Introduction

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Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline (Exton, 1997). Arachidonic acid is liberated from phosphatidic acid by phospholipase A₂ (PLA₂) or by the sequential action of phosphatidate phosphohydrolase and diacylglycerol lipase (Exton, 1997). Several cytokines, growth factors, hormones, or neurotransmitters stimulate PLD activity in various tissues or cell systems. PLD has been implicated in numerous cell functions, including signal transduction, cell proliferation, differentiation, and protein trafficking (Exton, 1997). Some of these cell functions have been proposed to be mediated by activation of protein kinase C (PKC) through generation of diacylglycerol (Exton, 1994). However, diacylglycerol derived from phosphatidic acid did not activate PKC in porcine aortic endothelial cells (Pettitt et al., 1997). Although PLD is activated by PKC in many cell types (Kiss, 1996), its activation is independent of PKC in other cell types such as Madin-Darby canine kidney cells and rat vascular smooth muscle cells (VSMCs) (Freeman et al., 1995; Balboa and Insel, 1998).

Activation of -adrenergic receptors by norepinephrine (NE), the principal neurotransmitter released from sympathetic neurons, has been shown to stimulate PLD activity in a variety of tissues, including rat tail artery, cerebral cortex, and cardiac myocytes (Llahi and Fain, 1992; Ye et al., 1994; Labelle et al., 1996). However, the mechanism by which PLD is regulated by hormones and neurotransmitters, including NE, is not known. Several signaling factors have been proposed to participate in PLD activation. PLD has been reported to be activated by GTP-binding proteins Rho and Rac (Malcolm et al., 1994), ADP-ribosylation factor (Arf) (Brown et al., 1993), and PKC (Kiss, 1996). PIP₂, Arf, and Rho have been shown to synergistically activate PLD in vitro (Hammond et al., 1997). Recently, it was reported that phosphorylation of PLD by PKC was correlated with inhibition of its activity (Sik Min et al., 1998b). There are also several reports demonstrating the involvement of a tyrosine kinase in the activation of PLD (Suzuki et al., 1996; Natarajan et al., 1996; Marcil et al., 1997; Sik Min et al., 1998a; Slaaby et al., 1998). PLD also has been shown to be activated by v-Src (Song et al., 1991) and Ras and the related small G protein Ral (Jiang et al., 1995a,b). Previously, we showed that in the signal transduction pathway of NE-induced arachidonic acid release in rabbit VSMC, mitogen-activated protein (MAP) kinase is involved and activation of MAP kinase is Ras-dependent (Muthalif et al., 1996, 1998). These observations raise the possibility that Ras/MAP kinase might be involved in stimulation of PLD by NE. In the present study, we demonstrate that NE-stimulated PLD activation in rabbit VSMC is dependent on Ras and MAP kinase but not on PKC or Rho. This novel mechanism of NE-stimulated PLD activation appears to involve phosphorylation of PLD by extracellular-regulated kinase 2 (ERK2) MAP kinase.

	Experimental Procedures

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Materials. [³H]Oleic acid (75 Ci/mmol) and L-[1-¹⁴C]phosphatidylcholine (57 mCi/mmol) were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO) and [³²P]orthophosphate and [-³²P]ATP (3000 Ci/mmol) from Amersham Life Sciences (Piscataway, NJ). Hanks' balanced salt solution, M-199, penicillin, streptomycin, fetal bovine serum, PBS, BSA, EGTA, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, nonionic detergent igepal CA-630, and NE were purchased from Sigma Chemical Co. (St. Louis, MO); PD-98059 and ERK2 MAP kinase from New England Biolabs (Beverly, MA); farnesyl protein transferase (FPT) inhibitor III, bisindolylmaleimide (BIM), phorbol-12-myristate-13-acetate (PMA), leupeptin, and aprotinin from Calbiochem (San Diego, CA). FPT inhibitor SCH-56582 was kindly provided by Dr. Robert Bishop (Schering-Plough Research Institute, Kenilworth, NJ). PD-98059 and SCH-56582 were dissolved in dimethylsulfoxide and other drugs were dissolved in double-distilled water.

Preparation of VSMCs. Male New Zealand White rabbits (1-2 kg) were anesthetized with 60 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL), and the thorax and abdomen were opened by a midline incision. The aorta was rapidly removed, and VSMCs were isolated as previously described (Nebigil and Malik, 1992). Cells between four and eight passages were plated in 12 or 24 wells or 100-mm plates. Cells were maintained under 5% CO₂ in M-199 medium with penicillin, streptomycin, and 10% fetal bovine serum.

Measurement of Changes in Cell Ca²⁺. The VSMCs cultured in six-well plates were arrested for 18 h and stimulated with NE (10 µM) in M-199 medium containing 5 µCi ⁴⁵Ca²⁺/ml in the presence and absence of FPT III, SCH-56582, and PD-98059. Ca²⁺ uptake was measured as described (Wang and Schneider, 1993).

Preparation of Dominant Negative Ras and Transfection of VSMCs. pZipneo-RasN17, wild-type Ras, and vector alone cDNAs were kindly provided by Dr. C. Der (University of North Carolina, Chapel Hill). Nonliposomal transfection reagent FuGENE 6 (Boehringer Mannheim, Indianapolis, IN), which enhances efficiency of transfection, was complexed with 4 µg of plasmid. VSMCs cultured on 100-mm plates (70% confluency) maintained in serum-free M-199 were transfected for 48 h according to the manufacturer's recommendations. The efficiency of transfection of VSMCs with dominant negative and wild-type Ras plasmids was estimated by cotransfection with -galactosidase.

-Galactosidase Assay. VSMCs (100-mm plates) were cotransfected with plasmids containing rasN17 and -galactosidase with FuGENE 6 transfection reagent. Expression of -galactosidase was determined by analyzing 10 µl of lysate with the reporter gene assay system (Boehringer Mannheim) according to the manufacturer's instructions. Cleavage of substrate orthonitrophenyl--D-galactoside by -galactosidase to the orthonitrophenyl anion, which produces a yellow color under basic conditions, was assayed. Changes in absorbance were monitored spectrophotometrically at 420 nm.

PLD₁ Antibody. The rabbit polyclonal PLD₁ antibodies (serum 03 and serum 04) were raised against a mixture of short PLD₁ peptides ¹MSLKNEPRVNTSALQK¹⁶, ¹⁴⁴RRQNVREEPREMPS¹⁶², ⁹⁶⁷DDPSEDIQDPVSDK⁹⁸¹, and ¹⁰²⁷KEDPIRAEEELKKI¹⁰⁴⁰ as previously described (Marcil et al., 1997).

General Experimental Protocol and PLD Assay. VSMCs that were arrested for 48 h with medium containing 0.05% fetal bovine serum were used for all studies. Cells were labeled during the last 18 h with [³H]oleic acid (1 µCi/ml), washed twice with balanced salt solution, and incubated with inhibitors of PKC (BIM), MAP kinase kinase (MEK) (PD-98059), and FPT (FPT III and SCH-56582), or their respective vehicles, and exposed to NE (10 µM) for an additional 10 min. The concentrations of inhibitors used in this study have been reported to be effective in blocking the activity of these enzymes in other cell systems. In one series of experiments, the VSMCs were treated with PMA for 24 h to deplete PKC levels and then exposed to NE for 15 min.

Cell Stimulation, Lysis, Immunoprecipitation, and Western Blotting. VSMCs (100-mm plates) were stimulated with NE (10 µM) for 10 min and lysed in nondenaturing lysis buffer containing 50 mM Tris-HCl buffered to pH 7.4, 150 mM NaCl, 1.5 mM MgCl₂, 5 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin. The lysates were incubated for 15 min before centrifugation at 13,000g for 15 min. The supernatant was incubated with 10 µl of anti-PLD₁ serum 03 and 1.2% igepal CA-630, 20 µg/ml aprotinin, and 20 µg/ml leupeptin for 3 h at 4°C. This was followed by an incubation with 60 µl of 50% slurry of protein A-agarose beads for 1 h at 4°C. The beads were washed three times with ice-cold lysis buffer containing 1% igepal and boiled for 7 min at 100°C in Laemmli sample buffer.

MAP Kinase Assay. Activation of MAP kinase was determined in cell lysates with BIOTRAK MAP kinase assay kit (Amersham) as previously described (Muthalif et al., 1996). Phosphorylation of MAP kinase was studied by Western blotting with phosphospecific MAP kinase antibody (New England Biolabs). This antibody detects phosphorylated Tyr residues of p44 and p42 MAP kinase but does not appreciably cross-react with the unphosphorylated forms.

PKC Assay. The activity of PKC in VSMCs was determined with the PKC assay system of Life Technologies (Bethesda, MD) according to the manufacturer's recommendations. A synthetic peptide from myelin basic protein (acetylated-myelin basic protein 4-14) was used as a specific substrate for PKC as described by Yasuda et al. (1990). PKC specificity was confirmed by using the PKC pseudosubstrate inhibitor peptide PKC residues 19-36. Cells were homogenized in extraction buffer (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 µg/ml each of aprotinin and leupeptin) and separated into cytosol and membrane fractions by centrifuging at 100,000g for 1 h. For each assay, 20 µg of protein and 10 µl of ³²P-containing substrate were mixed and incubated at 30°C for 5 min. The reactions were terminated by spotting 25 µl onto phosphocellulose discs. The phosphocellulose papers were washed twice with acid and then with double-distilled water and radioactivity was determined. PKC activity was expressed as picomoles per minute per 20 µg of protein.

Phosphorylation of PLD in Whole Cells. VSMCs were washed three times with phosphate-free Dulbecco's modified Eagle's medium and then prelabeled for 4 h with [³²P]orthophosphate (300 µCi/ml) along with inhibitors and treated with NE (10 µM) for 10 min. The cells were quickly washed three times with ice-cold PBS and immersed in a slurry of ice and ethanol. The cells were scraped and sonicated in nondenaturing lysis buffer. Protein concentration was adjusted to 1 mg/ml, and the lysate was incubated with anti-PLD₁ serum 03 for 4 h at 4°C and then with protein A-agarose beads for 1 h. The immunoprecipitate was centrifuged at 12,000 rpm for 5 min, and the pellet was washed with ice-cold PBS containing phosphatase inhibitors. The pellet was resuspended in Laemmli buffer, and the supernatants were subjected to SDS-PAGE and autoradiography.

In Vitro Phosphorylation. PLD was immunoprecipitated from VSMCs lysates with anti-PLD₁ serum 03 and washed twice with PBS and once with kinase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM EGTA, 2 mM dithiothreitol, 0.01% brij 35, pH 7.5). In vitro phosphorylation was conducted in 250 µl of kinase buffer supplemented with 100 µM ATP and 15 µCi [-³²P]ATP. Twenty-five units of activated ERK2 MAP kinase (New England Biolabs) was added and the reaction was performed at 30°C. The reaction was stopped at different time intervals by adding Laemmli buffer, and the phosphorylated proteins were separated by SDS-PAGE (8% gel), dried, and detected by autoradiography.

Data Analysis. The basal values of PLD activity were variable in different batches of cells. However, the effect of NE to increase PLD activity was consistent within each batch of cells. The results are expressed as means ± S.E.. The data were analyzed by one-way ANOVA; the Newman-Keuls multiple range test was applied to determine the difference among multiple groups; the unpaired Student's t test was applied to determine the difference between two groups. The null hypothesis was rejected at P < .05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Norepinephrine-Induced PLD Activation Is Dependent on Extracellular Ca²⁺. NE is known to increase PLD activity by stimulating -adrenergic receptors (Llahi and Fain, 1992; Labelle et al., 1996). In rabbit VSMCs, NE increases PLD activity via stimulation of both ₁- and ₂-adrenergic receptors (M.M.M. and K.U.M., unpublished data). NE increased PLD activity as measured by phosphatidyl ethanol (PEt) accumulation in a concentration-dependent manner with maximal effect at 10 µM NE (Fig. 1). The maximal accumulation of PEt in response to NE occured at 15 min. Therefore, this period of stimulation was used in the rest of the experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   NE stimulates PLD activity in rabbit VSMCs. VSMCs prelabeled with [3H]oleic acid were stimulated with NE and PLD activity was measured as accumulation of [3H]PEt. Data are shown as means ± S.E. from four experiments from two different batches of cells (100-mm plates). *, value significantly different from the basal (P < .05).

PKC Does Not Mediate NE-Stimulated PLD Activity. PKC activates PLD in many different types of cells (Kiss, 1996). However, there is evidence for receptor-mediated activation of PLD that is independent of PKC (Kiss, 1996; Balboa and Insel, 1998). To examine the role of PKC in NE-stimulated PLD, we first established the activation of PKC by NE. NE increased PKC activity 3-fold in membrane fractions and this effect was attenuated by the PKC inhibitor BIM (500 nM) (P < .05) (Fig. 2A). Chronic treatment of VSMCs with PMA (100 nM for 24 h) caused down-regulation of PKC and abolished PKC activity (data not shown). Moreover, PMA did not significantly decrease NE-stimulated PLD activation (104 ± 10% increase in PLD activity over vehicle with NE versus 87 ± 7% with PMA + NE) and NE-induced activation of PLD was not inhibited by BIM (Fig. 2B). Moreover, NE-induced phosphorylation of ERK1 and ERK2 MAP kinase was not reduced by BIM, and PMA treatment did not reduce NE-stimulated phosphorylation of ERK1 and ERK2 MAP kinases (Fig. 2C). These results show that PKC is not involved in NE-stimulated PLD activation in VSMCs.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   PKC does not mediate NE-stimulated PLD activity. Effect of PKC inhibitor BIM on NE-stimulated PKC (A) and PLD (B) activity. Cells labeled with [3H]oleic acid were preincubated with BIM for 30 min at 37°C and then stimulated with NE for 15 min. Membrane fractions (20 µg of protein) obtained from VSMCs were used to measure PKC activity. This assay is based on the phosphorylation of acetyl-myelin basic protein peptide (4-14) and its specific inhibition by the pseudosubstrate peptide (PKC 19-36). PLD activity was measured as described earlier. Data are expressed as ± S.E. of three or four experiments performed in different batches of cells. *, value significantly different from the corresponding values obtained in the presence of vehicle of NE, and , value different from that obtained with NE alone (P < .05). C, effect of BIM and PMA on phosphorylation of ERK1 and ERK2 MAP kinases in VSMCs. VSMC lysates (40 µg) were electrophoresed on 10% SDS-PAGE and transferred onto nitrocellulose membrane. The blot was then probed with phosphospecific MAP kinase antibody.

Ras Mediates NE-Induced PLD Activity. NE stimulates Ras activity in human VSMCs (Hu et al., 1996) and Ras is known to stimulate the Raf/MEK/MAP kinase pathway by associating with plasma membrane (Zhang et al., 1993). Stimulation of VSMCs with NE results in localization of Ras to the plasma membrane (Muthalif et al., 1998). The post-translational addition of a farnesyl moiety is required for recruitment of Ras to the plasma membrane and is critical for Ras activity. Inhibitors of protein farnesyl transferase have been shown to selectively block the function of Ras (Manne et al., 1995; Gibbs and Oliff, 1997). FPT III, which inhibits farnesyl transferase activity, also has been shown to block NE-stimulated localization of Ras to the plasma membrane (Muthalif et al., 1998). In this study, FPT III attenuated NE-stimulated MAP kinase activity (92 ± 10% above vehicle with NE versus 34 ± 3% above vehicle with FPT III + NE, P < .05). SCH-56582 (5 µM) and FPT III (25 µM), which inhibit farnesyl transferase, attenuated NE-stimulated PLD activity in VSMCs (Fig. 3). These data strongly suggest that NE stimulates PLD activity by a Ras-dependent pathway. Because the pharmacological agents may exert nonspecific effects, a more specific molecular tool, a dominant negative form of Ras, was used in the next series of experiments. VSMCs were transfected with wild-type Ras as a control. The efficiency of transfection of VSMCs with dominant negative and wild-type Ras was determined with -galactosidase assay. -galactosidase activity in VSMC lysate from the cells transfected with dominant negative Ras or wild-type constructs was 155.75 ± 48.99 U/mg protein, whereas in the cells transfected with vector alone, the -galactosidase activity was only 23.51 ± 5.51 U/mg protein. Consistent with the results of the pharmacological agents described above, expression of dominant negative but not wild-type Ras attenuated NE-stimulated PLD (Fig. 4A) and MAP kinase activity (Fig. 4B).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Effect of farnesyl transferase inhibitors on NE-stimulated PLD activity in VSMCs. Cells that were labeled with [3H]oleic acid were preincubated with indicated concentrations of SCH-56582 and FPT III for 4 h at 37°C and then stimulated with NE for 15 min. The inhibitors when used alone did not alter basal PLD activity. Data are expressed as means ± S.E. of four or five experiments performed on different batches of cells for assay of PLD activity. *, value significantly different from that obtained with vehicle, and , value different from that obtained with NE alone (P < .05).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of dominant negative Ras on NE-stimulated PLD (A) and MAP kinase (B) activities. VSMCs were transfected with vector alone, dominant negative (RasN17), or wild-type Ras for 48 h and then the cells were stimulated with NE for 15 min. Data are expressed as means ± S.E. of three experiments performed on different batches of cells. , value different from that obtained with cells transfected with vector alone (P < .05).

MAP Kinase Mediates NE-Induced PLD Activity. NE stimulates MAP kinase via activation of Ras (Muthalif et al., 1998). Hence, we tested the possible involvement of MAP kinase in NE-stimulated PLD activation. We have previously demonstrated that NE enhances both ERK1 and ERK2 MAP kinase activity in VSMCs and that this activation is blocked by the MEK inhibitor PD-98059 (Muthalif et al., 1996). To investigate the possible involvement of MAP kinase in NE-stimulated PLD activation, VSMCs were preincubated with PD-98059 for 4 h. Treatment of VSMCs with PD-98059 resulted in a concentration-dependent decrease in NE-stimulated PLD activity (Fig. 5). These results show that NE-stimulated PLD activation in VSMCs is mediated by MAP kinase.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of MEK inhibitor (PD-98059) on NE-stimulated PLD activity in VSMCs. Cells that were labeled with [3H]oleic acid were preincubated with indicated concentrations of PD-98059 for 4 h at 37°C and then stimulated with NE for 15 min. Data are expressed as means ± S.E. of four experiments on different batches of cells. *, value significantly different from the corresponding values obtained in the presence of vehicle of NE, and , value different from the corresponding values obtained with NE alone (P < .05).

ERK2 MAP Kinase Phosphorylates PLD. It has been shown that MAP kinase increases activity of cytosolic PLA₂ by phosphorylating this enzyme at Ser-505 (Lin et al., 1993). However, PLD has been shown to be tyrosine-phosphorylated and associated with several tyrosine-phosphorylated proteins (Marcil et al., 1997; Sik Min et al., 1998b; Slaaby et al., 1998). PLD₁ purified from rat was shown to be phosphorylated by PKC and -II (Suzuki et al., 1996). We tested whether the activation of PLD by MAP kinase involves phosphorylation. First, the presence of PLD in rabbit VSMCs was examined with antipeptide antibodies. VSMC lysates were immunoprecipitated with PLD antibody (serum 03) and immunoblotted with PLD antibody (serum 04). As shown in Fig. 6A, a band of ~115 kDa was observed. This band corresponded to the PLD band obtained in Sf9 cells overexpressing PLD. The specificity of the two anti-PLD sera was demonstrated by the immunodepletion of 115-kDa band in immunoblots carried out with antigen-preneutralized antibodies in VSMCs and Sf9 membranes (Fig. 6A). PLD was immunoprecipitated from rabbit VSMCs with anti-PLD₁ serum. NE increased incorporation of ³²P into PLD in VSMCs and this incorporation was attenuated by MEK inhibitor PD-98059 (Fig. 6B), suggesting a role for MAP kinase in NE-stimulated phosphorylation of PLD. Moreover, PLD immunoprecipitated from rabbit VSMCs was phosphorylated in vitro by ERK2 MAP kinase (Fig. 6C). This provides further confirmation that MAP kinase is involved in phosphorylation and activation of PLD in VSMCs.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Expression of PLD in Sf9 and VSMCs (A) and in vivo and in vitro phosphorylation of PLD (B and C). A, Sf9 cells were infected with PLD1b baculoviruses at a multiplicity of 10 and cultured for 48 h at 27°C. Sf9 cells were then scraped in ice-cold Hanks' buffered salt solution, centrifuged, and the cell pellet sonicated 2 × 20 s in ice-cold KCl-HEPES relaxation buffer (100 mM KCl, 50 mM HEPES, 5 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, 2.5 mM phenylmethylsulfonyl fluoride, pH 7.2). Unbroken cells and the nuclei were discarded by centrifugation and the supernatants ultracentrifuged at 180,000g for 45 min. Membrane pellets were washed once and resuspended in a small volume of ice-cold KCl-HEPES relaxation buffer and proteins from Sf9 membranes were mixed in equal volume of 2× Laemmli sample buffer, boiled for 7 min, electrophoresed, and transferred onto a nitrocellulose membrane. Immunobotting was performed with the anti-PLD1 serum 04 or the peptide neutralized anti-PLD1 serum (left). PLD1 was revealed with the enhanced chemiluminescence detection system. VSMC samples (right) were lysed in nondenatuting buffer as described in Experimental Procedures. Lysates (500 µg of proteins) were immunoprecipitated with 10 µl of anti-PLD serum 03 or the peptide neutralized anti-PLD serum 03, electrophoresed, and transferred to nitrocellulose membrane. The blot was probed with the anti-PLD serum 04. B, effect of PD-98059 on NE-stimulated 32P incorporation into PLD. VSMCs were metabolically labeled with [32P]orthophosphate with or without PD-98059 (20 µM) and lysates were obtained under denaturing conditions as described in Experimental Procedures. PLD was immunoprecipitated by anti-PLD1 serum 03, separated on an SDS-PAGE, and subjected to autoradiography. C, in vitro phosphorylation of immunoprecipitated PLD by ERK2 MAP kinase. PLD immunoprecipitated from rabbit VSMCs was in vitro phosphorylated with 25 U of ERK2 MAP kinase for indicated times as described in Experimental Procedures. Phosphorylated proteins were subjected to SDS-PAGE and autoradiography. Data are representation of two separate experiments performed on two different batches of cells.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Several agents, including growth factors, cytokines, reactive oxygen species, vasopressin, angiotensin II, acetylcholine, and NE, stimulate PLD activity (Exton, 1997). The mechanism by which neurotransmitters such as NE stimulate PLD activity has not yet been elucidated. The present study with rabbit VSMCs has led to the following conclusions: 1) NE-induced PLD activation is independent of PKC activity, 2) NE-induced PLD stimulation requires the activation of Ras/MAP kinase pathway, and 3) the mechanism of activation of PLD involves phosphorylation by ERK2 MAP kinase, a Ser/Thr kinase.

There are reports that PLD, in some cell systems, is regulated by PKC (Kiss, 1996). Our results provide evidence that NE-induced PLD activity in VSMCs is independent of PKC because the PKC inhibitor BIM blocked the increase in PKC but not PLD activity elicited by NE. Moreover, long-term treatment with PMA (24 h), which depleted PKC and abolished PKC activity, failed to alter NE-induced PLD activation in VSMCs. The increase in PLD activity produced by NE in rat tail artery (Gu et al., 1992) and brain slices (Llahi and Fain, 1992) also was not altered by PKC inhibitors. In Madin-Darby canine kidney cells, epinephrine-stimulated PLD activation is also PKC-independent (Balboa and Insel, 1998). Moreover, oxidized low-density lipoprotein-mediated activation of PLD in rabbit femoral arterial smooth muscle cells is PKC- and Ca²⁺-independent but was sensitive to the tyrosine kinase inhibitors genistein and vanadate (Natarajan et al., 1995).

Small GTP-binding proteins such as Ras and RhoA can activate PLD. Ras activates PLD in v-src- or v-ras-transformed cells, and this activation is mediated by ral GTPase with a direct interaction between ral and PLD (Kanaho et al., 1992; Jiang et al., 1995b). RhoA has been shown to mediate agonist stimulation of PLD via a direct interaction (Bae et al., 1998). In the present study, small GTP-binding protein Ras but not Rho is likely to be involved in NE-stimulated PLD activation due to the following reasons. First, farnesyltransferase inhibitors FPT III and SCH-56582, which were shown to inhibit NE-stimulated activation of Ras and MAP kinase (Muthalif et al., 1998)-attenuated NE-induced PLD activation. Second, Ras proteins are farnesylated, whereas other small G proteins such as rhoA, rac, cdc42, and ral GTPase are geranylgeranylated (Resh, 1996). Ras inhibitors used in this study, FPT III and SCH-56582, can inhibit geranylgeranylation only at concentrations much higher than those used in our study (Kelloff et al., 1997). Third, dominant negative Rho had no effect in NE-stimulated PLD activation (J.-H.P., M.M.M., and K.U.M., unpublished data). del Peso et al. (1996) suggested that Ras oncogenes activate PLD enzyme by a PKC-independent mechanism in 3T3 cell line. PLD activity is elevated in vRas-transfected cells (Carnero et al., 1994), suggesting that Ras may increase PLD activity in intact cells. Our data and results reported by other investigators (Carnero et al., 1994; del Peso et al. 1996) indicate that Ras is a component in the signaling machinery that results in PLD activation.

There are several reports suggesting activation of MAP kinase pathway by PLD and its product, phosphatidic acid. Ghosh et al. (1996) reported that phosphatidic acid interacts directly with the serine-threonine kinase Raf-1 translocation to the plasma membrane (Rizzo et al., 1999). There is evidence that tyrosine kinases are involved in PLD activation (Natarajan et al., 1996; Marcil et al., 1997; Sik Min et al., 1998b). However, the nature of this tyrosine kinase has not yet been established. In VSMCs, NE stimulates MAP kinase via a tyrosine kinase MEK (Muthalif et al., 1996). However, angiotensin II-stimulated PLD activity has been reported to be independent of MAP kinase in VSMCs derived from hypertensive rats (Wilkie et al., 1996). In A7r5, a rat VSMC cell line, activation of MAP kinase and PLD by vasopressin were shown to be concurrent but independent (Jones et al., 1994). The present study in rabbit VSMCs provides the first evidence that NE stimulates PLD by activating MAP kinase. This is based on our demonstration that PD-98059, an inhibitor of MEK, attenuated the increase in MAP kinase and PLD activity elicited by NE in VSMCs. The mechanism of PLD activation by MAP kinase involves phosphorylation (Fig. 6B) and this phosphorylation of PLD by MAP kinase appears to be direct (Fig. 6C). Phosphorylation of PLD by MAP kinase reported in this study is similar to that demonstrated by Sik Min et al. (1998b) who showed that PKC and PKCII phosphorylate the PLD in a time-dependent manner. Whether MAP kinase and PKC phosphorylates the same or distinct sites is not known. These results suggest that MAP kinase may phosphorylate PLD directly. However, we cannot rule out the possibility that MAP kinase may phosphorylate other proteins that may have stimulatory effects on PLD in vivo. In our study, the effect of MEK and farnesyl transferase inhibitors on PLD activation is unlikely to be due to their nonspecific actions on upstream signaling events because the effect of NE on ⁴⁵Ca uptake by VSMCs was not altered.

In conclusion, these results provide evidence for a novel signaling pathway for PLD activation by NE. This NE-induced PLD activation is mediated via Ras/MAP kinase in rabbit VSMCs. Further studies are needed to identify the sites of phosphorylation on PLD by MAP kinase.