not affected. Inositol bisphosphate and inositol trisphosphate were detected in extracts of neutrophil plasma membranes exposed to fluoride. To further explore the involvement of guanine nucleotides in functional responses of intact neutrophils triggered by fluoride, we preincubated cells with 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin), a selective inhibitor of inosine monophosphate dehydrogenase, to diminish guanine nucleotide synthesis and then compared superoxide generation induced by FMLP, PMA, digitonin, and 20.0 mM NaF to intracellular levels of guanine nucleotides. Preincubation of neutrophils for 2.5 h at 37 degrees C with tiazofurin resulted in dose-dependent depletion of GTP and GDP. Maximal depletion of guanine nucleotides required relatively high levels of tiazofurin (200 to 400 microM) and resulted in a 55 to 60% reduction of GTP and GDP. The effects of tiazofurin on guanine nucleotides levels were not observed when neutrophils were preincubated at 4 degrees C. AT 37 degrees C, tiazofurin also decreased intracellular ATP and ADP levels but adenine nucleotide depletion was less pronounced than guanine nucleotide depletion for each concentration of tiazofurin used. When tiazofurin was removed by washing cells after incubation, adenine nucleotide quickly returned to preincubation values but guanine nucleotide levels remained depressed. Addition of exogenous guanosine (200 microM) prevented tiazofurin-dependent depletion of guanine nucleotides but had no influence on adenine nucleotide depletion. Superoxide released triggered by FMLP and F- was inhibited to an extent similar to that of guanine nucleotide depletion under different conditions of preincubation. Inhibition of superoxide release was not observed if cells were preincubated at 4 degrees C, was not rapidly reversible, and was not observed when guanosine was added with tiazofurin.(ABSTRACT TRUNCATED AT 400 WORDS)