G-protein-stimulated phospholipase D activity is inhibited by lethal toxin from Clostridium sordellii in HL-60 cells.

Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4, 5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2 to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Calpha was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.     

On the substrate specificity of nitric oxide synthase.

Nitric oxide (.NO) synthase (NOS) activity in subcellular fractions from cultured endothelial cells (EC) and lipopolysaccharide-activated J774.2 monocyte/macrophages was investigated by monitoring the .NO-mediated increase in intracellular cyclic GMP in LLC-PK1 pig kidney epithelial cells. The constitutive NOS in EC (NOSc) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOSi) was equally distributed among cytosol and membrane(s). Both the cytosolic NOSc in EC and the membrane-bound NOSi in J774.2 cells were strictly Ca(2+)-dependent, whereas the membrane-bound NOSc in EC and the cytosolic NOSi in J774.2 cells were not. L-Homoarginine and L-arginine-containing small peptides, such as L-arginyl-L-phenylalanine, replaced L-arginine as a substrate for the NOSc in EC and the Ca(2+)-independent NOSi in J774.2 cells, but not the Ca(2+)-dependent NOSi. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca(2+)-dependency.     

The transphosphatidylation potential of a membrane-bound phospholipase D from poppy seedlings

Plant phospholipases D (PLDs) occur in a large variety of isoenzymes, which differ in Ca(2+) ion requirement, phosphatidylinositol-4,5-bisphosphate (PIP(2)) activation and substrate selectivity. In the present study a membrane-bound PLD has been identified in the microsomal fractions of poppy seedlings (Papaver somniferum). The maximum PLD activity is found after 2 days of germination in endosperms and after 3 days in developing seedlings. In contrast to the four poppy PLD isoenzymes described hitherto, the membrane-bound form is active at lower Ca(2+) ion concentrations (in the micromolar instead of millimolar range) and needs PIP(2) for hydrolytic activity. Remarkable differences are also observed in head group exchange reactions. The reaction rates of the transphosphatidylation of phosphatidylcholine by various acceptor alcohols follow the sequence glycerol>serine>myo-inositol>ethanolamine, whereas ethanolamine is preferred by most other PLDs. Despite the biocatalytic differences, the membrane-bound PLD interacts with polyclonal antibodies raised against α-type PLD, which reveals some structural similarities between these two enzymes.     

Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils

Agents which elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD) in human neutrophils. The PLD activity of human neutrophils requires protein factors in both membrane and cytosolic fractions. We have studied the regulation of PLD by the catalytic subunit of protein kinase A (cPKA) in a cell-free system. cPKA significantly inhibited GTPgammaS-stimulated PLD activity but had no effect on phorbol ester-activated PLD activity. Pretreatment of plasma membranes with cPKA and ATP inhibited subsequent PLD activation upon reconstitution with untreated cytosol. RhoA, which is known to be a plasma membrane activator of PLD, was dissociated from PKA-treated plasma membrane by addition of cytosol. Plasma membrane-associated RhoA in human neutrophils was phosphorylated by cPKA. The PKA-phosphorylated form of RhoA was more easily extracted from membranes by RhoGDI than the unphosphorylated form. These results suggest that inhibition of neutrophil PLD by PKA may be due to phosphorylation of RhoA on the plasma membrane.     

Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis

In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.     

Phospholipase D activity in nontransformed and transformed fibroblasts.

Rat embryo fibroblasts (REF52 cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and transformed cells. In cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatidylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [3H]myristoyl-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransformed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.     

Identification of phosphatidylcholine-selective and phosphatidylinositol-selective phospholipases D in Madin-Darby canine kidney cells.

Intact cells and cell-free systems were employed to characterize phospholipase D (PLD) activity in Madin-Darby canine kidney (MDCK) cells. In cells prelabeled with [3H]glycerol, 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited phosphatidylcholine (PC) hydrolysis by PLD, as shown by the prolonged formation of [3H]phosphatidylethanol (PEt) and an accompanying decrease in [3H]PC. In contrast, bradykinin elicited rapid formation of [3H]PEt (approximately 1 min) accompanied by a decrease in [3H]phosphatidylinositol (PI). When the agonists were administered simultaneously, [3H]PEt formation was biphasic. In cells prelabeled with [3H] choline, at times less than 1 min, bradykinin failed to induce significant change in [3H]choline release. Bradykinin-induced formation of [3H]PEt in the [3H]glycerol-labeled cells was strictly dependent on extracellular Ca2+, whereas TPA-induced formation of [3H]PEt did not require extracellular Ca2+. Cell-free assays for PLD were used to assess the enzyme location, substrate specificity, and cofactor requirements. The PC-PLD activity (PEt formation) against [3H]stearoyl-PC was primarily localized in the 440 x g pellet (membrane- and nuclear-associated), preferred PC as a substrate, required detergent, and was not influenced by Ca2+ at low concentrations but was inhibited by Ca2+ in excess of 0.5 mM. The PI-PLD activity against [3H]stearoyl-PI was found largely in the 100,000 x g supernatant (cytosol), was strictly Ca(2+)-dependent, and did not require detergent. From these data, we conclude that MDCK cells contain two PLD subtypes: 1) a membrane-associated, PC-selective enzyme that responds to TPA resulting in prolonged hydrolysis of PC (the PC-PLD is Ca(2+)-independent, but requires detergent); 2) a cytosolic, PI-selective enzyme that responds rapidly but transiently to bradykinin (the PI-PLD requires Ca2+ but not detergent).     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.     

Regulation of myeloid-specific calcium binding protein synthesis by cytosolic protein kinase C.

Two calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) is shown to enhance the inductive effect of RA and 1 alpha,25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1 alpha,25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1 alpha,25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.     

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.     

Cytosolic phospholipase A2-mediated regulation of phospholipase D2 in leukocyte cell lines.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.     

Phospholipase C, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells.

We show that epigallocatechin-3 gallate (EGCG), a major component of green tea, stimulates phospholipase D (PLD) activity in U87 human astroglioma cells. EGCG-induced PLD activation was abolished by the phospholipase C (PLC) inhibitor and a lipase inactive PLC-gamma1 mutant, which is dependent on intracellular or extracellular Ca(2+), with the possible involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). EGCG induced translocation of PLC-gamma1 from the cytosol to the membrane and PLC-gamma1 interaction with PLD1. EGCG regulates the activity of PLD by modulating the redox state of the cells, and antioxidants reverse this effect. Moreover, EGCG-induced PLD activation was reduced by PKC inhibitors or down-regulation of PKC. Taken together, these results show that, in human astroglioma cells, EGCG regulates PLD activity via a signaling pathway involving changes in the redox state that stimulates a PLC-gamma1 [Ins(1,4,5)P(3)-Ca(2+)]-CaM kinase II-PLD pathway and a PLC-gamma1 (diacylglycerol)-PKC-PLD pathway.     

Activation of phospholipase D by PKC and GTPgammaS in human neuroblastoma cells overexpressing MARCKS

Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCalpha). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPgammaS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPgammaS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPgammaS, stimulation of PLD mediated by GTPgammaS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPgammaS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.     

Phospholipase D modulation by ceramide in senescence

Phospholipase D (PLD) has been implicated in mitotic regulation and has been shown to be defective in cells following replicative senescence. We examined the source of changes in PLD activity in senescent human umbilical vein endothelial cells and in human diploid fibroblasts. Using fractionated cell components we found that the cytosolic components were necessary for maximum PLD activation. In comparison to low-passage cells, senescent cells showed a profound lack of PLD activatability. By recombining fractionated components from senescent and low-passage cells, we found that in senescence the membrane component is defective in activating PLD implicating either the PLD enzyme itself or its interaction with PKC and/or ARF. The sphingolipid ceramide has been implicated in mediating senescence. Treatment with ceramide resulted in a decrease in PLD activity, implicating ceramide as the mediator of the inhibition.     

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.     

Collapsin response mediator protein-2 inhibits neuronal phospholipase D(2) activity by direct interaction.

Although the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD(2)-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD(2)-interacting protein was detected by PLD(2) overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathione S-transferase fusion proteins, we found that two regions (amino acids 65-192 (the phagocytic oxidase domain) and 724-825) of PLD(2) and a single region (amino acids 243-300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed an in vivo interaction between CRMP-2 and PLD(2). Interestingly, CRMP-2 was found to potently inhibit PLD(2) activity in a concentration-dependent manner (IC(50) = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD(2) in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD(2) activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD(2) is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD(2), suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage.