2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils

Biochimica et Biophysica Acta 1573 (2002) 26 – 32 www.bba-direct.com

2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils Jih-Pyang Wang a,*, Ling-Chu Chang a, Mei-Feng Hsu b, Li-Jiau Huang c, Sheng-Chu Kuo c a

Department of Education and Research, Taichung Veterans General Hospital, 160, Chung Kang Road, Sec. 3, Taichung 407, Taiwan, ROC b Department of Biochemistry, China Medical College, Taichung 404, Taiwan, ROC c Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung 404, Taiwan, ROC Received 18 March 2002; received in revised form 27 June 2002; accepted 11 July 2002

Abstract 2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated phospholipase D (PLD)mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC50 values of 15.8 F 2.5 and 13.9 F 2.0 AM, respectively. The underlying cellular signaling mechanism of CCY1a inhibition was investigated. CCY1a inhibited the plateau phase but not the initial Ca2 + spike of fMLP-stimulated Ca2 + signal. CCY1a did not inhibit the [Ca2 + ]i change in Ca2 +-free medium in response to fMLP, but inhibited the [Ca2 + ]i change by the subsequent addition of Ca2 +. In addition, CCY1a treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCY1a in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPgS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCY1a. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca2 + entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Phospholipase D; Protein tyrosine phosphorylation; Intracellular free Ca2+; ADP-ribosylation factor; Rho A; Neutrophil

1. Introduction Phospholipase D (PLD) is a widely distributed enzyme. In mammalian cells, the hydrolysis of phosphatidylcholine by PLD is recognized as an important signaling pathway in different physiological processes such as secretion, vesicle trafficking, mitogenesis, and NADPH oxidase activation [1,2]. Phosphatidic acid (PA), the product of PLD, is able to alter the activities of many enzymes and proteins and can be further metabolized to diacylglycerol and lysophosphatidic acid. However, the role that PA and these lipid metabolites play in mediating the functional consequences of PLD activation is not yet clear. Two mammalian PLD isoenzymes, *

Corresponding author. Tel.: +886-4-2359-2525x4023; fax: +886-42359-2705. E-mail address: [email protected] (J.-P. Wang).

PLD1 and PLD2, which share about 50% homology, have been cloned [3,4]. PLD1 appears to be localized in Golgi, endoplasmic reticulum, and late endosomes, whereas PLD2 seems to be present in the plasma membrane and caveolae [3]. The PLD1 gene encodes two splice variants, PLD1a and PLD1b, of which PLD1b appears to be the predominant isoform in mammalian cells [5]. PLD activation occurs in many cell types in response to various agonists through G protein-coupled or tyrosine kinase receptors, suggesting that multiple pathways trigger the activation of PLD. The upstream regulation of PLD activation involves Ca2 +, protein kinase C (PKC), tyrosine kinase and small G proteins of Rho and ADP-ribosylation factor (ARF) families as reviewed previously [6]. PLD1 is under elaborate control whereas the PLD2 exhibits high basal activity and appears less subject to control in vitro [7,8]. However, Colley et al. [3] have proposed that PLD2 activity may normally be masked by

0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 3 2 9 - X

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associated inhibitors in vivo, and becomes activated by derepression when cells are stimulated. Neutrophil activation plays a central role in inflammatory reactions and constitutes the first line of host defense. Neutrophils stimulated with the chemoattractants display a variety of functional phenomena that include chemotaxis, degranulation, and the production of superoxide anion [9]. The signal transduction events pertaining to these specific functions remain elusive. Evidence has been accumulating to indicate that PLD plays an important role in the regulation of neutrophil functions [1,10]. Our previous reports demonstrate that a novel synthetic compound, 2-benzyloxybenzaldehyde (CCY1a), impaired the stimulation by formylmethionyl-leucyl-phenylalanine (fMLP) of superoxide anion generation in rat neutrophils [11]. It was clear that PLD is activated by fMLP in neutrophils and functionally linked to superoxide anion generation [12]. We recently found that CCY1a had very similar IC50 values in inhibition of both PLD activation and superoxide anion generation in response to fMLP. The present work was undertaken to determine the effect of CCY1a on fMLP-stimulated PLD activation.

2. Materials and methods 2.1. Materials Dextran T-500, enhanced chemiluminescence reagent, and 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine were

Fig. 2. Effect of CCY1a on [Ca2 + ]i. (A) Fluo-3-loaded cells were incubated with DMSO, 10 or 50 AM CCY1a for 3 min at 37 jC before stimulation with 0.3 AM fMLP in the presence of 1 mM Ca2 + in HBSS. (B) Fluo-3loaded cells in Ca2 +-free HBSS were stimulated with 0.3 AM fMLP in combination with DMSO, 10 or 50 AM CCY1a, and 0.3 mM Ca2 + was subsequently added. Results presented are representative of three independent experiments with similar results.

purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Hanks’ balanced salt solution was obtained from Gibco Life Technologies (Gaithersburg, MD, USA). Fluo-3/AM was purchased from Calbiochem-Novabiochem (San Diego, CA, USA). Mouse monoclonal antibodies to phosphotyrosine were purchased from BD Transduction Laboratories (Lexington, KY, USA). Rabbit polyclonal ARF and mouse monoclonal Rho A antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride membrane was obtained from Millipore (Bedford, MA, USA). CCY1a (purity>99%) was synthesized [11] and dissolved in dimethyl sulfoxide (DMSO). Other chemicals were purchased from Sigma Chemical (St. Louis, MO, USA). The final volume of DMSO in the reaction mixture was < 0.5%. Fig. 1. Effect of CCY1a on the formation of PLD-mediated products. Simultaneous addition of 5 Ag/ml of dhCB with DMSO (as control), 3 – 50 AM CCY1a in the presence or absence of 0.5% ethanol into 1-O[3H]octadecyl-sn-glycero-3-phosphocholine-loaded cell suspension for 3 min at 37 jC before stimulation with 1 AM fMLP for 0.5 min. Lipids in the reaction mixture were extracted and separated. The radioactivities of PEt and PA were counted with a PhosphorImager. Values are means F S.D. of percent inhibition of control values (1817 F 477 and 3809 F 951 counts, respectively) from four to six separate experiments.

2.2. Isolation of neutrophil Blood was collected from the abdominal aorta of anaesthetized rats (Sprague – Dawley), and the neutrophils were purified by dextran sedimentation, centrifugation through Ficoll-Hypaque and the hypotonic lysis of erythrocytes [13]. Purified neutrophils containing >95% viable cells were resuspended in Hanks’ balanced salt solution (HBSS) con-

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taining 10 mM HEPES, pH 7.4, and 4 mM NaHCO3, and kept in an ice bath before use. 2.3. Measurement of PLD-mediated products Neutrophils (4  107 cells/ml) were loaded with 10 ACi 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine in HBSS at 37 jC for 75 min, then washed. Cells were incubated with test drugs in the presence of 1 mM CaCl2 and 0.5% ethanol for 3 min at 37 jC before stimulation with fMLP in the presence of dihydrocytochalasin B (dhCB). Lipids in the reaction mixture were extracted, dried, and separated on silica gel 60 [14]. The plates were developed halfway by using the solvent system consisting of hexane/diethyl ether/ methanol/acetic acid (90:20:3:2, v/v/v/v), and then dried and developed again to the top using the upper phase of the solvent system consisting of ethylacetate/isooctane/acetic acid/water (110:50:20:100, v/v/v/v). The radioactivity of [3H]products was directly quantified with a PhosphorImager (Molecular Dynamics 445 SI) using ImageQuaNT software. 2+

2.4. [Ca ]i measurement Neutrophils (5  107 cells/ml) were loaded with 5 AM fluo-3/AM at 37 jC for 45 min. After being washed, the cells were resuspended in HBSS to 5  106 cells/ml. Fluorescence was monitored with a double-wavelength fluorescence spectrophotometer (PTI, Deltascan 4000) at 535 nm with excitation at 488 nm. [Ca2 + ]i was calibrated from the fluorescence intensity as follows: [Ca2 + ]i =Kd  [( F Fmin)/( Fmax F)], where F is the observed fluorescence intensity [15]. The values Fmax and Fmin were obtained at the end of experiments by the sequential addition of 0.33% Triton X-100 and 50 mM EGTA. The Kd was taken as 400 nM.

2.5. Protein tyrosine phosphorylation Cells (2  107 cells/ml) were preincubated with test drugs for the indicated time before stimulation with fMLP. Reactions were terminated by the addition of a stop solution (20% (w/v) trichloroacetic acid, 1 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide, 10 mM NaF, 2 mM Na3VO4, 2 mM p-nitrophenyl phosphate, 7 Ag/ml each of leupeptin and pepstatin). Proteins (60 Ag/ml per lane) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% (w/v) nonfat dried milk in TBST buffer (10 mM Tris – HCl, pH 7.5, 150 mM NaCl and 0.1% Tween 20) and probed with anti-phosphotyrosine antibody. Detection was performed with enhanced chemiluminescence reagent. Quantification was by densitometry. 2.6. ARF and Rho A membrane translocation Cells (5  107 cells/ml) were preincubated with test drugs for the indicated time before stimulation with fMLP, then washed and resuspended in disruption solution (0.34 M sucrose, 10 mM Tris – HCl, pH 7.0, 1 mM phenylmethylsulfonyl fluoride, 2 mM EGTA, 10 mM benzamidine, 10 Ag/ml each of leupeptin and pepstatin). After sonication, the lysate was centrifuged at 800  g for 30 min at 4 jC to remove the unbroken cells, then further centrifuged at 100,000  g for 30 min at 4 jC to collect pellets. In the cell-free system, cells (5  107 cells/ml) were washed with buffer B (25 mM HEPES, pH 7.4, 100 mM KCl, 3 mM NaCl, 5 mM MgCl2, 1 mM Mg – ATP, 1 mM EGTA, 5 mM dithiothreitol, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 10 Ag/ml of leupeptin), then resuspended in

Fig. 3. Effect of CCY1a on fMLP-stimulated protein tyrosine phosphorylation. Cells were preincubated with DMSO or 3 – 50 AM CCY1a in the presence of dhCB (5 Ag/ml) for 3 min, or with 50 AM genistein (GENI) for 30 min and dhCB (5 Ag/ml) for 3 min at 37 jC before stimulation with or without 1 AM fMLP. One minute later, protein tyrosine phosphorylation was detected by immunoblot analysis using anti-phosphotyrosine antibody, then quantified by densitometry. The data of Western blot analysis (left panel) are representative of three independent experiments with similar results. The right panel shows the densitometry results of lane 2 (a), lane 4 (b), lane 6 (c), and lane 8 (d).

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3. Results 3.1. Effect of CCY1a on PLD activation The catalytic mechanism of PLD involves two steps [17], the choline release from phosphatidylcholine and the transfer of the phosphatidyl moiety to water or primary alcohol to produce PA and phosphatidylalcohol, respectively. Addition of fMLP to 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine-loaded rat neutrophils in the presence of ethanol for 0.5 min significantly increased the formation of PA and phosphatidylethanol (PEt). CCY1a attenuated these effects in a concentration-dependent manner with IC50 values of 15.8 F 2.5 and 13.9 F 2.0 AM, respectively (Fig. 1). The presence of 1 mM EDTA greatly reduced the fMLP-stimulated PEt formation to less than 10% of the control value. Treatment of cells with 100 AM genistein for 3 min abolished the PLD activation. The viability was z 96% when cells were incubated with 100 AM CCY1a for 10 min at 37 jC as assessed by trypan blue exclusion and lactate dehydrogenase release. Fig. 4. Effects of CCY1a on the membrane translocation of Rho A and ARF in neutrophils. Cells were incubated with DMSO or 10 – 50 AM CCY1a in the presence of dhCB (5 Ag/ml) for 3 min at 37 jC before stimulation with or without 1 AM fMLP for 1 min, then disrupted by sonication. After sedimentation, the cytosol and membrane fractions were subjected to immunoblot analysis using anti-Rho A or anti-ARF antibody, then quantified by densitometry. (A) Represents the results of Western blot analysis and (B) shows means F S.D. of three to four independent experiments. * P < 0.05, * * P < 0.01, as compared with the corresponding control values (first column).

3.2. Effect of CCY1a on [Ca2+]i Addition of fMLP to the fluo-3-loaded cells stimulated [Ca2 + ]i elevation with two distinct phases, an initial transient Ca2 + spike followed by a long lasting plateau. CCY1a attenuated the plateau but not the initial phase of Ca2 + signal in a concentration-dependent manner (Fig. 2A). In the Ca2 +-free HBSS, fMLP evoked a small and rapid rising

0.5 ml of buffer B. After sonication, the lysate was centrifuged at 800  g for 5 min at 4 jC to remove the unbroken cells. The supernatants were preincubated with test drugs for the indicated time in the presence of 1 AM Ca2 + before stimulation with 10 AM GTPgS or 0.1 AM phorbol myristate acetate (PMA) [16]. Reaction was terminated by the addition of fivefold excess ice cold buffer B and centrifugation at 100,000  g for 30 min at 4 jC to collect pellets. Proteins (60 Ag/ml per lane) were resolved by 13% SDS-PAGE, then transferred to polyvinylidene difluoride membrane. The membranes were blocked with 1% (w/v) gelatin in TBST buffer and blotted using the anti-ARF antibody, or blocked with 5% (w/v) nonfat dried milk in TBST buffer and blotted using the anti-Rho A antibody. The labeled proteins were revealed using the enhanced chemiluminescence reagent. Quantification was by densitometry. 2.7. Statistical analysis Statistical analyses were performed using the Bonferroni t-test method after analysis of variance. A P value less then 0.05 was considered significant for all tests. Analysis of the regression line test was used to calculate IC50 values. Data are expressed as means F S.D.

Fig. 5. Effects of CCY1a on the membrane translocation of Rho A and ARF in a cell-free system. Cell lysates were incubated with DMSO or 10 – 50 AM CCY1a for 3 min at 37 jC in the presence of 1 AM Ca2 + before stimulation with or without (A) 10 AM GTPgS or (B) 0.1 AM PMA for 10 min. Reaction was stopped and membrane pellets were isolated. Membrane fractions were then subjected to immunoblot analysis using anti-Rho A or anti-ARF antibody. The data of Western blot analysis are representative of three to four independent experiments with similar results.

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phase of [Ca2 + ]i change. A subsequent increase in [Ca2 + ]i was observed following the reintroduction of Ca2 + in the medium. CCY1a treatment did not affect the initial phase, but suppressed the changes in [Ca2 + ]i caused by the subsequent addition of Ca2 + in a concentration-dependent manner (Fig. 2B). 3.3. Effect of CCY1a on protein tyrosine phosphorylation Protein tyrosine phosphorylation in rat neutrophils was assessed by immunostaining with anti-phosphotyrosine antibody after resolving the proteins of cell lysate by SDSPAGE. As shown in Fig. 3, several cellular proteins show increase in tyrosine phosphorylation following cell stimulation with fMLP. CCY1a treatment attenuated the immunointensities of prominent bands in a concentrationdependent manner. CCY1a was more active against the phosphorylation of 60– 66, 116 –118, and 133 kDa proteins than those of 50– 60, 77 –85, and 92– 97 kDa proteins. A considerable attenuation of the fMLP-induced response was also observed in genistein-treated cells. 3.4. Effect of CCY1a on membrane translocation of ARF and Rho A Two small G protein subfamilies, Rho and ARF, have been implicated in the regulation of PLD1 activation [6]. As assessed by immunoblot analysis of the subcellular distribution of these small G proteins, only slight immunointensity of ARF and Rho A was detected in the membrane fractions of unstimulated cells. Stimulation with fMLP resulted in a prominent membrane association of both ARF and Rho A. CCY1a treatment reduced the amounts of ARF recovered in the membrane fractions in a concentration-dependent manner with an IC50 value of 29.4 F 7.6 AM (Fig. 4A and B). However, CCY1a up to 50 AM only reduced the membrane association of Rho A to about 50% of the control value. In the cell-free system, GTPgS stimulated the translocation of Rho A and ARF from the cytosol to the membrane fraction by using immunoblot analysis. The membrane association of Rho A, but not ARF, was increased by the PMA treatment. Under these conditions, CCY1a (up to 50 AM) failed to exert any inhibitory effect on the membrane association of ARF and Rho A caused by GTPgS or PMA (Fig. 5A and B).

4. Discussion CCY1a inhibition of the fMLP-induced [3H]PA and [ H]PEt formation in 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine-loaded rat neutrophils was not attributable to the cytotoxic effect because cell viability remained relatively unchanged over the reaction time at the effective concentrations of CCY1a. Treatment of cells with PMA, in the 3

presence of ethanol, also increased the formation of PEt. However, CCY1a up to 50 AM had no appreciable inhibitory effect on PMA-induced response (data not shown). It is plausible that the CCY1a inhibition does not employ a direct suppression of PLD activity, but works through interaction with certain cellular signaling pathways. Previous studies in human and rabbit neutrophils have indicated that the elevation of [Ca2 + ]i by fMLP stimulation is the major factor for PLD activation [18,19]. The data that EDTA, to remove extracellular Ca2 +, strongly suppressed the PLD activation in rat neutrophils is consistent with this conclusion. It is believed that the initial Ca2 + spike is attributable to the internal Ca2 + release, whereas the extracellular Ca2 + entry contributed to the plateau phase of Ca2 + signal in response to fMLP. Inhibition of the plateau but not the initial phase by CCY1a implies the suppression of Ca2 + entry. In a Ca2 +-free medium, Ca2 + release from internal stores contributed to the observed fMLP-stimulated changes in [Ca2 + ]i. This was most likely mediated through the formation of inositol trisphosphate. In contrast, the [Ca2 + ]i change observed following the reintroduction of Ca2 + in the medium represented the entry of extracellular Ca2 + [20]. The data that the changes in [Ca2 + ]i caused by the subsequent addition of Ca2 + but not the initial rapid phase of fMLPinduced response was attenuated by CCY1a reaffirmed the earlier conclusion. However, the inhibition of [Ca2 + ]i alone might not play a critical role in the CCY1a inhibition of PLD because the effective concentrations required to significantly suppress [Ca2 + ]i change are higher than the IC50 for the inhibition of PLD. Consistent with the previous report that the fMLP-activated PLD is tyrosine kinase-dependent in human neutrophil [19], here the general tyrosine kinase inhibitor, genistein, abolished the fMLP-induced PLD activation in rat neutrophils. Although the activation of Gi proteincoupled receptor by fMLP stimulus the phosphorylation of protein tyrosine through the non-receptor tyrosine kinases has been proposed, the nature of the specific tyrosine kinases involved in the responses to PLD activation remains unclear. Src has been implicated in PLD activation [21] and the Ghg subunit derived from pertussis toxin-sensitive G protein has been shown to activate Src [22]. Treatment of cells with CCY1a attenuated the fMLP stimulation of tyrosine phosphorylation of several cellular proteins, although the nature of these proteins remains unclear. PLD activation does not involve a direct tyrosine phosphorylation of the PLD1 isoenzyme (120 kDa) in response to fMLP [23]. Our previous reports indicate that fMLP stimulates the phosphorylation of p42/44 and p38 mitogenactivated protein kinases (MAPK) in rat neutrophils [24,25]. Djerdjouri et al. [26] demonstrated the involvement of p42/44 MAPK cascade in PLD activation by fMLP in neutrophils. It is clear that cell stimulation induces a signaling cascade that leads to the activation of MAPK via phosphorylation on both tyrosine and threonine residues [27]. CCY1a inhibited the phosphorylation of p42/44

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MAPK with IC50 value of 31 AM; moreover, there is a lack of inhibition of p38 MAPK phosphorylation by CCY1a (data not shown). Nevertheless, it is assumed that CCY1a inhibition of PLD activation involves the attenuation of protein tyrosine phosphorylation. Because the fMLP-activated PLD has been reported to be Ca2 +- and tyrosine kinase-dependent [19], the attenuation of protein tyrosine phosphorylation accompanied by a concurrent blockade of Ca2 + entry may play a concert role in the CCY1a inhibition of PLD. Phorbol esters activate PLD in many cell types and phosphorylation does play a role in the in vivo regulation of PLD by PKC as reviewed previously [6]. However, in contrast to the PLD activation by phorbol ester, the fMLPactivated PLD has been reported to be independent of PKC activity [19]. Two small G protein subfamilies, Rho and ARF, regulate PLD1 activation. ARF and Rho A are predominantly cytosolic where they are present in an inactive (GDP-bound) state and must be changed into the active (GTP- or GTPgSbound) state to produce stimulation. Activation of neutrophils by fMLP stimulates PLD through Gi proteins, and Ghg targets the ARF and Rho exchange factors directly for recruitment of ARF and Rho to the plasma membrane where PLD is present [28], although CCY1a inhibited the membrane association of ARF and Rho A at effective concentrations higher than those required to perform PLD inhibition. These effects are probably contributed to the inhibition of PLD activation by CCY1a, because ARF and Rho A proteins act synergistically to regulate fMLP-stimulated PLD activity [28]. A 50-kDa factor has been shown to act synergistically with both ARF and Rho in stimulating PLD [29,30]. Whether CCY1a also inhibits the 50 kDa factor activity needs further investigation. Cell-free systems have advanced our understanding of the regulation of PLD. In the cell-free system, ARF and Rho A are required to reconstitute GTPgS-stimulated PLD activity in membrane fractions [29,31]. When GTPgS is used in a cell-free system, the exchange factors must be bypassed [28]. The nucleotide exchange, and therefore activation, results in the stable interaction of ARF and Rho with the membrane fractions [32]. The lack of appreciable inhibitory effect on GTPgS-induced membrane association of Rho A and ARF by CCY1a eliminates the possibility that this compound directly interferes with small G-proteins. PMA recruits both ARF and Rho to the membranes of human neutrophil [28]. However, in the cell-free system, PMA caused membrane translocation of Rho A, but not ARF. The inability of CCY1a to inhibit the membrane association of ARF and Rho A in a cell-free system raises the possibility that the implication of the cellular signaling mechanism in intact cells but not in a cell-free system is attributable to the CCY1a inhibition of PLD. cAMP-elevating agents are known to inhibit the activation of PLD in human neutrophils [33]. In general, cAMP acts through the cAMP-dependent protein kinase (PKA). It has been demonstrated that PKA inhibits GTPgS-stimulated

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PLD activity through the phosphorylation of membraneassociated Rho A, which leads to the dissociation of Rho A from the plasma membrane [34]. Our previous report demonstrated that CCY1a increases cellular cAMP via activation of adenylyl cyclase in rat neutrophils in a concentration- and time-dependent manner [11]. The result that CCY1a had no inhibitory effect on the membrane association of Rho A in a GTPgS-stimulated cell-free system implies the independence of cAMP. In addition, the PKA inhibitor, KT5720, failed to reverse the inhibition of PLD activation by CCY1a in neutrophils (data not shown), reaffirming this conclusion. These variations could be explained by the fact that signaling pathways besides cAMP play major roles in regulation of PLD activity after 3-min incubation with CCY1a, in which about twofold higher than the resting cAMP level was obtained (data not shown). The potential role of cAMP could be prominent when cAMP level gradually increased with time to a considerable amount. Previous reports have demonstrated that phosphatidylinositol 4,5-bisphosphate, ceramides, and presqualene diphosphate regulate PLD activation. The functional PH domain regulates PLD by mediating its interaction with phosphatidylinositol 4,5-bisphosphate-containing membranes; this might induce a conformational change, thereby regulating catalytic activity [35]. Ceramides prevent the activation of PLD via the inhibition of the translocation to membranes of ARF, Rho A, and PKC isoenzymes [36]. In addition, presqualene diphosphate directly inhibited PLD activity in neutrophil [37]. Whether the inhibition of PLD by CCY1a is also attributed to its ability to change the cellular phosphatidylinositol 4,5-bisphosphate levels or to increase the formation of presqualene diphosphate or ceramides awaits further investigation. In conclusion, the blockade of Ca2 + entry, protein tyrosine phosphorylation, and the membrane translocation of ARF and Rho A may be the cellular mechanisms underlying the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP.

Acknowledgements This work was supported by grants from the National Science Council (NSC90-2315-B-075A-001), Taiwan, Republic of China.

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