COMMUNICATION:
Phospholipase D Activity in PC12 Cells
EFFECTS OF OVEREXPRESSION OF alpha 2A-ADRENERGIC RECEPTORS*

(Received for publication, March 24, 1997)

Krishna M. Ella , Chen Qi , Anthony F. McNair , Jin-Hyouk Park , April E. Wisehart-Johnson and Kathryn E. Meier Dagger

From the Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

PC12 neuronal cells express a membrane phospholipase D (PLD) activity that is detected at similar levels in undifferentiated or differentiated cells. The regulation of this activity by agonists was explored. Membrane phospholipase D activity was increased by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or with nerve growth factor. The ability of PMA to activate PLD was confirmed in intact PC12 cells. Basal activity of PLD in membranes was reduced in RG20, a PC12 cell line overexpressing the human alpha 2A-adrenergic receptor. PMA did not increase PLD activity in RG20 cells, as assessed both in membrane preparations and in intact cells. Cyclic AMP levels did not regulate phospholipase D activity in either cell type. However, incubation of RG20 cells with the alpha 2-adrenergic antagonist rauwolscine or with pertussis toxin increased membrane PLD activity and restored activation of PLD by PMA. These data suggest that the effects of the overexpressed alpha 2A-adrenergic receptor on PLD activity are mediated by precoupling of the receptor to the heterotrimeric GTP-binding protein, Gi, but are independent of adenylate cyclase regulation. The results of this study suggest that membrane phospholipase D activity can be negatively regulated via Gi in PC12 cells.


INTRODUCTION

Phospholipase D (PLD)1 isoforms have been sequenced from yeast (1-3) and mammalian (4) cells. It appears that more than one form of PLD is expressed in mammals (5-8). PLDs can be activated in response to extracellular signals such as growth factors, hormones, and neurotransmitters (9) and by phorbol esters that stimulate protein kinase C (10). Hydrolysis of phosphatidylcholine (PC) by PLD produces phosphatidic acid (PA). PA is proposed to play a role in signal transduction as a lipid mediator or mediator precursor (11-14). Some PLDs are regulated by the small GTP-binding proteins ARF and/or Rho (15-17). However, the full range of effectors and mediators regulating different isoforms of PLD remains to be elucidated. This laboratory has characterized regulated PLDs in yeast and mammalian cells (10, 18-20). In this study, we explore the role of a heterotrimeric GTP-binding protein, Gi, in the regulation of PLD activity in a neuronal cell line.

Signal transduction pathways have been extensively studied in PC12, a rat phaeochromocytoma cell line that can be induced to differentiate to a neuronal phenotype. PC12 transfected with the alpha 2A-adrenergic receptor (alpha 2AAR) have been used to examine coupling of this receptor to its effectors (21). The alpha 2AAR couples to the heterotrimeric GTP-binding protein Gi, is widely expressed, and mediates the central hypotensive effects of alpha 2 agonists (22, 23). Gi proteins are heterotrimeric GTP-binding proteins containing an alpha i subunit. They are coupled to inhibition of adenylate cyclase as well as to pathways involving additional effectors, such as small GTP-binding proteins (24). In this study, alpha 2AAR-expressing PC12 cells were used to examine the potential role of Gi in regulating PLD activity.


MATERIALS AND METHODS

Cell Culture and Incubations

PC12 cell lines were maintained on Primaria plastic (Falcon) in Dulbecco's modified Eagle's medium (DME) supplemented with either 10% fetal calf serum or 7.5% fetal calf serum plus 2.5% horse serum. PC12K (a wild-type cell line) and RG20 (stably transfected with human alpha 2A/DAR) (21) were grown for 3-5 days in 35- or 100-mm dishes. Cytosolic and membrane extracts were prepared as described (18). Briefly, cells were incubated in culture medium at 37 °C, washed, resuspended in lysis buffer (20 mM HEPES (pH 7.5), 80 mM beta -glycerophosphate, 10 mM EGTA, 2 mM EDTA, and 2 mM dithiothreitol), sonicated, and sedimented at 100,000 × g for 20 min at 4 °C. The supernatant ("cytosol") was used for ERK assays and the pellet ("membranes") for PLD assays. Protein concentrations in cell extracts were determined using Coomassie reagent (Pierce).

Enzyme Assays

PLD activity was measured in vitro using the fluorescent substrate BODIPY-phosphatidylcholine (BPC), 2-decanoyl-1-(O-(11-(4, 4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)sn-glycero-3-phosphocholine (Molecular Probes). BODIPY is a trademark of Molecular Probes, Inc. The assay was performed, as described previously (18-20), using 10 µg membrane protein and 1% butanol (for transphosphatidylation) with incubation for 60 min at 30 °C. BODIPY-labeled products were separated by thin-layer chromatography and quantitated by Molecular Dynamics FluorImager or Helena Laboratories scanning densitometer. Product formation was calculated as percent of total fluorescence.

PLD activity was assessed in intact cells as described previously (18-20). Briefly, cells were incubated overnight with [3H]palmitate or [3H]oleate, washed, and incubated in triplicate with agonists and 0.5% ethanol at 37 °C in DME, 10 mM HEPES. Products were separated by TLC; PEt and PA were quantitated by liquid scintillation spectrometry after scraping from the plate. Product formation was calculated as a percent of total radioactivity recovered from each lane.

ERK activity was assessed in vitro as described previously (10, 20), using myelin basic protein as substrate. Results were normalized for cytosolic protein.


RESULTS AND DISCUSSION

A fluorescent in vitro assay using a BODIPY-labeled substrate, BPC, was used to screen for PLD activity in membranes prepared from a variety of cell types. PC12K cells were observed to express a very high level of membrane PLD activity as compared with other cell types (data not shown). This activity and its regulation were therefore further characterized. PC12K membranes convert BPC to PBt, the transphosphatidylation product generated by PLD in the presence of butanol (Fig. 1A). Most of the PLD activity was membrane-bound, with cytosol producing only 21% of the PBt seen in membranes with equal amounts of cellular protein (data not shown). PBt can be metabolized to lyso-PBt by a calcium-independent PLA2 in membranes from mammalian cells (18), including PC12K (Fig. 1A). PLD and PLA2 activities were similar between undifferentiated and NGF-differentiated cells.


Fig. 1. PLD activity in PC12K cells. A, membranes were prepared from PC12K cells incubated with or without 1 µg/ml NGF for 3 days. Generation of PLD products (PBt and lyso-PBt) was assessed, using an in vitro fluorescent assay. Each point represents the mean ± S.D. of values from duplicate dishes. B, PLD activity was assessed in intact PC12K cells labeled with [3H]oleate and then treated with 100 nM PMA in the presence of 0.5% ethanol for the indicated times. Each point represents the mean ± S.D. of values from triplicate dishes. PEt levels in untreated control cells remained stable for the duration of the incubation.
[View Larger Version of this Image (17K GIF file)]

The phorbol ester PMA generally increases PLD in mammalian cells. PMA activated PLD in intact undifferentiated PC12K cells, as seen using either [3H]oleate (Fig. 1B) or [3H]palmitate (data not shown) as metabolic label. The greatest accumulation of PEt occurred in the first 15 min after PMA addition. PEt did not accumulate in cells incubated without PMA (data not shown).

The potential influence of the alpha 2A/DAR, a Gi-coupled receptor, was examined in a PC12 cell line overexpressing this receptor. Radioligand binding, using the ligand [3H]RX 82102, confirmed that RG20 overexpressed the alpha 2A/DAR (7585 fmol/mg of protein in RG20 versus 78 in PC12K) (data not shown). Basal membrane PLD activity was substantially decreased in alpha 2AAR-expressing cells, as shown for two clonal RG20 lines (Fig. 2A). This decrease was not due to enhanced degradation of PBt by PLA2, since lyso-PBt production was not greater in RG20 than in PC12K. Calcium-independent PLA2 activity, as measured by lyso-PC production, was similar in both cell lines (Fig. 2A).


Fig. 2. PLD activity in PC12K and RG20 cells. A, membrane PLD activity was assessed in PC12K and two clonal RG20 lines. Each data point represents the mean ± S.D. of values from two separate experiments. Lyso-PBt and lyso-PC are PLA2 reaction products. B, PC12K and RG20 cells were incubated with 100 nM PMA or 1 µg/ml NGF for 15 min. Membrane PLD activity was assessed. Each point represents the mean ± S.D. of values from two separate experiments. C, PLD activity was assessed in intact [3H]palmitate-labeled PC12K and RG20 cells incubated in parallel with 0.5% ethanol in the absence and presence of 100 nM PMA for 30 min. Each point represents the mean ± S.D. of values from triplicate dishes. The numbers of cells/dish were 1.24 ± 0.2 × 106 for PC12K and 1.45 ± 0.4 × 106 for RG20.
[View Larger Version of this Image (31K GIF file)]

The abilities of PC12 and RG20 cells to activate PLD were compared. In PC12K, but not RG20, treatment with PMA or NGF increased membrane PLD activity (Fig. 2B). PMA treatment has been shown to activate ERK mitogen-activated protein kinases in PC12 cells (25). The lack of response to PMA was not due to an inability of RG20 cells to respond to phorbol ester, since a 15-min treatment with PMA activated cytosolic ERK mitogen-activated protein kinases to a similar extent in PC12K and RG20, as assessed by an in vitro assay using myelin basic protein as substrate (data not shown). The reduced ability of PMA to activate PLD in RG20 was also apparent in assays using intact cells (Fig. 2C). The intact cell assay could not be used to assess basal PLD activity, since radioactivity co-migrating with PEt was not significantly different for untreated cells incubated in the absence or presence of ethanol (data not shown). The ability of the in vitro assay to quantitate basal PLD activity is thus advantageous.

The mechanism by which by alpha 2AR expression inhibited PLD activity was explored. The alpha 2AR is coupled to inhibition of adenylate cyclase. However, dibutyryl cAMP (10 µM) had no effect on membrane or cytosolic PLD activity in PC12K or RG20 cells at times from 15 to 60 min (data not shown), suggesting that the effect of alpha 2AR expression was not due to decreased cAMP levels. Forskolin (10 µM) and epinephrine (1 µM) likewise had no effect on membrane PLD activity (data not shown). The lack of effect of epinephrine suggested that the overexpressed alpha 2AAR might be functionally "precoupled" to Gi in RG20, as indicated previously for this cell line (27, 28). PC12K and RG20 were therefore incubated with rauwolscine, an alpha 2AR antagonist, to uncouple the receptor from Gi. Rauwolscine increased membrane PLD activity in RG20, but had no significant effect in PC12K (Fig. 3A). One explanation for the fact that rauwolscine did not restore PLD levels in RG20 cells to that seen in PC12K cells is that a portion of the precoupled alpha 2AR receptors in RG20 cells are rauwolscine-resistant, as suggested previously (28). Incubation with pertussis toxin, an inhibitor of Galpha i-mediated signaling, likewise increased membrane PLD activity in RG20 (Fig. 3B). Rauwolscine and pertussis toxin had no effect on PLD activity in PC12K. These findings were confirmed by PLD assays using intact cells (Fig. 3C). Pertussis toxin alone slightly increased basal PLD activity in both PC12K and RG20. Neither pertussis toxin nor rauwolscine significantly increased activation of PLD in response to PMA in PC12K (Fig. 3C, left). In contrast, both pertussis toxin and rauwolscine enhanced PMA-induced PLD activation in RG2 (Fig. 3C, right). Rauwolscine alone had no effect on PLD activity in intact PC12K or RG20 cells (data not shown). These results support the hypothesis that the effect of alpha 2AR overexpression on PLD activity is mediated by precoupling of the receptor to Galpha i.


Fig. 3. Effects of various agents on PLD activity. A, PC12K and RG20 cells were incubated with or without 10 µM rauwolscine for 15 min. Membrane PLD activity was assessed. Each data point represents the mean ± S.D. of values from duplicate dishes. B, PC12K and RG20 cells were incubated with 100 ng/ml pertussis toxin for 60 min. Membrane PLD activity was assessed. Each data point represents the mean ± S.D. of values from two separate experiments. C and D, PLD activity was assessed in parallel in [3H]oleate-labeled PC12K (C) and RG20 (D) cells incubated for 30 min with 0.5% ethanol in the absence and presence of 100 nM PMA, 100 ng/ml pertussis toxin (2-h preincubation), and/or 10 µM rauwolscine (1 min preincubation). Each point represents the mean ± S.E. of values obtained from two separate experiments (expressed as percent of untreated control), each of which was performed using triplicate or quadruplicate dishes of cells.
[View Larger Version of this Image (29K GIF file)]

In summary, membrane PLD activity in PC12 cells is positively regulated by PMA and NGF and negatively regulated by the alpha 2AR. Previous reports have suggested that alpha 2-agonists, with protein kinase C co-activation, can stimulate PLD activity in myristate-labeled intact or broken PC12 cells transfected with the alpha 2AR (29, 30). We observed that an alpha 2-selective agonist did not significantly enhance the ability of PMA to activate PLD in intact oleate-labeled RG20 cells (data not shown). More than one form of PLD is expressed by mammalian cells (31, 32). In one study, PMA-activated PLD was detected in fibroblasts isotopically labeled with either a fatty acid or an alkyl-lyso-PC precursor, while v-Src-activated PLD could be detected only using the fatty acid precursor (14). In Madin-Darby canine kidney cells, PMA-activated PLD preferentially utilizes alkyl-PCs (33). The alkyl-PC substrate used in our in vitro assays may thus detect a PLD preferentially regulated by Gi. Other forms likely contribute to the activity measured in intact cells. Immunoblots obtained using an anti-PLD antibody (34) suggest that expression of PLD is similar in PC12K and RG20 (data not shown).

The mechanism of the novel negative interaction between Gi and PLD is unknown. Galpha i2, a pertussin toxin-sensitive G protein, can positively regulate PLA2 (35). Negative regulation of PLD by Gi could involve cross-talk between heterotrimeric Gi and small GTP-binding proteins (24). For example, the alpha 2AR can activate ras via a Gi-mediated pathway when transfected into fibroblasts (36). However, activation of rho is induced via the pertussis-toxin insensitive proteins Galpha 12 and Galpha 13, but not by Galpha i2 or Galpha q, in fibroblasts (37). Galpha 12 can also activate signaling mediated by Ras and Rac (38). Since rho activates some forms of PLD, a previously unidentified negative influence of a Gi component on the function of Rho (or another small GTP-binding protein) could potentially inhibit PLD activity. However, it should be noted that some agonists (e.g. LPA) bind to a Gi-coupled receptor that activates both rho (39) and PLD (40). The mechanism by which protein kinase C isoforms activate PLD remain to be defined, but may involve protein-protein interactions rather than phosphorylation (41-43). Interestingly, the effects of PMA on PLD activity in fibroblasts were recently shown to be independent of Rac (44), but partially dependent on Rho (45). The roles of heterotrimeric G-proteins in PLD regulation appear worthy of further study.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA58640-04 and HL07260, by the University Research Committee of the Medical University of South Carolina, and by National Science Foundation Grant EPS-9630167.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 803-792-5853; Fax: 803-792-2475.
1   The abbreviations used are: PLD, phospholipase D; alpha 2AR, alpha 2-adrenergic receptor; BPC (BODIPY-phosphatidylcholine), 2-decanoyl-1-(O(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)sn-glycero-3-phosphocholine; ERK, extracellular signal-regulated protein kinase; NGF, nerve growth factor; PA, phosphatidic acid; PBS, phosphate-buffered saline; PBt, phosphatidylbutanol; PC, phosphatidylcholine; PEt, phosphatidylethanol; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate; DME, Dulbecco's modified Eagle's medium.

ACKNOWLEDGEMENTS

We thank Dr. John Hildebrandt for helpful suggestions. The advice and assistance of Drs. Stephen Lanier, Motohiko Satoh, Qing Yang, and Sebastien Saulnier-Blache are gratefully acknowledged.


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