Phosphatidate Accumulation in Hormone-treated Hepatocytes via a Phospholipase D Mechanism*

Isolated rat hepatocytes responded to a variety of Ca2+-mobilizing agents (vasopressin, angiotensin 11, epinephrine, epidermal growth factor, ATP, and ADP) with a rapid increase in phosphatidate mass, as measured by a sensitive new method. When hepatocytes were incubated with vasopressin (lo-’ M), phosphati- date levels increased 2-%fold in 2 min, but there was no significant increase in diacylglycerol at this time. Changes in the fatty acid composition of phosphatidate also preceded those in diacylglycerol. De novo synthe- sis of phosphatidate from [3H]glycerol was unaffected by vasopressin in short-term incubation. Incubation of washed rat liver plasma membranes with GTP-yS caused a time-dependent increase in phos- phatidate. When membranes were incubated with GTP-yS and [-y-32P]ATP, no incorporation of “P into phosphatidate was observed. This excludes the phospholipase C-diacylglycerol kinase pathway and sug- gests that a phospholipase D activity produced the phosphatidate. At submaximal concentrations of GTP-yS, ATP and ADP stimulated membrane phosphatidate formation, presumably by acting through P2- purinergic receptors.

Isolated rat hepatocytes responded to a variety of Ca2+-mobilizing agents (vasopressin, angiotensin 11, epinephrine, epidermal growth factor, ATP, and ADP) with a rapid increase in phosphatidate mass, as measured by a sensitive new method. When hepatocytes were incubated with vasopressin (lo-' M), phosphatidate levels increased 2-%fold in 2 min, but there was no significant increase in diacylglycerol at this time. Changes in the fatty acid composition of phosphatidate also preceded those in diacylglycerol. De novo synthesis of phosphatidate from [3H]glycerol was unaffected by vasopressin in short-term incubation.
Incubation of washed rat liver plasma membranes with GTP-yS caused a time-dependent increase in phosphatidate. When membranes were incubated with GTP-yS and [-y-32P]ATP, no incorporation of "P into phosphatidate was observed. This excludes the phospholipase C-diacylglycerol kinase pathway and suggests that a phospholipase D activity produced the phosphatidate. At submaximal concentrations of GTP-yS, ATP and ADP stimulated membrane phosphatidate formation, presumably by acting through P2purinergic receptors. Only phosphatidylcholine, among the major phospholipids, decreased in the membranes in response to GTP-yS. The fatty acid composition of the phosphatidate produced in response to vasopressin in hepatocytes also suggests that phosphatidylcholine may be the source of hormonally elicited phosphatidate. We conclude that Ca2+-mobilizing hormones mainly increase phosphatidate levels in hepatocytes by a mechanism that does not involve phosphorylation of diacylglycerol or de novo synthesis but involves a guanine nucleotide-binding protein coupled to phospholipase D.
Phosphatidate has long been recognized as a central metabolite in both phospholipid and triglyceride metabolism but measurements of phosphatidate mass, which is only about 1% of total phospholipid, have been infrequent. Most studies of phosphatidate metabolism have employed radioisotopic labeling and two-dimensional thin layer chromatography of phospholipids. Ca2+-mobilizing hormones have been shown to increase [32P]P04 incorporation into phosphatidic acid in a mumber of cell types (for example, Ref. 1). This increase in labeling is thought to be a consequence of the phosphorylation * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Associate Investigator, Howard Hughes Medical Institute.
3 Investigator, Howard Hughes Medical Institute. To whom correspondence and reprint requests should be addressed.
by diacylglycerol kinase of diacylglycerol formed by the phosphodiesteratic cleavage of inositol phospholipids. In rat hepatocytes, Takenawa et al. (2) have reported that vasopressin (2 nM) elicits a transient increase in [32P]P04-labeled phosphatidate. Thomas et al. (3) have reported a similar increase in phosphatidate labeling following stimulation by vasopressin (10 nM). These workers found that the increase in ["PI phosphatidate coincided with a small early increase of [3H] arachidonyl-diacylglycerol labeling, but preceded the bulk of diacylglycerol labeling. This finding suggests that phosphatidate may be formed by a mechanism different from that involving phospholipase C and diacylglycerol kinase. Hokin-Neaverson and her co-workers (4) have suggested that a phospholipase D might be responsible for inositol production from phosphatidylinositol in response to acetylcholine in mouse pancreas. Cockcroft (5) has shown that, in neutrophils, f-met-leu-phe elicits a 6-fold increase in phosphatidate formation as measured by phosphate analysis. When neutrophils were labeled with ["P]PO,, the phosphatidate formed was found to have a much lower specific activity than that of the yPO4 of ATP. Cockcroft has taken this as evidence of either a direct formation of phosphatidate via phospholipase D or of a partial reversal of the CDP-diacylglycerol synthetase reaction. Phospholipase D has been widely studied in plants but was once believed not to occur in animals. Kanfer and his coworkers (6, 7) have characterized a phospholipase D from rat brain. This integral membrane protein will not hydrolyze exogenous substrates under most conditions, which probably explains why earlier workers did not observe it. Phospholipase D activity has also been observed in preparations from a variety of rat tissues including liver (7). With the exception of the work of Cockcroft and of Hokin-Neaverson, no studies have linked phospholipase D activity to hormonal action. In this report, we show that in hepatocytes, Ca2+-mobilizing hormones elicit an increase of phosphatidate mass as measured by a sensitive new procedure. This increase in phosphatidate precedes the hormone-elicited increase in diacylglycerol. Experiments with rat liver plasma membranes indicate that this increase in phosphatidate does not require ATP and proceeds via a phospholipase D mechanism that is stimulated by a guanine nucleotide-binding protein.
[T-~'P]ATP was a gift from Dr. C. Schworer of this department. R59 022 was a gift from Dr. D. de Chaffoy, Janssen Pharmaceutica, Beerse, Belgium.
One-ml samples were removed and extracted with 3.75 ml of CHC13/ MetOH 1:2 (9). Butylated hydroxytoluene (0.05%, w/v) was included in samples for fatty acid analysis. CHC13 (1.25 ml) and HzO (1.25 ml) were added with vigorous mixing. After 5 min, the samples were centrifuged (2000 X g for 5 min) and the aqueous layer was discarded. The CHCl3 layer was filtered through glass wool in a Pasteur pipette. The lipid extract was then stored at -20 "C under Nz until analysis.
Thin Layer Chromatography of Lipids-Phosphatidate was separated from other lipids by multidevelopment chromatography on 20 X 8-cm Silica Gel 60 F-254 plates (Merck). Samples (lipid from 3-4 mg of hepatocytes) and dipalmitoyl phosphatidate standard were applied in 20 pl of CHC13. The plates were developed with butanol/ acetic acid/HzO ( 6 l : l ) (10) to the top of the plate (8 cm). The plates were then dried for 40 min in a lyophilizer, and the top 1 cm of the plate containing the neutral lipids was removed. Thorough drying at all stages was necessary for optimal resolution and staining. The plates were turned 180 and then developed with CHC13/MetOH/ NH,OH (10:3:0.6) to the top of the plate. The plates were air-dried for 30 min, stained with Coomassie Brilliant Blue R250 (0.03% in 30% MetOH 100 mM NaCl) for 30 min and destained for 5 min in 30% MetOH 100 mM NaCl (11). The plates were air-dried and scanned with an LKB 2202 Ultroscan laser densitometer. The output was channelled to a Hewlett-Packard analogldigital converter and the data were stored in a Hewlett-Packard 3357 computer (Laboratory Automation System). Integration of thin layer chromatography bands was performed using the Hewlett-Packard CPLOT system. Standard curves of dipalmitoyl phosphatidate (0.25, 0.5, and 1 pg) were constructed for each plate. Recovery of added phosphatidate was 60% from hepatocyte extracts.
Fatty Acid Analysis-Phosphatide and diacylglycerol (from 70-90 mg of hepatocytes) were purified by thin layer chromatography as above in Nz-purged chambers. Plates were dried under Nz (-15 min between developments and before staining). The plates were lightly stained with Coomassie Blue, and the lipid-containing areas were scraped off and extracted with 2 ml of CHC13/MetOH (1:l) for diacylglycerol and with 2 ml of MetOH/l N HCl (201) for phosphatidate. The recoveries were about 70% for phosphatidate and 100% for diacylglycerol as assessed by fatty acid analysis of standards. The samples were evaporated under Nz, saponified, and the fatty acids were converted to 2-nitrophenylhydrazides by the method of Miwa et al. (12). The derivatized fatty acids were separated by HPLC using a Beckman Ultrasphere octyl column (5-pM particle size, 250 X 4.6 mm inner diameter), a Waters Associates M60000-A pump, a model U6K injector, and model 450 variable wavelength detector. The column was eluted isocratically with acetonitrile/HsO (85:15), and the nitrophenylhydrazides were detected by absorbance at 230 nm (12). Peaks were integrated using the Hewlett-Packard Laboratory Automation System as above. Contamination by plastics or by pencil lead was found to produce spurious peaks on the chromatogram.
Plusma Membrane Generation of Phosphatidate--Rat liver plasma membranes were prepared by the method of Prpic et al. (13) and resuspended at 1.5-2.5 mg of protein/ml in 50 mM HEPES, 1 mM EGTA, 200 pg/ml leupeptin, pH 7.5. The membrane suspension was added to an equal volume of 20 mM MgCl, and other additives where indicated. The mixtures were incubated for 10 min at 37 "C with shaking. Reactions were terminated by the addition of 1.5 ml of CHCWMetOH (1:2), followed by CHC13 (0.5 ml) and HzO (0.5 ml) (9). The samples were treated and assayed for phosphatidate as described above. Protein was measured by the bicinchoninic acid method (Pierce Chemical Co.) (14). Free CaZ+ in Ca2+-EGTA buffers was calculated by the COMICS program (15).
Membrane Phospholipid Determination-Membrane preparations were incubated as above and the extract equivalent to about 1 mg of membrane protein was applied to Silica Gel 60 F-254 plates. Phospholipids were separated using the two-dimensional system of Abdel-Latif (10). The plates were stained with Coomassie Blue and the areas containing the phospholipids were removed. A plate was run without sample to serve as a blank. Sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine were extracted with 2 ml of CHCl3/MetOH/HZ0, 5:5:1 plus 5 pl of NH,OH. Phosphatidylserine, phosphatidylinositol, and the phosphatidate were extracted with this solvent plus 20 pl of 1 N HC1. The samples were filtered and evaporated to dryness under N,. Perchloric acid (50 pl 11.6 N) and sulfuric acid (50 pl 5 N) were added, and the samples were heated at 200 "C for 60 min. 1 ml of water was added while the tubes were still hot, and the samples were assayed for PO, (16).
Choline and Phosphocholine Determination-Choline was assayed using choline kinase as described by Haubrich et al. (17). Bligh and Dyer aqueous supernatants were dried using a Speed Vac Concentrator (Savant) and 1 ml of H20 was added. Aliquots were treated with alkaline phosphatase (5 units/ml) for 30 min at 37 "C to convert phosphocholine to choline (18). The alkaline phosphatase was inactivated by boiling for 5 min before choline assay. Phosphocholine was taken as the difference between alkaline phosphatase-treated and untreated samples.
Determination of Alkyl and Alkenyl Phosphatidic Acid-"embranes were incubated with GTP+ as above. The phosphatidate was purified by thin layer chromatography as above and eluted with 4 ml of CHCl,/MetOH/H,O, 2:2:0.2. The phosphatidate was deacylated as described by Clarke and Dawson (19). Alkyl and alkenyl lipids are resistant to this deacylation procedure. The reaction products were separated on Silica Gel 60 with butanol/acetic acid/HzO, 6:l:l. Standards of lysophosphatidic acid and phosphatidic acid were also chromatographed. Lipids were visualized by a procedure that chars saturated and unsaturated lipids equally (20) and quantitated by densitometry.
Statistical Analysis-Values represent the mean k S.E. of three samples except where indicated in the figure legend.

RESULTS
Studies Using Intact Hepatocytes-The separation of phosphatidate from other lipids by thin layer chromatography is shown in Fig. 1, panel A. This multidevelopment chromatography was performed using the solvents from the two-dimensional system of Abdel-Latif et al. (10) and provides a clear resolution of phosphatidate from other lipids in one dimension. Other phospholipids are not well resolved by this system. Lipids were detected by the sensitive method of Nakamura and Handa (11) using Coomassie Blue staining. Separation of hepatocyte lipid extracts using this method followed by chromatography in a second dimension showed that the phosphatidate band consisted of 1 spot comigrating with phosphatidate standard (not shown). When 102 pg of phosphatidic acid purified by this chromatographic procedure was deacylated as under "Experimental Procedures," 1.4 pg of phosphatidate and less than 0.5 pg of lysophosphatidate remained. This sets an upper limit of approximately 2% for alkyl and alkenyl phosphatidate, in agreement with the low content of alkyl and alkenyl lipids found in liver (21). Fig. 1, panel B shows standard curves of dipalmitoyl phosphatidate, dilinoleoyl phosphatidate, and phosphatidate from egg lecithin measured by this method. The sensitivity of this method is such that we can routinely assay the phosphatidate content of extracts from 3-5 mg wet weight of hepatocytes.
The accumulation of phosphatidate was much more rapid than that of diacylglycerol in hepatocytes treated with vasopressin (IO-' M) as is shown in Fig. 2. Typically, both lipids

FIG. 1. Panel A, thin layer chromatography of hepatocyte extracts
and phosphatidate standard. Hepatocytes were exposed to albuminsaline (control) or vasopressin (lo-' M) for 5 min. Samples were processed and chromatographed, and dipalmitoyl phosphatidate (0.5 pg) standard was chromatographed as under "Experimental Procedures." Panel B, standard curve of phosphatidic acids (PA). The indicated amounts of dipalmitoyl phosphatidic acid, dilinoleoyl phosphatidic acid, and phosphatidic acid from egg yolk lecithin in 20 pl of CHCI, were applied to the thin layer plate which was developed, stained, and integrated as under "Experimental Procedures." The units are arbitrary densitometer units. increased 2-to 3-fold after incubation for 5 min with this vasopressin concentration. The control value for phosphatidate was 71 k 11 ng/mg wet weight (mean k S.E.; nine separate experiments). The time course and levels of diacylglycerol accumulation were very similar to those found in our earlier study using an HPLC method (22).
As shown in Table I, hepatocyte phosphatidate levels were increased by a variety of agents previously shown to mobilize Ca2+ (23). Vasopressin and ATP elicited a %fold increase in phosphatidate levels; angiotensin 11, epinephrine, epidermal growth factor, and ADP were slightly less efficacious. The calcium ionophore A23187 -M) also raised phosphatidate levels. Phosphatidate accumulation was stimulated by vasopressin in a concentration-dependent manner with an EC, of 1.4 nM similar to that previously found for diacylglycerol accumulation (1.2 nM) (22). These concentrations are higher than that needed for Ca2+ mobilization and phosphorylase activation (EC50 = 90 pM) (23). The depletion of hepatocyte calcium by a 15-min incubation with 5 mM EGTA reduced vasopressin-elicited phosphatidate accumulation by 50%, but had no effect on phosphatidate levels in control cells. Phorbol myristate acetate caused a slow increase in phosphatidate levels with a 1.6-fold stimulation at 10 min.
Since various hormone receptors have been shown to couple to phospholipases through guanine nucleotide-binding proteins, we incubated hepatocytes with toxins that modify certain of these coupling proteins (24,25). Cholera toxin (10 pg/ ml, 30 min) or pertussis toxin (2.5 pg/ml, 90 min) had no effect on phosphatidate levels in either control or vasopressintreated cells (data not shown).
We next investigated the source of the phosphatidate elicited by vasopressin. De mu0 synthesis of phosphatidate from glycerol is a major pathway in triglyceride synthesis (26). When hepatocytes were incubated with [3H]glycerol for 30 s prior to addition of vasopressin M), the label was incorporated rapidly into phosphatidate first and then into diacylglycerol, consistent with the de m u 0 pathway (Fig. 3) (26). Vasopressin had no effect on this labeling. In another experiment, the addition of 0.5 mM palmitate-bovine serum albu- min did not affect phosphatidate levels after vasopressin challenge (data not shown). These data suggest that vasopressin does not increase the de m u 0 synthesis of phosphatidate or diacylglycerol in the period of time (5 min) examined in these experiments.
Although phosphatidate accumulation preceded that of diacylglycerol (Fig. 2), phosphatidate could be derived from a rapidly phosphorylated pool of diacylglycerol. In this case we might expect to see a rapid change in the fatty acid composition of diacylglycerol as it is formed from a phospholipid substrate by phospholipase C. This change in diacylglycerol fatty acid composition should precede that of phosphatidate, much as in a radioactive labeling experiment. The fatty acid composition of diacylglycerol did change in response to vasopressin (Fig. 4 and Ref. 22), with an increase in arachidonate and stearate and a parallel decline in palmitate and linoleate. Phosphatidic acid had a different fatty acid composition under basal conditions but underwent a similar change in fatty acid composition after vasopressin with arachidonate and stearate increasing rapidly and palmitate and linoleate declining (Fig.  4). The change in phosphatidate fatty acid composition preceded that in diacylglycerol composition suggesting that the phosphatidate was not derived from diacylglycerol. Diacylglycerol mass at 5 min was 1.9 X control; phosphatidate mass at 5 min was 2.3 X control as estimated by total fatty acid recovery. Using these values, the fatty acid composition of the lipid produced in response to vasopressin can be calculated. Vasopressin-elicited diacylglycerol (at 5 min) had the following fatty acid composition (mol %): 22:6, 4.1%; 20:4, 29.5%; 182, 15.7%; 16:0, 18 Studies Using Isolated Plasma Membranes-To assess the mechanism of phosphatidate accumulation, we incubated washed isolated liver plasma membranes with varying amounts of GTPyS. This nucleotide has been used widely to activate guanine nucleotide-binding proteins and alter membrane processes in the absence of hormone. GTPyS stimulated phosphatidate formation by the plasma membranes (Fig.  5). High concentrations of GTPyS (10 PM) caused a 5.7-fold increase in membrane phosphatidate; half-maximal stimulation was seen at 1.5 pM GTPyS. GDPBS (250 WM) was without effect. It should be noted that this experiment was performed without added ATP. At high concentrations of GTPyS (20 PM), ATP had no effect on the accumulation of phosphatidate (data not shown) which argues against a role for diacylglycerol kinase in phosphatidate formation in this system. When plasma membranes were incubated with [y-32P]ATP there was no incorporation of 32P into membrane phosphatidate under conditions where 4.9 nmol of phosphatidate was formed in response to GTPyS at 10 FM (Table 11). The time course of membrane phosphatidate formation is shown in Fig. 6. After an initial lag, phosphatidate formation proceeded for at least 10 min. It is very unlikely that sufficient carryover of ATP exists in the washed membrane preparation to phosphorylate 9 nmol of diacylglycerol to phosphatidate. In addition, in intact hepatocytes oleoyl-acetyl glycerol and dioctanoyl glycerol were not phosphorylated to a detectable extent (by mass) and the diacylglycerol kinase inhibitor (27)   intact hepatocytes (data not shown). These findings, when taken together, exclude a role for diacylglycerol kinase in phosphatidate formation by hepatic plasma membranes. An attractive alternative hypothesis is that hepatic plasmic membranes possess a phospholipase D activity.

Effect of incubation o f p h m u membranes with or without CTPyS on formation of f2P]phosphutidic acid
The effects of various Ca2+-mobilizing agents on membrane phosphatidate accumulation at a low concentration of GTPyS (0.3 p~) is shown in Table 111. Preliminary experiments with Ca2+-EGTA buffers showed that EGTA inhibited membrane phosphatidate accumulation by about 33%; this inhibition was reversed by Ca2+ at a calculated free concentration of 72 nM (data not shown). Most experiments were conducted with EGTA because the stimulation by GTPyS of the hydrolysis of inositol phospholipids by phospholipase C is totally inhibited under this condition (24). Hormone effects on phosphatidylcholine hydrolysis have been shown to require Ca2+ (31), and we included Ca2+ (free concentration = 192 nM) in experiments with hormones. At 0.3 p~ GTP-yS, only ATP and ADP increased phosphatidate formation significantly (Table  111). These agents mobilize Ca2+ and cause phosphatidate accumulation in isolated hepatocytes (Ref. 29 and Table I).
They are presumably acting through Pz-purinergic receptors, but ATP may also prevent the breakdown of GTPyS.
F-was found to be a powerful inhibitor of phosphatidate production in plasma membranes. Both control and GTP-ySstimulated phosphatidate accumulation were profoundly (about 90%) inhibited by 6.8 mM NaF (data not shown). In intact hepatocytes, 5 mM NaF plus 100 p M AICls did not cause phosphatidate accumulation at 5 min (not shown), in contrast to the elevation of diacylglycerol content and cystolic Caz+ seen under these conditions (30). It seems likely that Fhas a direct inhibitory action on the phospholipase D, but an inhibitory guanine nucleotide-binding protein has not been ruled out.
Potential phospholipid sources of phosphatidate were investigated by incubating plasma membranes with GTPyS (20 p~) in the absence of Ca2+ (Table IV). The major phospholipids were separated as described under "Experimental Procedures,'' ashed, and assayed for PO, content. Phosphatidate rose from 3.8 nmol/mg protein to 11.6 nmol/mg protein (Table IV). The only lipid that declined was phosphatidylcholine, which was reduced from 146.3 to 130.2 nmol/mg by the inclusion of GTPyS. This experiment does not rule out other sources for phosphatidate, but it should be observed that, in plasma membranes, phosphatidylserine and phosphatidylinositol are present in amounts only 4-and 2-fold greater, respectively, than phosphatidate and probably do not contribute to its formation.
To explore the effect of GTPyS on phosphatidylcholine breakdown, we measured the production of choline, phosphocholine, phosphatidate, and diacylglycerol by plasma membranes (Fig. 7). Both choline and phosphocholine were produced in response to GTPyS (Fig. 7A) in agreement with the findings of Irving and Exton (31), but choline was always increased to a larger extent than phosphocholine. Phospha-   tidate and diacylglycerol were also formed (Fig. 7B) suggesting that both phospholipase D and C are acting on phosphatidylcholine in this preparation. The larger mass of choline + phosphocholine found in response to GTPyS (19.2 nmol/mg) relative to phosphatidate + diacylglycerol (9.4 nmol/mg) may reflect breakdown of the lipid products under the conditions of the assay. We have observed production of monoacylgly-cero1 under these conditions, possibly formed by diacylglycerol lipase. The increment in choline + phosphocholine formed in this experiment (19.2 nmol/mg with GTPyS) is similar to the values found for phosphatidylcholine breakdown in response to GTPyS (16.1 nmol/mg, Table IV) in four other experiments.

DISCUSSION
Phosphatidate has been thought to function as part of the "phosphoinositide cycle" in which inositol phospholipids are hydrolyzed to diacylglycerol, which is then phosphorylated by diacylglycerol kinase (1). The phosphatidate formed is converted to CDP-diacylglycerol and ultimately back to inositol lipid. The evidence for this scheme rests almost entirely on radioisotopic labeling experiments and in uitro enzyme activities. The results we report here do not support the phospholipase C-diacylglycerol kinase pathway as a major source of phosphatidate in hormone-stimulated rat hepatocytes. We find that increases in phosphatidate mass and changes in phosphatidate fatty acid composition precede similar changes in diacylglycerol (Figs. 2 and 4). Furthermore, the fatty acid composition of phosphatidate and diacylglycerol formed in response to vasopressin more closely resembles that of phosphatidylcholine than that of inositol lipids ( Fig. 4 and text). Rat liver inositol lipids typically have a stearoyl-arachidonyl glycerol backbone with no other fatty acid comprising more than 5% of total fatty acids (32). Phosphatidylcholines, too, have a high proportion of stearate (25%) and arachidonate (25%) but also contain palmitate (22%) and linoleate (12%) (32). The de novo synthesis of phosphatidate and diacylglycerol from glycerol was not stimulated by vasopressin (Fig. 3). These findings raise the intriguing possibility that the Ca2+mobilizing hormones increased phosphatidate levels through a phospholipase D mechanism.
We have performed experiments with rat liver plasma membranes that strongly suggest that a phospholipase D activity is indeed present and is regulated by a guanine nucleotidebinding protein. GTPyS, a nonhydrolyzable GTP analogue, stimulated the formation of phosphatidate by washed plasma membranes; this formation did not require ATP (Fig. 5). In addition, when membranes were incubated with [32P]ATP and with GTPyS, no incorporation of 32P into phosphatidate was observed (Table 11). These experiments effectively excluded phosphorylation of diacylglycerol by diacylglycerol kinase in the formation of phosphatidate in isolated liver membranes.
Recently, Irving and Exton (31) reported an activity in rat liver plasma membranes that hydrolyzed phosphatidylcholine. Diacylglycerol, phosphocholine, and choline were formed in response to GTPyS, and the enzyme activity was characterized as a phospholipase C. The formation of phosphatidate under identical reaction conditions (reported here) suggests that phosphatidylcholine is also hydrolyzed by phospholipase D to form phosphatidate. Both activities do not require Ca2+ for GTPyS activation (in contrast to the hydrolysis of inositol lipids) (24), are stimulated by ATP and ADP when GTP+ is submaximal (Table 111), and are resistant to both cholera and pertussis toxins (31).
In addition both activities are stimulated by phorbol myristate acetate. When plasma membranes were incubated with GTPyS, phosphatidate increased and phosphatidylcholine decreased to a similar extent (Table  IV). When intact hepatocytes are labeled with [3H]choline, free choline is released in response to vasopressin.' Experiments with exogenous substrates are underway to assess the substrate specifity of the phospholipase D activity.
All of the Ca2+-mobilizing hormones tested stimulated phosphatidate accumulation in intact hepatocytes (Table I).
In contrast, only ATP and ADP were effective in isolated plasma membranes (Table 111). We have no explanation for the discrepancy at this time, but it should be noted that ATP and ADP are also superior to vasopressin as activators of hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis in liver plasma membranes (31).
The nature of the guanine nucleotide-binding protein(s) involved in activation of the phospholipase D activity is unknown. It appears that G, and Gi are not involved as cholera toxin, and pertussis toxin did not alter phosphatidate production. F-, which as been used widely to activate guanine nucleotide-binding proteins, inhibited phospholipase D activity. Whether this is a result of an inhibitory coupling protein or direct inhibition of phospholipase D is uncertain. Since Fproduces Ca2+ mobilization and diacylglycerol accumulation (30) without phosphatidate accumulation, it may be a valuable probe into the function of hormone-elicited phosphatidate.
The relationship of this phospholipase D activity to that found previously in rat liver and other tissues is unknown (6, 7). These activities may represent basal states of hormonestimulated activities. Similarly the function of the phosphatidate formed is obscure. We have found that incubation of hepatocytes with physiologically relevant concentrations of phosphatidate (from egg yolk lecithin, Sigma) or phospholipase D from Streptomyces chromofuscus produces Ca2+ influx and accumulation and increases cytosolic free Ca", in agreement with the findings of many others (33, 34). In addition, we find that phosphatidate inhibits glucagon stimulation of CAMP production (see Ref. 35) and activates phosphorylase through an undetermined mechani~m.~ The notion that phosphatidate functions as a second messenger is not new (33,34) but deserves re-examination in light of these and other recent findings (33, 36).