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Characterization of cytosolic phospholipases C from porcine aortic endothelial cells
Thrombosis Research. Vol. 73, No. 6, pp. 405-417, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the.USA. All rights reserved W49-3848/‘94 $6.00 + .oO
Pergamon
CHARACTERIZATION
OF CYTOSOLIC
PHOSPHOLIPASES
PORCINE AORTIC ENDOTHELIAL
C FROM
CELLS
Yigong Fu, Jin-xuan Cheng, and Suchen L. Hong Division of Cardiology, New England Deaconess Hospital and Harvard Medical School, Boston, Massachusetts 02215
(Received 3 February 7992 by Editor J. L. Moake; accepted 14 January 1994)
Abstract
Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells are involved in myriad normal and pathophysiologic functions, Although it is known that agonists activate PLCs in endothelial cells, second messengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosolic PLCs from porcine aortic endothelial cells were partially purified by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B and heparin-agarose. Three PLC isozymes immunologically similar to bovine brain PLC-p, PLC-Y, and PLC-c were identified. The relative levels of PLC activities in the cytosol were: PLC-p, 50%; PLC-y, 44%; PLC-S, 6%. The level of PLC-p activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may play a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P,) as substrate were similar to those with phosphatidylinositol (Ptdlns) as substrate, indicating that cytosolic PLCs hydrolyze both Ptdlns and Ptdlns(4,5)P, and no Ptdlns(4,5)P,-specific PLC was present in the cytosol. The catalytic properties of the partially purified PLC isozymes from porcine endothelial cells were similar to their counterparts from bovine brain. These include the dependence of hydrolysis of Ptdlns on Ca2+, the optimal Ca2+ concentrations for the hydrolysis of Ptdlns and Ptdlns(4,5)P,, the pH optima, and the stimulatory effects of deoxycholate.
Key Words:
phospholipase
C, isozymes, endothelial 405
cells
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PLCs cleave the phosphoinositides, giving rise to inositol phosphates and 1,2-diacylglycerol. Both 1,2-diacylglycerol(1) and inositol 1,4,5trisphosphate (2), one of the inositol phosphates, are second messengers in the interaction of ligands and cell surface receptors. PLCs from bovine brain (3, 4), sheep seminal vesicles (5), guinea pig uterus (6), and human platelets (7) have been purified to homogeneity. Amino acid sequences of PLCs, classified into three isozyme types: PLC-8, PLC-y and PLC-&i (1 l), have been deduced from cDNAs cloned from bovine brain (8, 9). Endothelial cells line the interior surface of the blood vessel wall and are important in the maintenance of normal hemostasis and in the pathogenesis of thrombosis, atherosclerosis and inflammation. Upon stimulation, these cells release von Willebrand factor and synthesize prostacyclin (12), cytokines (13) and adhesive proteins (14). Although it is known that agonist activation of endothelial cells leads to PLC activation, second messenger formation, and cellular responses (1520), little is known of the types of PLCs present in endothelial cells. PLC-y participates in the signal transduction of growth factors that have receptors containing tyrosine kinase (32-34). PLC-8 may be activated by G-protein and mediate the sigal transduction of agonists that interact with G-protein coupled receptors (35, 36). Knowledge of the PLCs in endothelial cells will further the understanding of endothelial functions. We partially purified and characterized the PLCs in the cytosol of porcine aortic endothelial cells and identified them as immunologically and catalytically similar to PLC-p, PLC-y, and PLC-6 of bovine brain. MATERIALS AND METHODS m Primary porcine endothial cells were isolated from porcine aortae and cultured in minimal essential medium containing 10% fetal calf serum, penicillin, and streptomycin as previously described (21). Endothelial cells were identified by their characteristic microscopic appearance, a monolayer of polygonal cobblestone-like morphology, and by the presence of angiotensin I converting enzyme (21). To obtain sufficient amounts of cells for PLC isolation, cells were passed and grown to passages 12-l 6. Cells from confluent cultures were rinsed with phosphate bufferred saline (PBS), and detached from the substratum by rubber-policemen into overlaying PBS. The cells were pelleted and washed twice with PBS by centrifugation at 200 xg for 10 minutes and stored at -70” C. Partial Purification of Phospholipase C The purification procedures were adapted from those described for bovine brain (3,4). Unless indicated otherwise, all steps were carried out at o-4 ac. Step 7. Preparation of Cvtosol Frozen cell pellets prepared from 150 roller bottles (-5 x 10’ cells) were thawed, suspended in 75 ml of Buffer-A (20 mM TriseHCI, pH 7.4, 0.5 mM dithiothreitol, 0.2 mM p-phenylmethylsulfonylfluoride, 5 PM leupeptin, and 0.5 mM EDTA), and disrupted by sonication with a Branson sonifier (model 250) for 50 sec. The sonicated suspension was centrifuged first at 10,000 xg for 10 minutes, and the resultant supernatant was then centrifuged at 100,000 xg for 60 min. Step 2. Ammonium
Sulfate fractionation
The high-speed
supernatant
from Step 1 was first
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brought to 25% saturation by adding solid ammonium sulfate while the pH was maintained at 7.0 with 1 M ammonium hydroxide. The precipitate was collected by centrifugation at 10,000 xg for 20 minutes. The supernatant was then brought to 65% ammonium sulfate saturation at pH 7.0 and stirred for 1 hr. The suspension was again centrifuged at 10,000 xg for 20 min, and the pellet was redissolved three buffer changes.
in and dialyzed overnight
against Buffer-A with
The dialyzed fraction (23.5 ml) from Step Ster, 3, DEAE-SeDharose CL-6f3 Chromatoamh\l 2 was applied to a DEAE-Sepharose CL-6B column (2.2 x 40 cm) pre-equilibrated with Buffer-A. The column was washed with 150 ml of the same buffer and eluted with a linear NaCl gradient from 0 to 0.8 M over 1.2 liter, followed by 1 M NaCI. Fractions (5 ml) were collected at a flow rate of 1 ml/min and assayed for PLC activity using either Ptdlns or Rdlns(4,5)P, as substate. Those fractions with PLC activity were pooled (Peak-l, fractions 99 to 111; Peak-2, fractions 112 to 117; Peak-3, fractions 118 to 127; Peak-4, fractions 128 to 135, Fig. 1) and dialyzed against Buffer-A containing 10% glycerol. Step 4. HeDarin-aoarose Chromatourmhv Each of the four pools of PLC from Step 3 was applied to a heparin-agarose column (0.7 x 5.2 cm) pre-equilibrated with Buffer-A containing 10% glycerol. The column was washed with 20 ml of the equilibrating buffer and eluted with a linear NaCl gradient of 0 to 0.8 M over 50 ml, followed by 1 M NaCI. Fractions of 1 ml were collected at a flow rate of 5 ml/hr. The fractions containing PLC activity were pooled and designated H-l (fractions 79-87, Fig. 2A), H-2 (fractions 91-101, Fig. 2B), H-3 (fractions 86-97, Fig. 2C), and H-4 (fractions 91-100, Fig. 2D) derived from Peak-l, 2, 3 and 4, respectively. Phospholipase C Activitv Assavs ptdlns Ptdlns-hydrolyzing activity was measured in reaction mixtures (100 ~1): 0.2 mM phospholipids (in rat liver total lipids), Ptd[2-3H]Ins (-10,000 cpm), 3 mM CaCI,, 0.5 mM EDTA, 0.5 mM sodium deoxycholate, and 100 mM HEPES and enzyme (final pH 6.0). Total lipids were prepared from rat liver by Folch extraction (22). Phospholipid content therein was quantitated by phosphate determination (39). The final concentration of free Ca*+ in the assay reaction was calculated to be 2.5 mM according to Perrin and Sayce (30) using published values for the binding constants (31). The assay mixture without enzyme was prepared as follows: the desired aliquots of the lipid and radioactive substrate were combined, dried under a stream of nitrogen, and then dispersed in assay reaction mixture (minus enzyme) in a sonicator bath until the suspension was clear. Aliquots of this suspension were then transferred to glass tubes, and after the addition of enzyme, the reaction mixtures were incubated with gentle shaking at 37” for 15 min. The inositol phosphate was separated from Ptdlns by a procedure modified from Agranoff et al. (37). The reaction was stopped by the addition of 1.l ml of chIoroform/methanol/6N HCI (15/15/2, v/v/v), followed by 0.275 ml of chloroform and 0.375 ml of water. The tubes were vortexed vigorously, and centrifuged at 700 xg for 10 min. Aliquots of the resultant upper phase containing inositol phosphate were transferred to counting vials, flushed with air to remove chloroform and methanol, and counted for radioactivity in a Packard 2000CA Liquid Scintillation Counter. Rd/r&4,5)P, PLC activity using Ptdlns(4,5)P, as substrate was assayed in reaction mixtures (100 ~1): 0.2 mM phospholipids (in rat liver total lipids), 5 PM Ptdlns(4,5)P,,
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Ptd[2-3H]lns(4,5)P, (-5,000 cpm), 1 mM sodium deoxycholate, 200 mM KCI, 100 mM HEPES buffer, pH 6.0, 0.525 mM EDTA and 0.5 mM CaCI, and enzyme. The reaction was stopped and processed as described above. Final concentration of free Ca2+ was 100 PM which was determined as described above. Polvacrvlamide Gel Analvsis and lmmunoblottinq PLC preparations from heparin-agarose column chromatography were electrophoresed on SDS-polyacrylamide gradient gels (6 to 16%) (23). The proteins were electrophoretically transferred to nitrocellulose membranes in 25 mM TrisaHCI, pH 8.3, 192 mM glycine, and 20% methanol. The nitrocellulose was treated with 10 mM TrisHCI, pH 8.0, 150 mM NaCI, and 0.05% Tween-20 containing 1% bovine serum albumin, and then subjected to immunoblotting with monoclonal antibodies to purified bovine brain PLC-p (mixture of clones K-32-3, K-82-3, K-92-3, L-7-4), monoclonal antibodies to purified bovine brain PLC-y (mixture of clones B-2-5, C-l-6, D-7-3, E-8-4, E-9-4, F-7-2), or monoclonal antibodies to purified bovine brain PLC-6 (mixture of clones R-39-2, S-l 1-2,Z-785) (3,4). The antibodies bound to nitrocellulose were visualized by using alkaline phosphatase-conjugated goat antimouse IgG. Pre-stained molecular weight standards, myosin, p-glactosidase, bovine serum albumin, and ovalbumin were used for calibration. Materials Ptd[2-3H]Ins (specific activity, 9.2 Ci/mmol) and Ptd[2-3H]InsP(4,5)P2 (specific activity, 4.7 Cilmmol) were purchased from Du Pont. Ptdlns and Ptdlns(4,5)P, were from Sigma Chemical Co; nitrocellulose membranes from Schleicher & Schuell; monoclonal antibodies specific for PLC-p, PLC-y, and PLC-6 were generously provided by Dr. Sue Goo Rhee, National Institute of Health; alkaline phosphatase-conjugated goat antimouse IgG from Promega Co; pre-stained molecular weight standards from BioRad. RESULTS Partial Purification of Phospholipase C Upon chromatography on DEAE-sepharose CL-6B column, cytosolic PLC were separated into two major Ptdlns-PLC activity peaks (peak-l and peak-3, eluting at 0.16 M and 0.25 M NaCI), each being followed by a shoulder, peak-2 and peak-4, respectively (Fig. 1A). When Ptdlns(4,5)P, was used as a substrate, the profile of PLC activity was very similar to that using Ptdlns, suggesting that cytosolic PLCs hydrolyzed both substrates (Fig. 1B). Peak-l and Peak-2 were separated by subsequent heparin-agarose column chromatography. Peak-l eluted at -0.25 M (Fig. 2A) and Peak-2 at -0.45 M NaCl (Fig. 2A and 28). However, heparin-agarose column chromatography did not separate peak-3 and peak-4; both were eluted at 0.2-0.3 M NaCl (Figs, 2C and 2D). Purification of PLC from 100,000x g supernantant achieved after heparin-agarose chromatography was: 60 fold for H-l, 35 fold for H-2, 72 fold for H-3, and 7 fold for H-4. The relative levels of activities present in the cytosol were 50%, 6%, and 44% for H-l, H-2, and H-3 and H-4, respectively. H-l preparation was immunoreactive only to SDS-PAGE and lmmunoblot Analvsis antibodies specific for PLC+ with a molecular mass of -150 kDa, similar to that of bovine PLC-13 (9). H-2 preparation was immunoreactive only with PLC-c antibodies with a band at -85 kDa, whereas H-3 and H-4 preparations were reactive only to PLC-y antibodies, each with one single band at - 145 kDa.
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4000
3000
2000
1000
0
0
,
0
100
200
300
Fraction Number
FIG. 1
DEAE-Sepharose CL-6B Column Chromatography of Cytosolic Phospholipases C. Panel A: PLC activity using Ptdlns as substrate (0) was assayed every fraction; &8o Cm (A); NaCl (0). Panel B: PLC activity using Ptdlns(4,5)P, as substrate was assayed every three fractions.
Properties of Endothelial Phospholipase C Using partially purified PLC preparations from the heparin-agarose column chromatography step (H-l, H-2, H-3 and H-4), the endothelial PLC isozymes were characterized with respect to pH, deoxycholate and Ca2+ concentrations, andsubstrate types, since earlier work has shown that these parameters have different effects on the catalytic activities of PLC isozymes from bovine brain (3-5). The Effect of Ca2+ on /XC Acfivitv All four preparations of PLC required Ca2+ ions for activity when using Ptdtlns as substrate. There was no detectable activity when free Ca2+ was below 1 FM (Fig. 4A). The optimal concentration of Ca2+ for activity was at l-5 mM for all four preparations. At higher concentrations, stimulation by Ca2+ was slightly reduced. The profiles of Ca2+ concentration effect on H-3 and H-4 preparations were similar. The optimal Ca2+ concentration for endothelial PLC isozymes, using Ptdlns(4,5)P, as substrate, is broad (20-500 PM, Fig. 48) and lower than that for the hydrolysis of Ptdlns. The
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so0
0
. B
/
,..-• -*-*-,-._.-
loo0 0300
.
A’
1000 -
!ioo
a
-a
0
1W
100
50
~o.oooLo
T
150
1000 0.300
I
o.ooo 100
50
Fraction Number
150
0
FIG. 2
Heparin-agarose Column Chromatography of Cytosolic Phospholipase C. The four pools of PLC from Step 3 DEAE-column chromatography (A: Peak-l, 6: Peak-e, C: Peak-3 and D: Peak-4) were each applied to a heparin-agarose column and eluted as described in “METHODS.” PLC activity using Ptdlns as substrate (o), A2801rm(A) and NaCl (0).
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FIG. 3
HI
PLC-B,
150 kDa
_
PLC-y,
145 kDa
-
PLC-6,
85 kDa
_
H2 H3 H4
lmmunoblot Analysis of Cytosolic Phospholipase C. The PLC preparations partially purified from heparin-agarose column chromatographic step (H-l, H-2, H-3 and H-4) were separated by SDS polyacrylamide gradient gels. The proteins were electrophoretically transferred to nitrocellulose membranes and immunobloted with monoclonal antibodies specific for A: PLC-p; 9: PLC-y; and C: PLC-6; as described in “METHODS”.
FIG. 4
,OO
Effect of Free Ca2+ Concentration on Phospholipase C Activity. The effect of calcium was examined with PLC
A. Ptdlns
preparations, H-l (O), H-2 (O), H-3 (A) and H-4 (0). PLC activity using Rdlns (A) or Ptdlns(4,5)P, (9) as substrate was assayed as described in “METHODS”, except that free Ca2+ concentration was varied by addition of CaCI, to the assay reaction mixtures, PLC activity is expressed as % of that obtained at the optimal Ca2+ concentration for each enzyme preparation. The values shown are from the averages of duplicates which agree within 10% of the average, Three separate experiments all produced similar results, Free Ca*+,
M
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difference in Ca2+ requirement for these two substrates is similar to that for the bovine brain PLCs (4). Unlike PLC activities using Ptdlns as substrate, there were basal levels of activity toward Ptdlns(4,5)P, at Ca2+ concentration below 1 PM. Activities in the absence of added Ca2+ were 45%, 6%, 15%, and 16% of those at 20 PM Ca2+ for H-l, H-2, H-3 and H-4, respectively. The Effect ofn# on PLC activitv All four preparations of PLC had optimal pH between 5 and 6 for activity using Ptdlns as substrate (Fig. 5A). Optimal pH range was narrow for H-l, H-3 and H-4, with little activity below pH 4 or above pH 7, whereas optimal pH for H-2 was broader, with 50% activity remaining at pH 8. The broader pH optimum for H-2 (a PLC-6) was similar to that for bovine brain PLC-6 (4). With Ptdlns(4,5)P, as substrate, the pH optima were also between 5 and 6 (Fig. 5B). Again, H-2 has a broader optimal pH range with higher residual activity at pH 8 than the other three preparations. The Effect of Deoxvcholate Deoxycholate stimulated the PLC activity; the stimulation was highest for H-4, followed by H-3, H-2, and H-l. The optimal concentration range of deoxycholate was narrow: at -0.5 mM with Ptdlns as substrate (Fig. 6A), and at 1 mM with Ptdlns(4,5)P, (Fig 6B). The higher stimulatory effect of deoxycholate on H-4 and H-3 (PLC-y) than H-l (PLC-8) is consistent with that reported for the respective bovine brain isozymes (3). Deoxycholate at concentrations higher than l-2 mM inhibit PLC activities. Again, these effects of deoxycholate on endothelial PLC isozymes are similar to those from bovine brain (3,4). DISCUSSION The objective of this study was to identify the PLC isozymes present in endothelial cells, to characterize their properties, and to determine if a novel PLC might be present in endothelial cells. Column chromatographic separations of cytosol indicated that endothelial cells contained three PLC isozymes, which were identified by immunoblotting with monoclonal antibodies specific for bovine brain PLC-p, PLC-y and PLC-6. The relative levels of enzyme activities in the cytosol of endothelial cells were close to those of bovine brain enzymes (24) and different from those of several established cell lines, For example, in mouse fibroblast NIH 3T3, glioma CGBul, and neuroblastoma PC-12, PLC-y was the major isozyme and PLC-p was very low or barely detectable by radioimmunoassays (27). Although enzyme activity levels and enzyme protein levels are not always directly comparable, the particularly high level of PLC-p activity in endothelial cells may be of as yet unknown significance. In DEAE column chromatography, elution profile of PLC with Ptdlns(4,5)P, as substrate is very similar to that with Ptdlns, indicating that all PLC species in endothelial cell cytosol hydrolyze both Ptdlns and Ptdlns(4,5)P,. This is consistent with the findings by other investigators that PLC isozymes use Ptdlns and Ptdlns(4,5)P, as well as phosphatidylinositol 4-phosphate as substrates (3,25,26). Endothelial PLC activity toward phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate or phosphatidylinositol 3,4,5_trisphosphate was not examined in this study, because other investigators have demonstrated that the polyphosphoinositides produced by phosphatidylinositol3-kinase are poor substrate for PLC (38). The properties
of partially purified endothelial
PLCs were similar to those of bovine brain
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FIG. 5
3
2
,x .> z
4
5
6
7
8
Effect of pH on Phospholipase C Activitiy. The effect of pH was examined with PLC preparations, H-l (0) H-2 (Cl), H-3 (A) and H-4 (0). PLC activity using Ptdlns (A) or Ptdlns (4,5)P, (B) as substrate was assayed as described in “METHODS”, except that pH was varied as indicated. PLC activity is expressed as % of that obtained at the optimal pH for each enzyme preparation. The values shown are from the averages of duplicates which agree within 10% of the average. The experiment was repeated once and a similar result was obtained.
910
B. loo-.
Q
07
:
2
3
:
4
:
:
5
6
:
7
:
:
8
910
PH
FIG. 6
Effect
of
Deoxycholate
on
Phospholipase C Activitiy. The effect of deoxycholate was examined with PLC preparations, H-l (O), H-2 (II), H-3 (A) and H-4 (0). PLC activity using Ptdlns (A) or Ptdins (4,5)P, (B) as substrate was assayed as described in “METHODS”, except that deoxycholate level was varied. PLC activity is expressed as % of that obtained in the absence of deoxycholate for each enzyme preparation. The values shown are from the averages of duplicates which agree within 10% of the average. The experiment was repeated once and a similar result was obtained. 0
2
4
Deoxycholate,
8
6
mM
10
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enzymes. The pH optima were similar to bovine brain enzymes when either Ptdlns or Ptdlns(4,5)P, was used as substrate. Further, the slightly higher pH optimum and residual activity of H-2 at high pH were very similar to those of purified PLC-6 from bovine brain (4) and consistent with the identification by immunoblotting of H-2 as PLC-6. In addition, endothelial PLC isozymes are similar to the corresponding brain PLC isozymes in the dependence of hydrolysis of Ptdlns on Ca2’ (3,4), the effect of Ca2+ concentration on activities towards Ptdlns and Ptdlns(4,5), (3,4), and the different stimulatory effects of deoxycholate on PLC-8 and PLCy (3). In conclusion, three types of PLC isozymes (PLC-8, PLC-y and PLC-C) are present in the cytosol of porcine aortic endothelial cells. The shoulder (Peak-4, Fig. 1) observed during DEAE chromatography might be either a chromatographic abnormality or an additional isozyme. Findings that support the former notion are: the similarity of H-3 and H-4 preparations in the elution conditions from heparin-agarose column; the profiles of the effects of Ca2+, pH and deoxycholate; the molecular weights and immunological reactivity. Several subsequent attempts of partial purification, however, failed to yield a similar shoulder in DEAE-chromatography. Thus it is likely that only three types of PLC are present in the cytosol of porcine aortic endothelial cells. Nevertheless, we could not rule out the alternative possibility that Peak-4 may be an isoform of PLC with molecular weight, immunological reactivity, and catalytic properties very similar to PLC-y. The rationale behind this speculation is that a subtype of PLC-y has been demonstrated by molecular cloning in rat muscle (28) and in Epstein-Barr virus transformed human lymphocytes (29). The possibility of a variant of PLC-y in porcine endothelial cells awaits confirmation in future studies and is beyond the scope of the current effort. Acknowledgements We thank Dr. Sue Goo Rhee for the generous gift of monoclonal antibodies against phospholipases C isozymes, Dr. Hava Avraham for her assistance in immunoblotting experiments and Dr. Zenon Grabarek for his assistance in the estimation of calcium ion concentration. This work was supported by Research Grant HL28778 from the National Institute of Heart, Lung and Blood and by an American Heart Association Established Investigator Award to S.L.H. REFERENCES NISHIZUKA, Y. The role of protein kinase C in cell surface signal transduction 1. tumour promotion. Nature, 308, 693-698, 1984. BERRIDGE, M.J. and IRVINE, R.F. lnositol trisphosphate, 2. cellular sinal transduction. Nature, 372, 315321, 1984.
and
a novel second messenger
in
3. RYU, S.H., CHO, K.S., LEE, K.Y., SUH, P.G. and RHEE, S.G. Purification and Characterization of two immunologically distinct phosphoinositide-specific phospholipase C from bovine brain. J. Biol. Chem. 262, 12511-12518, 1987.
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RYU, S.H., SUH, P.G., CHO, KS., LEE, K.Y. and RHEE, S.G. Bovine brain cytosol 4. contains three immunologically distinct forms of inositol phospholipid- specific phospholipase C. Proc. Nat/. Acad. Sci. 84, 6649-6653, 1967. HOFMANN, S.L. and MAJERUS, P.W. Identification and properties of two distinct 5. phosphatidylinositol-specfic phospholipase C enzymes from sheep seminal vesicular glands, J. Ho/. Chem. 257, 6461-6469, 1982. BENNETT, C.F. 6. phosphoinositide-specific
and CROOKE, phospholipase
ST. Purification and Characterization of a C from guinea pig uterus. phosphorylation by
protein kinase C in vivo. J.Bio/. Chem. 262, 13789-l 3797, 1987. BANNO, Y., YADA, Y. and NOZAWA, Y. Purification and Characterization of Membrane7. bound Phospholipase C Specific for Phosphoinositides from Human Platelets. J. Biol. Chem. 263, 11459-l 1465, 1988. 8. KATAN, M. and PARKER, P.J. Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain. Eur. J. Chem. 768, 413-418, 1987. 9. SUH, P.G., RYU, S.H., MOON, K.H., SUH, H.W. and RHEE S.G. Cloning and Sequence of Multiple Forms of Phospholipase C. Cell, 54, 161-169, 1988.
10. BENNET, C.F., BALCAREK, J.M. VARRICHIO, A. and CROOKE, S.T. Molecular Cloning and Complete Amino Acid Sequence of Form-l Phosphoinositide-specific Phospholipase C. Nature, 334, 268-270, 1988. 11. RHEE, S.G., Phospholipid-Specific
SUH, P.G., RYU, S.H., and LEE, S.Y. Studies Phospholipase C. Science, 244, 546-550, 1988.
12. JAFFE, E.A. Ceil Biology of Endothelial
Cells. Human Pathology,
of
lnositol
78, 234-239, 1987.
13. MANTOVANI, A. and DEJANA, E. Cytokines as Communication Signals Leukocytes and Endothelial Cella. immunology Today, 70, 370-375, 1989.
between
14. BEVILACQUA, M.P., STENGELIN, S., GIMBRONE, M.A. Jr. and SEED, B. Endothelial Leukocyte Adhesive Molecule 1: An Inducible Receptor for Neutrophils Related to Complement Regulatory Proteins and Lectins. Science, 243, 1160-l 165, 1989. 15. KAYA, H., PATTON, G.M. and HONG, S.L. Bradykinin-induced Activation Phospholipase 4 Is Independent of the Activation of Polyphosphoinositide-hydrolyzing Phospholipase C. J. Ho/. Chem. 264, 4972-4977, 1989.
of
16. MOSCAT, J., MORENO, F., HERRERO, C., LOPEZ, C. and GARCIA-BARRENO, P. Endothelial Cell Growth Factor and lnonophore A231 87 Stimulation of Production of lnositol Phosphates in Porcine Aorta Endothelial Cells. Proc. Nat/. Acad. Sci. U.S.A. 85, 659-663, 1988.
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17. BARTHA, K., MULLER-PEDDINGHAUS, Ft. and VAN ROOIJEN, L.A.A. Bradykinin and Thrombin Effects on Polyphosphoinositide Hydrolysis and Prostacylin Production in Endothelial Cells. Biochem. J. 263, 149-155, 1989. 18. LAMBERT, T.L., KENT, R.S. and WHORTON, A.R. Bradykinin Stimulation of lnositol Polyphosphate Production in Porcine Aortic Endothelial Cells. J. Biol. Chem. 267, 1528815293, 1988. 19. DERIAN, C.K. and MOSKOWITZ, M.A. Polyinositide Hydrolysis Carotid Artery Segments. J. 5iol. Chem. 267, 3831-3837, 1986.
in Endothelial
Cells and
20. JAFFE, E.A., GRULICH, J., WEKSLER, B.B., HARPEL, G. and WATANABE, K. Correlaion betweem Thrombin-induced Prostacyclin Produciton and lnositol Trisphosphate and Cytosolic Free Calcium Levels in Cultured Human Endothelial Cells. J. Biol. Chem. 262, 8557-8565, 1987. 21. HONG, S.L. Effect of Bradykinin and Thrombin on Prostacyclin Synthesis in Endothelial Cells from Calf and Pig Aorta and Human Umbilical Cord Vein. Thrombosis Research, 78, 787-95, 1980. 22. FOLICH, J., LEES, M. and STANLEY, S. A Simple Method for the Isolation Purification of Total Lipides from Animal Tissues. J. Biol. Chem. 226, 497-509, 1957. 23. LAEMMLI, U.K. Cleavage of Structural Protein during the Assembly Bacteriophage T4. Nature, 227, 680-685, 1970.
and
of the Head of
24. SUH, P.G., RYU, S.H., CHOI, W.C., LEE, K.Y. and RHEE, S.G. Monoclonal antibodies to Three Phospholipase C lsozymes from Bovine Brain. J, 5iol. Chem. 263, 14497-14504, 1988. 25. WILSON, D. B., BROSS, T.E., HOFMANN, S.L. and MAJERUS, Polyphosphoinositides by Purified Sheep Seminal Vesicle Phospholipase Chem. 259, 11718-l 1724, 1984.
P.W. Hydrolysis of C Enzymes. J. 5io.
26. REBECCHI, M.J. and ROSEN, O.M. Purification of a Phosphoinositide-specific Phospholipase C from Bovine Brain. J. Biol. Chem. 262, 12526-12532, 1987. 27. SMITH, M.R., LIU, Y.L., KIM, H., RHEE, S.H. and KUNG, H.F. Inhibition of Serum- and Res-Stimulated DNA Synthesis by Antibodies to Phospholipase C. Science 247, 1074-l 077, 1990. 28. EMORI, Y., HOMMA, Y., SORIMCACHI, H., KAWASAKI, H., NAKANISH, O., SUZUK, K. and TAKENAWA, T. A Second Type of Rat Phosphoinositide Phospholipase C Containing a src-related Sequence Not Essential for Phosphoinositide-hydrolyzing Activity. J. 5iol. Chem. 264, 21885-21890 , 1989.
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29. OHTA, S., MATSUI, A., NAZAWA, Y. and KAGAWAN, Y. Complete cDNA Encoding a Putative Phospholipase C from Transformed Human Lymphocytes. FEBS 242, 31-35, 1988. 30. PERRIN, D.D. and SAYCE, LG. Computer Calculation of Equilibrium Concentrations Mixture of Metal Ions and Complexing Species. Talanta, 74, 833-842, 1967.
in
31. SILLEN, L.G. and MARTEL, A.E. Stability Constants of Metal Complexes. 2nd edn, Spec. Publ. No 17 (The Chemical society, London), 1967. 32.
Wahl, M.I., NISHIBE, S, SUH, P, RHEE, S.G., CARPENTER, G. Epidermal Growth Factor
Stimulates Tyrosine Phosphorylation of Phospholipase C-II Independently of Receptor Internalization and Extracellular Calcium. Proc. Nat/. Acad. Sci. USA, 86, 1568-l 572, 1989. 33. MARGOLIS, B., RHEE, S.G., FELFRT, S., MERVIC, M., LYALL, R., LEVITZKI, A., ULLRICH, A., ZILBERSTEIN, A. and SCHLESSINGER, J. EGF Induces Tyrosine Phosphorylation of Phospholipase C-II: a Potential Mechanism for EGF Receptor Signaling. Cell 57, 1101-l 107, 1989. 34. MEISENHELDER, J., SUH, P.G., RHEE, S.R. and HUNTER, T. Phospholipase C-y Is a Substrate for the PDGF and EGF Receptor Protein-tyrosine Kinases in Vivo and in Vitro. Cell 57, 1109-l 122, 1989. 35. SMRCKA, A.V., HEPLER, J.R., BROWN, K.O. and STERNWIES, P.C. Regulation of Polyphosphoinositide-specific Phospholipase C Activity by Purified Gq. Science 251,804-807, 1991. 36. TAYLOR, S.J., CHAE, H.Z., RHEE, S.G. and EXTON, J.H. Activation of pl lsozyme of Phospholipase C by CYSubunits of the Gq Class of G Proteins. Nature 350, 516-518, 1991. 37. AGRANOFF, B.W., MURTHY, P. andSEQUIN, E.B.Thrombin-induced Phosphodiesteratic Cleavage of Phosphotidylinositol Bisphosphate in Human Platelets. J. Biol. Chem. 258, 20762078, 1983. 38. SERUNIAN, L.A., HABER, M.T., FUKUI, T., KIM, J.W., RHEE, S.G., LOWENSTEIN, J.M., and CANTLEY L.C. Polyphosphoinositides Produced by Phosphatidylinositol 3-Kinase Are Poor Substrates for Phospholipases C from Rat Liver and bovine Brain. J. Biol. Chem. 264, 17809-l 5, 1989. 39. KAYA, H., PATTON, G.M., and HONG, S.L. Bradykinin-induced Activation Phospholipase & Is Independent of the Activation of Polyphosphoinositide-hydrolyzing Phospholipase C. J. Ho/. Chem. 264, 4972-4977, 1989.
of
Pergamon
CHARACTERIZATION
OF CYTOSOLIC
PHOSPHOLIPASES
PORCINE AORTIC ENDOTHELIAL
C FROM
CELLS
Yigong Fu, Jin-xuan Cheng, and Suchen L. Hong Division of Cardiology, New England Deaconess Hospital and Harvard Medical School, Boston, Massachusetts 02215
(Received 3 February 7992 by Editor J. L. Moake; accepted 14 January 1994)
Abstract
Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells are involved in myriad normal and pathophysiologic functions, Although it is known that agonists activate PLCs in endothelial cells, second messengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosolic PLCs from porcine aortic endothelial cells were partially purified by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B and heparin-agarose. Three PLC isozymes immunologically similar to bovine brain PLC-p, PLC-Y, and PLC-c were identified. The relative levels of PLC activities in the cytosol were: PLC-p, 50%; PLC-y, 44%; PLC-S, 6%. The level of PLC-p activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may play a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P,) as substrate were similar to those with phosphatidylinositol (Ptdlns) as substrate, indicating that cytosolic PLCs hydrolyze both Ptdlns and Ptdlns(4,5)P, and no Ptdlns(4,5)P,-specific PLC was present in the cytosol. The catalytic properties of the partially purified PLC isozymes from porcine endothelial cells were similar to their counterparts from bovine brain. These include the dependence of hydrolysis of Ptdlns on Ca2+, the optimal Ca2+ concentrations for the hydrolysis of Ptdlns and Ptdlns(4,5)P,, the pH optima, and the stimulatory effects of deoxycholate.
Key Words:
phospholipase
C, isozymes, endothelial 405
cells
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PLCs cleave the phosphoinositides, giving rise to inositol phosphates and 1,2-diacylglycerol. Both 1,2-diacylglycerol(1) and inositol 1,4,5trisphosphate (2), one of the inositol phosphates, are second messengers in the interaction of ligands and cell surface receptors. PLCs from bovine brain (3, 4), sheep seminal vesicles (5), guinea pig uterus (6), and human platelets (7) have been purified to homogeneity. Amino acid sequences of PLCs, classified into three isozyme types: PLC-8, PLC-y and PLC-&i (1 l), have been deduced from cDNAs cloned from bovine brain (8, 9). Endothelial cells line the interior surface of the blood vessel wall and are important in the maintenance of normal hemostasis and in the pathogenesis of thrombosis, atherosclerosis and inflammation. Upon stimulation, these cells release von Willebrand factor and synthesize prostacyclin (12), cytokines (13) and adhesive proteins (14). Although it is known that agonist activation of endothelial cells leads to PLC activation, second messenger formation, and cellular responses (1520), little is known of the types of PLCs present in endothelial cells. PLC-y participates in the signal transduction of growth factors that have receptors containing tyrosine kinase (32-34). PLC-8 may be activated by G-protein and mediate the sigal transduction of agonists that interact with G-protein coupled receptors (35, 36). Knowledge of the PLCs in endothelial cells will further the understanding of endothelial functions. We partially purified and characterized the PLCs in the cytosol of porcine aortic endothelial cells and identified them as immunologically and catalytically similar to PLC-p, PLC-y, and PLC-6 of bovine brain. MATERIALS AND METHODS m Primary porcine endothial cells were isolated from porcine aortae and cultured in minimal essential medium containing 10% fetal calf serum, penicillin, and streptomycin as previously described (21). Endothelial cells were identified by their characteristic microscopic appearance, a monolayer of polygonal cobblestone-like morphology, and by the presence of angiotensin I converting enzyme (21). To obtain sufficient amounts of cells for PLC isolation, cells were passed and grown to passages 12-l 6. Cells from confluent cultures were rinsed with phosphate bufferred saline (PBS), and detached from the substratum by rubber-policemen into overlaying PBS. The cells were pelleted and washed twice with PBS by centrifugation at 200 xg for 10 minutes and stored at -70” C. Partial Purification of Phospholipase C The purification procedures were adapted from those described for bovine brain (3,4). Unless indicated otherwise, all steps were carried out at o-4 ac. Step 7. Preparation of Cvtosol Frozen cell pellets prepared from 150 roller bottles (-5 x 10’ cells) were thawed, suspended in 75 ml of Buffer-A (20 mM TriseHCI, pH 7.4, 0.5 mM dithiothreitol, 0.2 mM p-phenylmethylsulfonylfluoride, 5 PM leupeptin, and 0.5 mM EDTA), and disrupted by sonication with a Branson sonifier (model 250) for 50 sec. The sonicated suspension was centrifuged first at 10,000 xg for 10 minutes, and the resultant supernatant was then centrifuged at 100,000 xg for 60 min. Step 2. Ammonium
Sulfate fractionation
The high-speed
supernatant
from Step 1 was first
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brought to 25% saturation by adding solid ammonium sulfate while the pH was maintained at 7.0 with 1 M ammonium hydroxide. The precipitate was collected by centrifugation at 10,000 xg for 20 minutes. The supernatant was then brought to 65% ammonium sulfate saturation at pH 7.0 and stirred for 1 hr. The suspension was again centrifuged at 10,000 xg for 20 min, and the pellet was redissolved three buffer changes.
in and dialyzed overnight
against Buffer-A with
The dialyzed fraction (23.5 ml) from Step Ster, 3, DEAE-SeDharose CL-6f3 Chromatoamh\l 2 was applied to a DEAE-Sepharose CL-6B column (2.2 x 40 cm) pre-equilibrated with Buffer-A. The column was washed with 150 ml of the same buffer and eluted with a linear NaCl gradient from 0 to 0.8 M over 1.2 liter, followed by 1 M NaCI. Fractions (5 ml) were collected at a flow rate of 1 ml/min and assayed for PLC activity using either Ptdlns or Rdlns(4,5)P, as substate. Those fractions with PLC activity were pooled (Peak-l, fractions 99 to 111; Peak-2, fractions 112 to 117; Peak-3, fractions 118 to 127; Peak-4, fractions 128 to 135, Fig. 1) and dialyzed against Buffer-A containing 10% glycerol. Step 4. HeDarin-aoarose Chromatourmhv Each of the four pools of PLC from Step 3 was applied to a heparin-agarose column (0.7 x 5.2 cm) pre-equilibrated with Buffer-A containing 10% glycerol. The column was washed with 20 ml of the equilibrating buffer and eluted with a linear NaCl gradient of 0 to 0.8 M over 50 ml, followed by 1 M NaCI. Fractions of 1 ml were collected at a flow rate of 5 ml/hr. The fractions containing PLC activity were pooled and designated H-l (fractions 79-87, Fig. 2A), H-2 (fractions 91-101, Fig. 2B), H-3 (fractions 86-97, Fig. 2C), and H-4 (fractions 91-100, Fig. 2D) derived from Peak-l, 2, 3 and 4, respectively. Phospholipase C Activitv Assavs ptdlns Ptdlns-hydrolyzing activity was measured in reaction mixtures (100 ~1): 0.2 mM phospholipids (in rat liver total lipids), Ptd[2-3H]Ins (-10,000 cpm), 3 mM CaCI,, 0.5 mM EDTA, 0.5 mM sodium deoxycholate, and 100 mM HEPES and enzyme (final pH 6.0). Total lipids were prepared from rat liver by Folch extraction (22). Phospholipid content therein was quantitated by phosphate determination (39). The final concentration of free Ca*+ in the assay reaction was calculated to be 2.5 mM according to Perrin and Sayce (30) using published values for the binding constants (31). The assay mixture without enzyme was prepared as follows: the desired aliquots of the lipid and radioactive substrate were combined, dried under a stream of nitrogen, and then dispersed in assay reaction mixture (minus enzyme) in a sonicator bath until the suspension was clear. Aliquots of this suspension were then transferred to glass tubes, and after the addition of enzyme, the reaction mixtures were incubated with gentle shaking at 37” for 15 min. The inositol phosphate was separated from Ptdlns by a procedure modified from Agranoff et al. (37). The reaction was stopped by the addition of 1.l ml of chIoroform/methanol/6N HCI (15/15/2, v/v/v), followed by 0.275 ml of chloroform and 0.375 ml of water. The tubes were vortexed vigorously, and centrifuged at 700 xg for 10 min. Aliquots of the resultant upper phase containing inositol phosphate were transferred to counting vials, flushed with air to remove chloroform and methanol, and counted for radioactivity in a Packard 2000CA Liquid Scintillation Counter. Rd/r&4,5)P, PLC activity using Ptdlns(4,5)P, as substrate was assayed in reaction mixtures (100 ~1): 0.2 mM phospholipids (in rat liver total lipids), 5 PM Ptdlns(4,5)P,,
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Ptd[2-3H]lns(4,5)P, (-5,000 cpm), 1 mM sodium deoxycholate, 200 mM KCI, 100 mM HEPES buffer, pH 6.0, 0.525 mM EDTA and 0.5 mM CaCI, and enzyme. The reaction was stopped and processed as described above. Final concentration of free Ca2+ was 100 PM which was determined as described above. Polvacrvlamide Gel Analvsis and lmmunoblottinq PLC preparations from heparin-agarose column chromatography were electrophoresed on SDS-polyacrylamide gradient gels (6 to 16%) (23). The proteins were electrophoretically transferred to nitrocellulose membranes in 25 mM TrisaHCI, pH 8.3, 192 mM glycine, and 20% methanol. The nitrocellulose was treated with 10 mM TrisHCI, pH 8.0, 150 mM NaCI, and 0.05% Tween-20 containing 1% bovine serum albumin, and then subjected to immunoblotting with monoclonal antibodies to purified bovine brain PLC-p (mixture of clones K-32-3, K-82-3, K-92-3, L-7-4), monoclonal antibodies to purified bovine brain PLC-y (mixture of clones B-2-5, C-l-6, D-7-3, E-8-4, E-9-4, F-7-2), or monoclonal antibodies to purified bovine brain PLC-6 (mixture of clones R-39-2, S-l 1-2,Z-785) (3,4). The antibodies bound to nitrocellulose were visualized by using alkaline phosphatase-conjugated goat antimouse IgG. Pre-stained molecular weight standards, myosin, p-glactosidase, bovine serum albumin, and ovalbumin were used for calibration. Materials Ptd[2-3H]Ins (specific activity, 9.2 Ci/mmol) and Ptd[2-3H]InsP(4,5)P2 (specific activity, 4.7 Cilmmol) were purchased from Du Pont. Ptdlns and Ptdlns(4,5)P, were from Sigma Chemical Co; nitrocellulose membranes from Schleicher & Schuell; monoclonal antibodies specific for PLC-p, PLC-y, and PLC-6 were generously provided by Dr. Sue Goo Rhee, National Institute of Health; alkaline phosphatase-conjugated goat antimouse IgG from Promega Co; pre-stained molecular weight standards from BioRad. RESULTS Partial Purification of Phospholipase C Upon chromatography on DEAE-sepharose CL-6B column, cytosolic PLC were separated into two major Ptdlns-PLC activity peaks (peak-l and peak-3, eluting at 0.16 M and 0.25 M NaCI), each being followed by a shoulder, peak-2 and peak-4, respectively (Fig. 1A). When Ptdlns(4,5)P, was used as a substrate, the profile of PLC activity was very similar to that using Ptdlns, suggesting that cytosolic PLCs hydrolyzed both substrates (Fig. 1B). Peak-l and Peak-2 were separated by subsequent heparin-agarose column chromatography. Peak-l eluted at -0.25 M (Fig. 2A) and Peak-2 at -0.45 M NaCl (Fig. 2A and 28). However, heparin-agarose column chromatography did not separate peak-3 and peak-4; both were eluted at 0.2-0.3 M NaCl (Figs, 2C and 2D). Purification of PLC from 100,000x g supernantant achieved after heparin-agarose chromatography was: 60 fold for H-l, 35 fold for H-2, 72 fold for H-3, and 7 fold for H-4. The relative levels of activities present in the cytosol were 50%, 6%, and 44% for H-l, H-2, and H-3 and H-4, respectively. H-l preparation was immunoreactive only to SDS-PAGE and lmmunoblot Analvsis antibodies specific for PLC+ with a molecular mass of -150 kDa, similar to that of bovine PLC-13 (9). H-2 preparation was immunoreactive only with PLC-c antibodies with a band at -85 kDa, whereas H-3 and H-4 preparations were reactive only to PLC-y antibodies, each with one single band at - 145 kDa.
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4000
3000
2000
1000
0
0
,
0
100
200
300
Fraction Number
FIG. 1
DEAE-Sepharose CL-6B Column Chromatography of Cytosolic Phospholipases C. Panel A: PLC activity using Ptdlns as substrate (0) was assayed every fraction; &8o Cm (A); NaCl (0). Panel B: PLC activity using Ptdlns(4,5)P, as substrate was assayed every three fractions.
Properties of Endothelial Phospholipase C Using partially purified PLC preparations from the heparin-agarose column chromatography step (H-l, H-2, H-3 and H-4), the endothelial PLC isozymes were characterized with respect to pH, deoxycholate and Ca2+ concentrations, andsubstrate types, since earlier work has shown that these parameters have different effects on the catalytic activities of PLC isozymes from bovine brain (3-5). The Effect of Ca2+ on /XC Acfivitv All four preparations of PLC required Ca2+ ions for activity when using Ptdtlns as substrate. There was no detectable activity when free Ca2+ was below 1 FM (Fig. 4A). The optimal concentration of Ca2+ for activity was at l-5 mM for all four preparations. At higher concentrations, stimulation by Ca2+ was slightly reduced. The profiles of Ca2+ concentration effect on H-3 and H-4 preparations were similar. The optimal Ca2+ concentration for endothelial PLC isozymes, using Ptdlns(4,5)P, as substrate, is broad (20-500 PM, Fig. 48) and lower than that for the hydrolysis of Ptdlns. The
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PORCINE ENDOTHELIAL CELLS
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so0
0
. B
/
,..-• -*-*-,-._.-
loo0 0300
.
A’
1000 -
!ioo
a
-a
0
1W
100
50
~o.oooLo
T
150
1000 0.300
I
o.ooo 100
50
Fraction Number
150
0
FIG. 2
Heparin-agarose Column Chromatography of Cytosolic Phospholipase C. The four pools of PLC from Step 3 DEAE-column chromatography (A: Peak-l, 6: Peak-e, C: Peak-3 and D: Peak-4) were each applied to a heparin-agarose column and eluted as described in “METHODS.” PLC activity using Ptdlns as substrate (o), A2801rm(A) and NaCl (0).
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411
FIG. 3
HI
PLC-B,
150 kDa
_
PLC-y,
145 kDa
-
PLC-6,
85 kDa
_
H2 H3 H4
lmmunoblot Analysis of Cytosolic Phospholipase C. The PLC preparations partially purified from heparin-agarose column chromatographic step (H-l, H-2, H-3 and H-4) were separated by SDS polyacrylamide gradient gels. The proteins were electrophoretically transferred to nitrocellulose membranes and immunobloted with monoclonal antibodies specific for A: PLC-p; 9: PLC-y; and C: PLC-6; as described in “METHODS”.
FIG. 4
,OO
Effect of Free Ca2+ Concentration on Phospholipase C Activity. The effect of calcium was examined with PLC
A. Ptdlns
preparations, H-l (O), H-2 (O), H-3 (A) and H-4 (0). PLC activity using Rdlns (A) or Ptdlns(4,5)P, (9) as substrate was assayed as described in “METHODS”, except that free Ca2+ concentration was varied by addition of CaCI, to the assay reaction mixtures, PLC activity is expressed as % of that obtained at the optimal Ca2+ concentration for each enzyme preparation. The values shown are from the averages of duplicates which agree within 10% of the average, Three separate experiments all produced similar results, Free Ca*+,
M
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difference in Ca2+ requirement for these two substrates is similar to that for the bovine brain PLCs (4). Unlike PLC activities using Ptdlns as substrate, there were basal levels of activity toward Ptdlns(4,5)P, at Ca2+ concentration below 1 PM. Activities in the absence of added Ca2+ were 45%, 6%, 15%, and 16% of those at 20 PM Ca2+ for H-l, H-2, H-3 and H-4, respectively. The Effect ofn# on PLC activitv All four preparations of PLC had optimal pH between 5 and 6 for activity using Ptdlns as substrate (Fig. 5A). Optimal pH range was narrow for H-l, H-3 and H-4, with little activity below pH 4 or above pH 7, whereas optimal pH for H-2 was broader, with 50% activity remaining at pH 8. The broader pH optimum for H-2 (a PLC-6) was similar to that for bovine brain PLC-6 (4). With Ptdlns(4,5)P, as substrate, the pH optima were also between 5 and 6 (Fig. 5B). Again, H-2 has a broader optimal pH range with higher residual activity at pH 8 than the other three preparations. The Effect of Deoxvcholate Deoxycholate stimulated the PLC activity; the stimulation was highest for H-4, followed by H-3, H-2, and H-l. The optimal concentration range of deoxycholate was narrow: at -0.5 mM with Ptdlns as substrate (Fig. 6A), and at 1 mM with Ptdlns(4,5)P, (Fig 6B). The higher stimulatory effect of deoxycholate on H-4 and H-3 (PLC-y) than H-l (PLC-8) is consistent with that reported for the respective bovine brain isozymes (3). Deoxycholate at concentrations higher than l-2 mM inhibit PLC activities. Again, these effects of deoxycholate on endothelial PLC isozymes are similar to those from bovine brain (3,4). DISCUSSION The objective of this study was to identify the PLC isozymes present in endothelial cells, to characterize their properties, and to determine if a novel PLC might be present in endothelial cells. Column chromatographic separations of cytosol indicated that endothelial cells contained three PLC isozymes, which were identified by immunoblotting with monoclonal antibodies specific for bovine brain PLC-p, PLC-y and PLC-6. The relative levels of enzyme activities in the cytosol of endothelial cells were close to those of bovine brain enzymes (24) and different from those of several established cell lines, For example, in mouse fibroblast NIH 3T3, glioma CGBul, and neuroblastoma PC-12, PLC-y was the major isozyme and PLC-p was very low or barely detectable by radioimmunoassays (27). Although enzyme activity levels and enzyme protein levels are not always directly comparable, the particularly high level of PLC-p activity in endothelial cells may be of as yet unknown significance. In DEAE column chromatography, elution profile of PLC with Ptdlns(4,5)P, as substrate is very similar to that with Ptdlns, indicating that all PLC species in endothelial cell cytosol hydrolyze both Ptdlns and Ptdlns(4,5)P,. This is consistent with the findings by other investigators that PLC isozymes use Ptdlns and Ptdlns(4,5)P, as well as phosphatidylinositol 4-phosphate as substrates (3,25,26). Endothelial PLC activity toward phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate or phosphatidylinositol 3,4,5_trisphosphate was not examined in this study, because other investigators have demonstrated that the polyphosphoinositides produced by phosphatidylinositol3-kinase are poor substrate for PLC (38). The properties
of partially purified endothelial
PLCs were similar to those of bovine brain
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FIG. 5
3
2
,x .> z
4
5
6
7
8
Effect of pH on Phospholipase C Activitiy. The effect of pH was examined with PLC preparations, H-l (0) H-2 (Cl), H-3 (A) and H-4 (0). PLC activity using Ptdlns (A) or Ptdlns (4,5)P, (B) as substrate was assayed as described in “METHODS”, except that pH was varied as indicated. PLC activity is expressed as % of that obtained at the optimal pH for each enzyme preparation. The values shown are from the averages of duplicates which agree within 10% of the average. The experiment was repeated once and a similar result was obtained.
910
B. loo-.
Q
07
:
2
3
:
4
:
:
5
6
:
7
:
:
8
910
PH
FIG. 6
Effect
of
Deoxycholate
on
Phospholipase C Activitiy. The effect of deoxycholate was examined with PLC preparations, H-l (O), H-2 (II), H-3 (A) and H-4 (0). PLC activity using Ptdlns (A) or Ptdins (4,5)P, (B) as substrate was assayed as described in “METHODS”, except that deoxycholate level was varied. PLC activity is expressed as % of that obtained in the absence of deoxycholate for each enzyme preparation. The values shown are from the averages of duplicates which agree within 10% of the average. The experiment was repeated once and a similar result was obtained. 0
2
4
Deoxycholate,
8
6
mM
10
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enzymes. The pH optima were similar to bovine brain enzymes when either Ptdlns or Ptdlns(4,5)P, was used as substrate. Further, the slightly higher pH optimum and residual activity of H-2 at high pH were very similar to those of purified PLC-6 from bovine brain (4) and consistent with the identification by immunoblotting of H-2 as PLC-6. In addition, endothelial PLC isozymes are similar to the corresponding brain PLC isozymes in the dependence of hydrolysis of Ptdlns on Ca2’ (3,4), the effect of Ca2+ concentration on activities towards Ptdlns and Ptdlns(4,5), (3,4), and the different stimulatory effects of deoxycholate on PLC-8 and PLCy (3). In conclusion, three types of PLC isozymes (PLC-8, PLC-y and PLC-C) are present in the cytosol of porcine aortic endothelial cells. The shoulder (Peak-4, Fig. 1) observed during DEAE chromatography might be either a chromatographic abnormality or an additional isozyme. Findings that support the former notion are: the similarity of H-3 and H-4 preparations in the elution conditions from heparin-agarose column; the profiles of the effects of Ca2+, pH and deoxycholate; the molecular weights and immunological reactivity. Several subsequent attempts of partial purification, however, failed to yield a similar shoulder in DEAE-chromatography. Thus it is likely that only three types of PLC are present in the cytosol of porcine aortic endothelial cells. Nevertheless, we could not rule out the alternative possibility that Peak-4 may be an isoform of PLC with molecular weight, immunological reactivity, and catalytic properties very similar to PLC-y. The rationale behind this speculation is that a subtype of PLC-y has been demonstrated by molecular cloning in rat muscle (28) and in Epstein-Barr virus transformed human lymphocytes (29). The possibility of a variant of PLC-y in porcine endothelial cells awaits confirmation in future studies and is beyond the scope of the current effort. Acknowledgements We thank Dr. Sue Goo Rhee for the generous gift of monoclonal antibodies against phospholipases C isozymes, Dr. Hava Avraham for her assistance in immunoblotting experiments and Dr. Zenon Grabarek for his assistance in the estimation of calcium ion concentration. This work was supported by Research Grant HL28778 from the National Institute of Heart, Lung and Blood and by an American Heart Association Established Investigator Award to S.L.H. REFERENCES NISHIZUKA, Y. The role of protein kinase C in cell surface signal transduction 1. tumour promotion. Nature, 308, 693-698, 1984. BERRIDGE, M.J. and IRVINE, R.F. lnositol trisphosphate, 2. cellular sinal transduction. Nature, 372, 315321, 1984.
and
a novel second messenger
in
3. RYU, S.H., CHO, K.S., LEE, K.Y., SUH, P.G. and RHEE, S.G. Purification and Characterization of two immunologically distinct phosphoinositide-specific phospholipase C from bovine brain. J. Biol. Chem. 262, 12511-12518, 1987.
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RYU, S.H., SUH, P.G., CHO, KS., LEE, K.Y. and RHEE, S.G. Bovine brain cytosol 4. contains three immunologically distinct forms of inositol phospholipid- specific phospholipase C. Proc. Nat/. Acad. Sci. 84, 6649-6653, 1967. HOFMANN, S.L. and MAJERUS, P.W. Identification and properties of two distinct 5. phosphatidylinositol-specfic phospholipase C enzymes from sheep seminal vesicular glands, J. Ho/. Chem. 257, 6461-6469, 1982. BENNETT, C.F. 6. phosphoinositide-specific
and CROOKE, phospholipase
ST. Purification and Characterization of a C from guinea pig uterus. phosphorylation by
protein kinase C in vivo. J.Bio/. Chem. 262, 13789-l 3797, 1987. BANNO, Y., YADA, Y. and NOZAWA, Y. Purification and Characterization of Membrane7. bound Phospholipase C Specific for Phosphoinositides from Human Platelets. J. Biol. Chem. 263, 11459-l 1465, 1988. 8. KATAN, M. and PARKER, P.J. Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain. Eur. J. Chem. 768, 413-418, 1987. 9. SUH, P.G., RYU, S.H., MOON, K.H., SUH, H.W. and RHEE S.G. Cloning and Sequence of Multiple Forms of Phospholipase C. Cell, 54, 161-169, 1988.
10. BENNET, C.F., BALCAREK, J.M. VARRICHIO, A. and CROOKE, S.T. Molecular Cloning and Complete Amino Acid Sequence of Form-l Phosphoinositide-specific Phospholipase C. Nature, 334, 268-270, 1988. 11. RHEE, S.G., Phospholipid-Specific
SUH, P.G., RYU, S.H., and LEE, S.Y. Studies Phospholipase C. Science, 244, 546-550, 1988.
12. JAFFE, E.A. Ceil Biology of Endothelial
Cells. Human Pathology,
of
lnositol
78, 234-239, 1987.
13. MANTOVANI, A. and DEJANA, E. Cytokines as Communication Signals Leukocytes and Endothelial Cella. immunology Today, 70, 370-375, 1989.
between
14. BEVILACQUA, M.P., STENGELIN, S., GIMBRONE, M.A. Jr. and SEED, B. Endothelial Leukocyte Adhesive Molecule 1: An Inducible Receptor for Neutrophils Related to Complement Regulatory Proteins and Lectins. Science, 243, 1160-l 165, 1989. 15. KAYA, H., PATTON, G.M. and HONG, S.L. Bradykinin-induced Activation Phospholipase 4 Is Independent of the Activation of Polyphosphoinositide-hydrolyzing Phospholipase C. J. Ho/. Chem. 264, 4972-4977, 1989.
of
16. MOSCAT, J., MORENO, F., HERRERO, C., LOPEZ, C. and GARCIA-BARRENO, P. Endothelial Cell Growth Factor and lnonophore A231 87 Stimulation of Production of lnositol Phosphates in Porcine Aorta Endothelial Cells. Proc. Nat/. Acad. Sci. U.S.A. 85, 659-663, 1988.
416
PORCINE ENDOTHELIAL CELLS
Vol. 73, No. 6
17. BARTHA, K., MULLER-PEDDINGHAUS, Ft. and VAN ROOIJEN, L.A.A. Bradykinin and Thrombin Effects on Polyphosphoinositide Hydrolysis and Prostacylin Production in Endothelial Cells. Biochem. J. 263, 149-155, 1989. 18. LAMBERT, T.L., KENT, R.S. and WHORTON, A.R. Bradykinin Stimulation of lnositol Polyphosphate Production in Porcine Aortic Endothelial Cells. J. Biol. Chem. 267, 1528815293, 1988. 19. DERIAN, C.K. and MOSKOWITZ, M.A. Polyinositide Hydrolysis Carotid Artery Segments. J. 5iol. Chem. 267, 3831-3837, 1986.
in Endothelial
Cells and
20. JAFFE, E.A., GRULICH, J., WEKSLER, B.B., HARPEL, G. and WATANABE, K. Correlaion betweem Thrombin-induced Prostacyclin Produciton and lnositol Trisphosphate and Cytosolic Free Calcium Levels in Cultured Human Endothelial Cells. J. Biol. Chem. 262, 8557-8565, 1987. 21. HONG, S.L. Effect of Bradykinin and Thrombin on Prostacyclin Synthesis in Endothelial Cells from Calf and Pig Aorta and Human Umbilical Cord Vein. Thrombosis Research, 78, 787-95, 1980. 22. FOLICH, J., LEES, M. and STANLEY, S. A Simple Method for the Isolation Purification of Total Lipides from Animal Tissues. J. Biol. Chem. 226, 497-509, 1957. 23. LAEMMLI, U.K. Cleavage of Structural Protein during the Assembly Bacteriophage T4. Nature, 227, 680-685, 1970.
and
of the Head of
24. SUH, P.G., RYU, S.H., CHOI, W.C., LEE, K.Y. and RHEE, S.G. Monoclonal antibodies to Three Phospholipase C lsozymes from Bovine Brain. J, 5iol. Chem. 263, 14497-14504, 1988. 25. WILSON, D. B., BROSS, T.E., HOFMANN, S.L. and MAJERUS, Polyphosphoinositides by Purified Sheep Seminal Vesicle Phospholipase Chem. 259, 11718-l 1724, 1984.
P.W. Hydrolysis of C Enzymes. J. 5io.
26. REBECCHI, M.J. and ROSEN, O.M. Purification of a Phosphoinositide-specific Phospholipase C from Bovine Brain. J. Biol. Chem. 262, 12526-12532, 1987. 27. SMITH, M.R., LIU, Y.L., KIM, H., RHEE, S.H. and KUNG, H.F. Inhibition of Serum- and Res-Stimulated DNA Synthesis by Antibodies to Phospholipase C. Science 247, 1074-l 077, 1990. 28. EMORI, Y., HOMMA, Y., SORIMCACHI, H., KAWASAKI, H., NAKANISH, O., SUZUK, K. and TAKENAWA, T. A Second Type of Rat Phosphoinositide Phospholipase C Containing a src-related Sequence Not Essential for Phosphoinositide-hydrolyzing Activity. J. 5iol. Chem. 264, 21885-21890 , 1989.
Vol. 73, No. 6
PORCINE ENDOTHELIAL CELLS
417
29. OHTA, S., MATSUI, A., NAZAWA, Y. and KAGAWAN, Y. Complete cDNA Encoding a Putative Phospholipase C from Transformed Human Lymphocytes. FEBS 242, 31-35, 1988. 30. PERRIN, D.D. and SAYCE, LG. Computer Calculation of Equilibrium Concentrations Mixture of Metal Ions and Complexing Species. Talanta, 74, 833-842, 1967.
in
31. SILLEN, L.G. and MARTEL, A.E. Stability Constants of Metal Complexes. 2nd edn, Spec. Publ. No 17 (The Chemical society, London), 1967. 32.
Wahl, M.I., NISHIBE, S, SUH, P, RHEE, S.G., CARPENTER, G. Epidermal Growth Factor
Stimulates Tyrosine Phosphorylation of Phospholipase C-II Independently of Receptor Internalization and Extracellular Calcium. Proc. Nat/. Acad. Sci. USA, 86, 1568-l 572, 1989. 33. MARGOLIS, B., RHEE, S.G., FELFRT, S., MERVIC, M., LYALL, R., LEVITZKI, A., ULLRICH, A., ZILBERSTEIN, A. and SCHLESSINGER, J. EGF Induces Tyrosine Phosphorylation of Phospholipase C-II: a Potential Mechanism for EGF Receptor Signaling. Cell 57, 1101-l 107, 1989. 34. MEISENHELDER, J., SUH, P.G., RHEE, S.R. and HUNTER, T. Phospholipase C-y Is a Substrate for the PDGF and EGF Receptor Protein-tyrosine Kinases in Vivo and in Vitro. Cell 57, 1109-l 122, 1989. 35. SMRCKA, A.V., HEPLER, J.R., BROWN, K.O. and STERNWIES, P.C. Regulation of Polyphosphoinositide-specific Phospholipase C Activity by Purified Gq. Science 251,804-807, 1991. 36. TAYLOR, S.J., CHAE, H.Z., RHEE, S.G. and EXTON, J.H. Activation of pl lsozyme of Phospholipase C by CYSubunits of the Gq Class of G Proteins. Nature 350, 516-518, 1991. 37. AGRANOFF, B.W., MURTHY, P. andSEQUIN, E.B.Thrombin-induced Phosphodiesteratic Cleavage of Phosphotidylinositol Bisphosphate in Human Platelets. J. Biol. Chem. 258, 20762078, 1983. 38. SERUNIAN, L.A., HABER, M.T., FUKUI, T., KIM, J.W., RHEE, S.G., LOWENSTEIN, J.M., and CANTLEY L.C. Polyphosphoinositides Produced by Phosphatidylinositol 3-Kinase Are Poor Substrates for Phospholipases C from Rat Liver and bovine Brain. J. Biol. Chem. 264, 17809-l 5, 1989. 39. KAYA, H., PATTON, G.M., and HONG, S.L. Bradykinin-induced Activation Phospholipase & Is Independent of the Activation of Polyphosphoinositide-hydrolyzing Phospholipase C. J. Ho/. Chem. 264, 4972-4977, 1989.
of