(Received for publication, February 12, 1997, and in revised form, April 28, 1997)
From the Environmental and Health Sciences Division,
Oak Ridge Associated Universities, Oak Ridge, Tennessee 37831-0117
and § Department of Biochemistry and Biophysics "F.
Cedrangolo," Second University of Naples,
Naples 80138, Italy
Acyl analogs of platelet-activating factor (PAF)
(1-acyl-2-acetyl-sn-glycero-3-phosphocholine,
acylacetyl -GPC) are the predominant products synthesized during
thrombin or ionophore A23187-mediated activation of endothelial cells.
However, the biosynthetic pathway responsible for the production of
acylacetyl-GPC is not well understood. In the present investigation, we
have demonstrated that the acyl analogs of PAF are also the major
products from calf pulmonary artery endothelial cells in response to a
time-dependent stimulation of ATP (103
M), bradykinin (10
8 M), or
ionophore A23187 (2 µM). In addition, we have found that the CoA-independent PAF:acyllyso-GPC transacetylase recently identified by us is concurrently and transiently induced with maximal 4-fold enhancement at 5 min and returned to near basal level by 10 min treatment of endothelial cells with ATP. Acid phosphatase reduces the
increased PAF:acyllyso-GPC transacetylase activity from the homogenates
of ATP-activated endothelial cells. Reduced PAF:acyllyso-GPC transacetylase activity can be restored by incubating the acid phosphatase-treated homogenates with ATP (5 mM) and
Mg2+ (10 mM). Furthermore, okadaic acid, a
protein phosphatase 1 and 2A inhibitor, incubated with endothelial
cells in a dose-dependent manner (1-100 nM)
for 10-min potentiates and sustained the stimulation of
PAF:acyllyso-GPC transacetylase activity by ATP. On the other hand,
genistein, tyrphostin-25 (inhibitors of tyrosine-specific protein
kinase), and calphostin C (an inhibitor of protein kinase C) block the
activation of PAF:acyllyso-GPC transacetylase by ATP. These results are
consistent with the notion that ATP regulates the transacetylase
activity by reversible activation and inactivation via the
phosphorylation and dephosphorylation cycle. ATP also augments the
activities of alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase.
However, the activation of the acetyltransferases precedes that of the
transacetylase with peak activation occurring at 1-2 min of the ATP
treatment. In addition, sodium vanadate, also an inhibitor of protein
phosphatase, stimulates the increase in the incorporation of
[3H]acetate into acyl[3H]acetyl-GPC of the
ATP-treated endothelial cells. Collectively, our data show that both
acetyltransferases and transacetylase participate in and
contribute to the biosynthesis of acyl analogs of PAF in a coordinate
fashion in endothelial cells.
Platelet-activating factor (PAF)1 is a potent lipid mediator with diverse pathophysiological responses such as inflammation, allergic reactions, and reproduction (see reviews Refs. 1-3). Different cell types from various species activated by specific stimuli are able to produce PAF (4). The chemical structure of PAF is originally elucidated as 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, 5-7). However, it recently becomes apparent that acylacetyl-GPC can be the predominant product in certain cell types. For instance, the major compound produced by human umbilical vein endothelial cells activated with thrombin and ionophore A23187 (8-11) or bovine pulmonary artery endothelial cells stimulated by ionophore A23187 (12) is acylacetyl-GPC. Also, depending on the agonist used, either alkylacetyl-GPC or acylacetyl-GPC can be synthesized within the same cell type. For example, the human basophils generate mainly acylacetyl-GPC in response to anti-IgE and mostly alkylacetyl-GPC in response to ionophore A23187 (13).
Although the biological activities of acylacetyl-GPC are >500-fold
less potent than alkylacetyl-GPC in lowering the blood pressure of
spontaneous hypertensive rats, in releasing serotonin from rabbit
platelets (14), in increasing the intracellular Ca2+
concentrations, and in activating microtubule-associated protein-2 kinase activity (15), however, acylacetyl-GPC can act as a specific noncompetitive inhibitor of alkylacetyl-GPC-induced activation of the
human neutrophils and leukotriene C4 release from the human leukocytes (10, 16). Furthermore, 1-acyl analogs of PAF at nM concentrations can prime polymorphonuclear leukocytes
for enhanced O2 production after
stimulation with fMet-Leu-Phe or human recombinant C5a (17).
Additionally, acylacetyl-GPC has the ability to decrease the
susceptibility of the low density lipoprotein particles to oxidative
modification mediated by copper ions, monocytes, or endothelial cells,
whereas PAF has no effect (18).
The pathways leading to the accumulation of acylacetyl-GPC are not completely understood. It is suggested that the remodeling pathway (3) is responsible for the biosynthesis of alkylacetyl-GPC/acylacetyl-GPC during short term stimulation by agonists in endothelial cells (19). The first step of the remodeling pathway involved the conversion of alkylarachidonoyl-GPC/acylarachidonoyl-GPC to alkyllyso-GPC/acyllyso-GPC by a putative phospholipase A2 or/and a CoA-independent transacylase (20-22). The alkyllyso-GPC/acyllyso-GPC formed is subsequently acetylated by the alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase (19, 23, 24). Increases in intracellular calcium concentration and protein kinase C activate the phospholipase A2 that is involved in the synthesis of PAF (25). The alkyllyso-GPC:acetyl-CoA acetyltransferase is regulated by phosphorylation and dephosphorylation (26-29).
Alkylacetyl-GPC and acylacetyl-GPC have different catabolic routes. In most cell types, alkylacetyl-GPC is hydrolyzed by acetylhydrolase to form alkyllyso-GPC and then the alkyllyso-GPC is reacylated to produce alkylacyl-GPC (see reviews herein). On the other hand, the major metabolic pathway for the acylacetyl-GPC is the removal of acyl group at the sn-1 position by a yet to be characterized 1-acylhydrolase (30). The acetyl-GPC formed from this reaction is further degraded to glycerophosphocholine, choline, or phosphocholine (30). However, it is reported that lysophospholipase can also catalyze the stoichiometric conversion of acylacetyl-GPC to acetyl-GPC (31, 32). In addition, the activity of lysophospholipase may control the level of acyl analogs of PAF, because peritoneal macrophages that preferentially synthesize acyl analogs of PAF contain only one-sixth of the lysophospholipase activity in comparison to that of alveolar macrophages which primarily make PAF (33). Besides, the amounts of acylacetyl-GPC produced within a specific cell type can be increased by blocking the phenylmethylsulfonyl fluoride-sensitive phospholipase A1 or lysophospholipase activity (31, 34, 35).
We have previously identified a novel CoA-independent and PAF-dependent transacetylase that transfers the acetate group from PAF to a variety of lysophospholipids; acyllyso-GPC is the most active acceptor that converts to acylacetyl-GPC by the transacetylase (36). In the present studies, we investigate the possibility that PAF-dependent transacetylase may be involved in the biosynthesis of acyl analogs of PAF. Our results demonstrate that the PAF:acyllyso-GPC transacetylase is induced severalfold during agonist-coupled activation of the endothelial cells and is most likely to be regulated via covalent modification of the enzyme through phosphorylation and dephosphorylation. Based on the temporal relationships that exist between the synthesis of acylacetyl-GPC and induction of the transacetylase activity in stimulus-activated cells, our findings suggest that the PAF-dependent transacetylase participates in the production and controls the level of acylacetyl-GPC in endothelial cells.
1-Hexadecyl-2-[3H]acetyl-GPC (7.1 Ci/mmol), [3H]acetate (1.9 Ci/mmol), and
[3H]acetyl-CoA (1.54 Ci/mmol) were purchased from NEN
Life Science Products. Hexadecylacetyl-GPC, hexadecyllyso-GPC,
palmitoyllyso-GPC, acetyl-CoA, phospholipase C type V from
Bacillus cereus, acid phosphatase from potato, ionophore
A23187, thrombin, bradykinin, ATP, genistein, and tyrphostin 25 were
the products of Sigma. Sodium salt of okadaic acid, calphostin C,
KT5720, H-89 dihydrochloride, and
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) were
from Calbiochem. Alkylacetyl-GPC and oleoylacetyl-GPC were obtained from Avanti Polar Lipids Inc. All cell culture reagents were from Life
Technologies, Inc.
Calf pulmonary artery endothelial cells (CCL-209) were obtained from the American Type Culture Collection and grown in Eagle's minimum essential medium with 20% fetal bovine serum. Cells were cultured in 75-cm2 flasks, and only the confluent monolayers between passages 20 and 25 were used for experiments. Unless indicated otherwise, the cells were washed twice with 10 ml of Hank's balanced salt solutions (HBSS) before starting the experiments.
Determination of the Rate of Incorporation of [3H]Acetate into Radyl[3H]acetyl-GPC and Its SubclassesWashed monolayers of calf pulmonary artery
endothelial cells were incubated with 25 µCi of
[3H]acetate in the presence of 10 ml of HBSS, 10 mM Hepes (pH 7.4), and with or without agonist, thrombin (2 units/ml), bradykinin (108 M), ATP
(10
3 M), or ionophore A23187 (2 µM) at 37 °C for various times as indicated in the
figures. At the end of incubations, the media were removed, and the
cells were rinsed twice with 5 ml of HBSS, 10 mM Hepes (pH
7.4) before scraping into 3 ml of methanol. The cellular lipids were
extracted by the method of Bligh and Dyer (37) except that 2% of
acetic acid was included in the methanol. The thin layer
chromatographic solvent system of chloroform/methanol/acetic acid/H2O (50:25:8:6, v/v/v/v) was used to isolate the
radyl[3H]acetyl-GPC fraction. Radioactivity in lipid
fractions separated by thin layer chromatography (TLC) was determined
by area or zonal scraping of the silica gel into vials for liquid
scintillation counting.
The purity of the isolated radyl[3H]acetyl-GPC was further confirmed by using a TLC solvent system of chloroform/methanol/concentrated NH4OH/H2O (60:35:8:2.3; v/v/v/v). The purified samples of radyl[3H]acetyl-GPC were then treated with phospholipase C to form radyl[3H]acetylglycerols that were derivatized with benzoic anhydride. The benzoate derivatives of radyl[3H]acetylglycerols were separated into acylacetyl-, alkylacetyl-, and alk-1-enylacetylglycerol benzoates on silica G TLC plates as described (38). The radioactivities in each subclass were determined by liquid scintillation spectrometry.
Enzyme AssaysPAF:acyllyso-GPC transacetylase and PAF:lysoplasmalogen transacetylase were determined as reported earlier by us (36) with minor modification. Briefly, standard incubations consisted of 50 µM PAF (0.5 µCi in 50 µl of 0.1% bovine serum albumin/saline), 300 µM palmitoyllyso-GPC or lysoplasmalogen (in 50 µl of 0.1% bovine serum albumin/saline), 5 mM EDTA, 1 mM sodium acetate, 100 mM Tris-HCl (pH 7.4), and 100 µg of homogenate protein in a final volume of 0.50 ml. The incubations were carried out at 37 °C for 15 and 30 min to ensure that the enzyme activity is measured under zero order kinetic. The cell homogenates were prepared by scraping agonist-treated or nontreated cells from the surface of the flasks into 2 ml of homogenizing buffer (0.25 M sucrose, 100 mM Tris-HCl (pH 7.3), 1 µg/ml leupeptin, 1 mM dithiothreitol, 50 mM NaF). The cell suspensions were homogenized by using an ultrasonic cell disruptor (MicrosonTM) at 30% power output for 15 s × 5 times. The extracted lipid product (>95% as radyl[3H]acetyl-GPC) was treated by phospholipase C. The resulting radyl[3H]acetylglycerols were converted to their respective benzoate derivatives and analyzed by a combination of TLC and liquid scintillation counting as described in the above section.
Alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase and PAF acetylhydrolase activities were measured according to the methods we previously described (39, 40) except that the cell homogenates were used as the enzyme sources in most of the experiments. The protein content of the enzyme preparations was determined by the method of Lowry et al. (41).
To study the possible
involvement of PAF-dependent transacetylase in the
biosynthesis of acylacetyl-GPC of endothelial cells, it was necessary
to establish in our laboratory that the stimulus-treated endothelial
cells produced acyl analogs of PAF as the predominant product. We chose
bovine pulmonary artery endothelial cells as a model system for the
convenience. Data in Fig. 1A showed that the
incorporation of [3H]acetate into
radyl[3H]acetyl-GPC was increased in a
time-dependent fashion with noticeable rise at 1 min and
approaching plateau at 10 min, when the endothelial cells were
incubated with ATP (103 M). Treatment of
endothelial cells with bradykinin (10
8 M) or
ionophore A23187 (2 µM) yielded similar time course
responses (data not shown). However, the increase in radylacetyl-GPC
synthesis was minimal in thrombin-treated (2 units/ml) endothelial
cells (data not shown). These results were consistent with the findings demonstrated by Garcia et al. (12). They showed that 1 µM bradykinin or 10 µM ionophore A23187
enhanced the [3H]acetate incorporation into
radylacetyl-GPC in bovine pulmonary artery endothelial cells. Besides,
our data were similar to that reported by Whatley et al.
(42) in that cultured endothelial cells from various bovine blood
vessels responded to ATP, bradykinin, or ionophore A23187 but not to
thrombin stimulation in generating an increase in PAF production.
Garcia et al. (12) described that when calf pulmonary artery endothelial cells were treated with ionophore A23187 (10 µM), the primary product formed under the conditions was acyl analogs of PAF rather than PAF. We found that acyl analogs of PAF were also the major product when calf pulmonary artery endothelial cells were stimulated with ATP (approximately 85% as acylacetyl-GPC) (Fig. 1B), bradykinin (about 67% as acylacetyl-GPC), or ionophore A23187 (nearly over 80% as acylacetyl-GPC) (data not shown). The kinetics of the time-dependent incorporation of [3H]acetate into the subclasses of radylacetyl-GPC were similar among the three agonists tested. On the other hand, the kinetic of the time-dependent incorporation of [3H]acetate into the subclasses of radylacetyl-GPC treated with thrombin was different from that treated with ATP, bradykinin, or ionophore A23187. It is possible that the kinetics of the time-dependent incorporation of [3H]acetate in thrombin-treated endothelial cells reflected the turnover of the endogenous subclasses of radylacetyl-GPC, since bovine pulmonary artery endothelial cells did not respond to thrombin activation.
Induction of PAF:Acyllyso-GPC Transacetylase by ATP and Its MechanismSince ATP caused the highest stimulation on the
incorporation of [3H]acetate into
radyl[3H]acetyl-GPC among the physiological agonists we
tested, ATP was chosen as the agonist in the subsequent experiments.
Fig. 2 depicted that ATP induced a rapid and transient
increase in PAF:acyllyso-GPC transacetylase activity with maximal
activation at 5 min. The activity of PAF:acyllyso-GPC transacetylase
returned to near basal levels after 10 min of ATP treatment. The
increase in transacetylase activity at 5 min incubation of ATP with
endothelial cells was about 4-fold over that in the untreated
cells.
It was unlikely that the increase in PAF:acyllyso-GPC transacetylase
activity by ATP was due to de novo protein synthesis, because the induction of the transacetylase activity by ATP was a rapid
process (within 2 min). Therefore, we investigated the possibility that
the activity of PAF:acyllyso-GPC transacetylase may be regulated by ATP
through covalent modifications, specifically through phosphorylation
and dephosphorylation. When the homogenates of ATP-stimulated
endothelial cells were treated with an acid phosphatase from potato for
15 min, a decrease of 61% in transacetylase activity was observed
(Fig. 3, 2nd column versus 1st column). The
percent of decrease in transacetylase activity depended on the
concentrations of acid phosphatase used and the lengths of the
preincubation times of the homogenates with acid phosphatase (data not
shown). Furthermore, preincubation of the homogenates with boiled acid
phosphatase (at 100 °C for 10 min) had no effect on the
transacetylase activity (Fig. 3, 3rd column versus 1st column). On the other hand, addition of ATP (5 mM)
plus Mg2+ (10 mM) to the homogenates of
ATP-treated endothelial cells (Fig. 3, 4th column versus 1st
column) or to the homogenates of ATP-stimulated endothelial cells
that were pretreated with acid phosphatase (Fig. 3, 5th column
versus 2nd column) could further potentiate the transacetylase
activity. These data from in vitro experiments suggested
that the transacetylase activity is activated/inactivated through
phosphorylation/dephosphorylation.
To further substantiate the fact that the transacetylase activity is regulated through phosphorylation and dephosphorylation, several following experiments using whole cell systems were performed.
Endothelial cells were preincubated with various concentrations of
okadaic acid (1 to 100 nM) for 30 min and then incubated with ATP (103 M) for an additional 10 min.
The activity of transacetylase was closed to near basal levels when
endothelial cells were treated with ATP for 10 min (see Fig. 2).
Okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A (43),
induced a 2-fold increase in PAF:acyllyso-GPC transacetylase activity
over that of the 10-min ATP-treated control (Fig. 4).
Optimal concentrations of okadaic acid that potentiated the
ATP-augmented transacetylase activity occurred between 10 and 100 nM (Fig. 4). Therefore, these results were consistent with
the notion that by blocking the dephosphorylation of the proteins with
okadaic acid, the transacetylase activity is amplified. A different
protein phosphatase inhibitor, sodium orthovanadate (1 mM),
likewise sustained and prolonged the activation of transacetylase in
calf pulmonary artery endothelial cells treated with ATP for 10 min
(data not shown).
When endothelial cells were preincubated with a selective inhibitor of
tyrosine-specific protein kinase, tyrphostin-25 (44, 45) for 10 min,
and incubated with ATP for additional 5 min, tyrphostin-25 blocked the
induction of PAF:acyllyso-GPC transacetylase activity by ATP in a
dose-dependent manner with close to total inhibition at 50 µM (Fig. 5). Similar results were obtained
when another inhibitor of tyrosine-specific protein kinase, genistein (46), was used (data not shown). These data provide added support that
PAF:acyllyso-GPC transacetylase activity is controlled by reversible
activation and inactivation through phosphorylation and
dephosphorylation.
To gain insight as to which of the protein kinase(s) may be involved in the activation of PAF:acyllyso-GPC transacetylase besides tyrosine-specific protein kinase, we tested the effects of inhibitors for three different kinds of protein kinases, namely protein kinase C, protein kinase A, and casein kinase II, on both PAF:lysoplasmalogen transacetylase and PAF:acyllyso-GPC transacetylase activities (Table I). Based on the results we obtained previously from the mixed substrate experiments, the transfer of the acetate group from PAF to either lysoplasmalogen or acyllyso-GPC is catalyzed by a single enzyme (47). Therefore, it is not surprising that the effects of various protein kinase inhibitors are similar for both PAF:lysoplasmalogen transacetylase and PAF:acyllyso-GPC transacetylase activities (Table I). Only calphostin C, a specific inhibitor for protein kinase C, exerted inhibitory effect on both transacetylase activities, whereas the inhibitors for protein kinase A (KT 5720 or H-89) and casein kinase II (DRB) did not block neither ATP-induced transacetylase activities. Thus, our data suggest that tyrosine protein kinase and protein kinase C are directly or indirectly involved in the activation of the transacetylase activity through protein phosphorylation.
|
To determine which of the amino acids (i.e. tyrosine, serine, and/or threonine) in the transacetylase is/are phosphorylated by the protein kinase(s), we carried out several experiments similar to that described by Whitman et al. (48) to show the phosphorylation of tyrosine in the phosphatidylinositol 3-kinase. When ATP-treated or control untreated endothelial cell lysates were prepared and immunoprecipitated with polyclonal antibody against either phosphotyrosine, phosphoserine, or phosphothreonine (Zymed Laboratories Inc.) using a combined method specified by Whitman et al. (48), Domin et al. (49), and manufacturer's instructions, our preliminary data showed that the PAF:lysoplasmalogen transacetylase activity in ATP-treated endothelial cell lysates was enriched in part in the immunocomplex precipitated by anti-phosphotyrosine antibody in comparison to that of the untreated control endothelial cell lysates. Similarly, the PAF:lysoplasmalogen transacetylase activity was higher in anti-phosphotyrosine-treated immunoprecipitate of the ATP-stimulated endothelial cell lysates than that in antigen affinity purified IgG antibody precipitated immunocomplex of the same cell lysates (a kind gift from Dr. Steve Kennel, Oak Ridge National Laboratory). These results suggest that PAF:lysoplasmalogen transacetylase is phosphorylated by a tyrosine kinase. However, we are currently attempting to purify the transacetylase and prepare the antibody against the purified protein to conclusively address the questions concerning the specific protein kinase(s) and the phosphorylation site of the amino acids involved in the activation of the transacetylase.
A Protein Phosphatase Inhibitor, Sodium Vanadate, Potentiates the Incorporation of [3H]Acetate into Radyl[3H]acetyl-GPC in ATP-stimulated Endothelial CellsIt is reasonable to assume that if PAF:acyllyso-GPC transacetylase activity is reversibly activated/inactivated through phosphorylation/dephosphorylation and is involved in the synthesis of acyl analogs of PAF, then a protein phosphatase inhibitor that potentiates the activation of transacetylase activity by ATP (see Fig. 4 and above) should also augment the incorporation of [3H]acetate into acylacetyl-GPC. Results in Table II indeed showed that the incorporation of [3H]acetate into radyl[3H]acetyl-GPC was increased severalfold by vanadate in both ATP-treated and untreated endothelial cells. However, vanadate had no effect on the percent of [3H]acetate incorporated into each subclass of radyl[3H]acetyl-GPC (data not shown).
|
To determine the role of
alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferases in the
synthesis of acylacetyl-GPC, we measured the Km and
Vmax values, and the time course of induction by
ATP for the acetyltransferases in the membrane fraction of the calf
pulmonary artery endothelial cells. The Vmax was
only slightly less when palmitoyllyso-GPC was the substrate in
comparison with that when hexadecyllyso-GPC was the substrate (7.6 nmol/min·mg protein versus 9.2 nmol/min·mg protein).
However, the acetyltransferase had a slightly higher substrate affinity
for the palmitoyllyso-GPC than that of hexadecyllyso-GPC (8.3 versus 9.1 µM). Additionally, both
alkyllyso-GPC:acetyl-CoA acetyltransferase and acyllyso-GPC:acetyl-CoA acetyltransferase in the homogenates of calf pulmonary artery endothelial cells responded to a time-dependent ATP
activation with maximal increase within 1-2 min after the addition of
ATP (Fig. 6). The stimulation in activity by ATP was
higher for acyllyso-GPC:acetyl-CoA acetyltransferase (190% over basal
at 1 min) than that for alkyllyso-GPC:acetyl-CoA acetyltransferase
(147% over basal at 2 min). Because the induction of
alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase by ATP (peak
increase in activity occurred between 1 and 2 min) preceded that of
PAF:acyllyso-GPC transacetylase (maximal increase in activity occurred
at 5 min) (also see Fig. 3 versus Fig. 6), our data implied that both acetyltransferases and transacetylase participated in the
synthesis of acylacetyl-GPC. Holland et al. (19) have shown that the alkyllyso-GPC:acetyl-CoA acetyltransferase was transiently induced in thrombin-exposed human endothelial cells and in
bradykinin-treated bovine pulmonary artery endothelial cells and
concluded that the synthesis of PAF in intact cells was regulated by
the alkyllyso-GPC:acetyl-CoA acetyltransferase. Furthermore, they found
that the alkyllyso-GPC was a 2-fold superior substrate than
acyllyso-GPC in both control and thrombin-activated human endothelial
cells (19).
Effect of ATP on the PAF Acetylhydrolase Activity in Endothelial Cells
Since the incubations of [3H]acetate and ATP with endothelial cells were short durations (1-10 min), the rates of incorporation of [3H]acetate into radyl[3H]acetyl-GPC that we measured should reflect mostly as the rates of synthesis of radyl[3H]acetyl-GPC. Nevertheless, we did assess the possible effects ATP may have on the PAF acetylhydrolase activity. Results (data not shown) indicated that ATP had minimal effect on the PAF acetylhydrolase activity.
We have established that ATP (103 M),
bradykinin (10
8 M), and ionophore A23187 (2 µM) stimulate the incorporation of
[3H]acetate into radyl[3H]acetyl-GPC of
calf pulmonary artery endothelial cells in a time-dependent manner (see Fig. 1A). In addition, the majority of the
radyl[3H]acetyl-GPC produced under these conditions is
acylacetyl-GPC (67-87%) (see Fig. 1B). It is known that
the synthesis of PAF is increased in human umbilical vein endothelial
cells in response to interleukin-1 (50), tumor necrosis factor (51),
histamine, bradykinin, ATP, peptide leukotrienes LTD4 and
LTC4, thrombin (52-54), and calcium ionophore A23187 (55)
and in bovine blood vessel endothelial cells in response to ATP,
bradykinin, angiotensin II, and ionophore A23187 (12, 42). However,
only the subclasses of radylacetyl-GPC formed in thrombin- and
ionophore A23187-stimulated human umbilical vein endothelial cells and
ionophore A23187-treated bovine pulmonary artery endothelial cells are
analyzed and found to consist mostly as acylacetyl-GPC (8, 9, 11-13).
Our results suggest that acylacetyl-GPC also is the major product in
ATP- and bradykinin-activated endothelial cells.
The intriguing observations of our work are the induction of PAF:acyllyso-GPC transacetylase activity (Fig. 2) and the modulation of PAF:acyllyso-GPC transacetylase activity by ATP through covalent modification of phosphorylation and dephosphorylation (Figs. 3, 4, 5). In addition, the maximal activation of PAF:acyllyso-GPC transacetylase activity by ATP (occurred at 5 min, Fig. 2) lags behind that of alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase activities (maximum at 1-2 min, Fig. 6). These results indicate that the activities of acetyltransferases and transacetylase are stimulated by ATP in a coordinated fashion. It is reported that PAF stimulates the alkyllyso-GPC:acetyl-CoA acetyltransferase activity in neutrophils (56). Nevertheless, PAF:acyllyso-GPC transacetylase is not induced or augmented by incubating the endothelial cells with PAF (1 µM) for 3 min or PAF (1 µM) plus ATP for 5 min, respectively (data not shown). Furthermore, the increase of PAF:acyllyso-GPC transacetylase was returned to near basal level after 10 min treatment of the endothelial cells with ATP (Fig. 2), whereas the incorporation of [3H]acetate into acyl[3H]acetyl-GPC remained elevated at 10 min (Fig. 1). These data suggest the possibility that other catabolic enzymes (i.e. 1-acylhydrolase and/or lysophospholipase) may be involved in regulating the level of acyl analogs of PAF; however, these issues are beyond the scope of the present investigation.
It is logical that the stimulation of alkyllyso-GPC:acetyl-CoA acetyltransferase activity by ATP precedes prior to the increase in the transacetylase activity, because alkyllyso-GPC:acetyl-CoA acetyltransferase supplies PAF which is one of the co-substrates for the PAF:acyllyso-GPC transacetylase. Also, Prescott et al. (57) have shown that essentially all of the newly synthesized PAF remains associated with the endothelial cells so that PAF:acyllyso-GPC transacetylase will have easy access to PAF.
Our in vitro experiments showed that the PAF:acyllyso-GPC transacetylase activity was reduced by treatment of the homogenates of the ATP-stimulated endothelial cells with acid phosphatase. Reduction in PAF:acyllyso-GPC transacetylase activity could be reversed by incubating the acid phosphatase-treated and reconstituted (acid phosphatase-treated membrane fraction plus acid phosphatase-untreated soluble fraction) homogenates with ATP (Fig. 3). These results indicate that PAF:acyllyso-GPC transacetylase activity is activated and inactivated through reversible phosphorylation and dephosphorylation. It is not clear at present which types of protein kinase(s) and phosphatase(s) are involved in the direct phosphorylation and dephosphorylation of the transacetylase. Since both tyrosine kinase inhibitors (genistein and tyrphostin-25 (Fig. 5)) and protein kinase C inhibitor (calphostin C (Table I)) inhibit the stimulation of PAF:acyllyso-GPC transacetylase by ATP, these results suggest that the phosphorylation of the tyrosine residue(s), and/or serine and threonine residue(s) of the proteins, is involved in the signal transduction transmitted by ATP for the activation of the transacetylase activity. However, we cannot totally rule out the possibility that phosphorylation/dephosphorylation of another protein is affecting the transacetylase activity. Investigations are currently underway in our laboratory to determine the type(s) of protein kinase(s) and phosphatase(s) that mediate the direct activation and inactivation of the transacetylase activity.
We and others (19, 23, 24, 58) have shown that alkyllyso-GPC:acetyl-CoA acetyltransferase can use acyllyso-GPC as a substrate, but at a reduced rate (50%), to generate acylacetyl-GPC. This is consistent with the findings reported in this work except that the differences in Vmax between alkyllyso-GPC:acetyl-CoA acetyltransferase and acyllyso-GPC:acetyl-CoA acetyltransferase are less in calf pulmonary artery endothelial cells than in other cell systems previously observed. Further studies are needed to discern the possibility for the existence of two isoforms of acetyltransferase. Importantly, based on the kinetic patterns of the [3H]acetate incorporation into acyl[3H]acetyl-GPC and the inductions of acetyltransferases and transacetylase activities by ATP, and the potentiation effect of sodium vanadate on the increased synthesis of acylacetyl-GPC by ATP (Figs. 1, 2, and 6 and Table II), our studies establish that activations of alkyllyso-GPC:acetyl-CoA acetyltransferase, acyllyso-GPC:acetyl-CoA acetyltransferase, and PAF:acyllyso-GPC transacetylase by ATP contribute to the stimulated synthesis of acylacetyl-GPC in calf pulmonary artery endothelial cells.