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Arachidonate incorporation prostaglandin production in cultured endothelial and smooth muscle cells from pig aorta
Prostaglandins Leukotrienes and Medicine 11: 63-72,
1983
C. Ody+ and D. Duval**. *Respiratory Diseases, Hcpital Cantonal Universitaire, 1211 Geneva 4, Switzerland. **Department of Physiology and Pharmacology, INSERT U7, HijpitalNecker, Paris, France. (reprint requests to D. Duval) SUMARY In order to investigate the utilization of arachidonic acid by vasth muscle cular cells, we have compared cultured endothelial and cells from p glet aorta, after a 24 hour incubation with [y M-arachidonit acid ( f 4H] -AA). Ue studied both the release of labeled cyclo-oxygenase products, and the distribution of the radioactive fatty acid among lipids as determined by thin layer chromatography. As already described by others the main prostaglandins (PG) released by endothelial -cells were PGF2a and 6 keto-PGFIa whereas smooth muscle cells mainly produced PGE2. These differences wre associated with marked modification of the radioactivity distributi n among the various lipid classes. In particular, the proportion of [ sH] -AA incorporated into neutral lipids was much more important in smooth muscle than in endothelial cells ( -40% of the total incorpor ted radioactivity versus 8%). On the other hand, the distribution of [ 9 H 1 -oleic acid into cell lipids after a 24 hour incorporation was very similar in both types of cells. These results suggest the existence of precise mechanisms controlling the incorporation and the availability of the PG precursor in the various types of cells. This might in part explain the differences observed for different cell types in the PG secretion pattern. INTRODUCTION The metabolic pathways leading to the synthesis of eicosanoids have been extensively investigated over the past few years. It is now generally accepted that arachidonate, the fatty acid precursor of eicosanoids, is essentially derived from membrane phospholipid stores and then converted into various derivatives under the action of cyclooxygenase or lipoxygenate enzymes (l-3). However, many aspects of the intracellular regulation of arachidonic acid metabolism remain unclear as recently reviewed by Irvine (4). First, in many cell types, only a minimal proportion of the liberated arachidonate is converted into prostaglandins and hydroxyacids whereas the major part of the precursor 63
is either released in the extracellular medium, as unmetabolized fatty acid, or reincorporated into phospholipids under the action of acyltransferases. Several authors have in addition presented evidence for the existence of different pools of esterified arachidonate with different sensitivities to hormonal stimuli (5-7). Second, there are still controversies concerning the nature of the phospholipid species providing arachidonate for prostaglandin synthesis as well as the mechanisms of membrane lipid deacylation. Since arachidonate is generally esterified in the 2 position of phospholipids it was assumed that phospholipase A2 represents the major enzyme leading to arachidonate liberation (8). Nevertheless, there is now some evidence that this precursor can also be released from membrane phospholipids, mainly from phosphatidyl-inositol (PI) through the combined action of phospholipase C and diglyceride-lipase (9, 10). On the other hand, the pattern of incorporation of labelled arachidonic acid into cellular lipids varies greatly from one cell type to another : rat peritonal macrophages and MI myeloid cells show a preferential incorporation into phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE), platelets show a preferential incorporation into PE, whereas 3T3 cells show an exclusive incorporation into PC (11-14). In order to investigate the possible relationship between the pattern of fatty acid incorporation and that of prostaglandin production we have studied in parallel these two parameters in cultured endothelial and smooth muscle cells from the same origin. Our results demonstrate that the different profiles of PG production, which are observed in endothelial and smooth muscle cells, are associated with different distributions of arachidonate incorporation among the various lipid classes. MATERIAL AND METHODS Young piglets (large white strain, l-3 weeks old) were obtained from an agriculture center. All culture media and antibiotics were obtained from Gibco Biocult (Glasgow, Scotland). [3H] -arachidonic acid (78 Ci/mmole) was obtained from New England Nuclear (Paris, France) and 3H ] -oleic acid (504 Ci/mmole) was purchased from the Radiochemicaf Center (Amersham, UK). Organic solvents were bought from Merck (Darmstadt, Germany). Silica gel plates were Whatman LK6DF plates. Cell cultures were carried out as described previously (15). For endothelial cells first passage cultures were used, whereas for smooth muscle cells, cultures were taken from the first to the fifth passage. Incorporation of T3H1 -arachidonic acid and [3H] -oleic acid Confluent cell cultures were incubate for one hour with either 2 uCi/ml of r3H ] -arac idonate (2.5 x lo-6 ) or 1 u Ci/ml of [3H j oleic acid (2 x lo-'4M) in Dulbecco's medium. Fetal bovine serum ’
64
was then added to achieve a 2% final concentration, and the cells were further incubated for 23 more hours in the presence of the precursor. At the end of this incubation, the cells were washed three times with Hanks balanced salt solution (HBSS) at 37-C and received fresh mediun for an additional 2 hours incubation period. At the end of this incubation period, we measured in parallel the appearance of labelled PG into the medium and the incorporation into cellular lipids. Analysis of cellular lipids The cells were washed three times with cold HBSS, detached in 1 ml H20 with a rubber policeman and sonicated twice for 10 set at 4'C using a Branson 812 sonicator. Aliquots of the suspension were then taken for determination of radioactivity and protein contents. The rest of the sonicate was extracted twice by 3 ml of chloroform-methanol 2:l v/v. Lipid extracts were evaporated under N and separated on silica gel plates using chloroform-methanol-H20 $5: 25:4 (v/v/v) as a solvent system. After exposure to iodine vapor the radioactive lipids identified by comparison with known standards run in parallel, and the spaces in between were scraped off and counted by liquid scintillation spectrometry. For some samples, the identity of the different phospholipids was verified with a two dimensional thin layer chromatography system according to Abd el latif et al. (16) Prostaglandins were determined by following the release in the culture medium of radio-labelled materials or by radioimmunoassay. Previous experiments (15) have shown that the major prostaglandins produced by our cell cultures were 6 keto-PGF1, PGE2 and PGF2a and therefore, radioimmunoassays were carried out 50 measure these three compounds.
RESULTS Distribution of L3Hl-arachidonate among cellular lipids (a) Endothelial cells Arachidonic acid was readily incorporated into cellular lipids during the 24 h incubation period. The mean incorporation was 8115 + 875 dpm/ug prot. (mean + SD, n = 6) and represented more than 50% 07 the initially added rad?oactivity. As shown on the upper panel of Figure 1 the major incorporation was associated with phospholipix 2,8Q%, whereas only a small proportion was recovered in neutral lipids (free arachidonate + glycerides). The two dimensional thin layer chromatography analysis allowing the separation between phosphadidyl-
65
origin
front
I
I 0
PS
s
PE
CI;; 25c 0 I-
?C
;; s
--
Is1
I
o-
5o
b
t
I
IL --
.=.
.
r-
Ot origin
Figure 1: Distribution of radioactivity among cellular lipids after prelabelling with [3H]-arachidonic acid. Each value (mean + SD, n= 6) is expressed as a percentage of the total radioactivity hyered on the silicagel plate. (a) endothelial cells, (b) smooth muscle cells. PC: phosphatidylcholine, PE: phosphatidylPS: phosphatidylserine, ethanolamine, NL: neutral lipids. serine (PS) and phosphatidylinositol (PI) indicated that PI did not contain more than 2% of radioactive arachidonate (results not shown).
66
b) Smooth muscle cells Under similar experimental conditions, 3Hl carachidonic acid incorporation was lower in smooth muscle cclc s and amounted to 4450 + 786 dpm/ g prot. (mean + SD, n = 6). In addition, the distribution 07 the incorporated radioaztivity among the various lipids differed markedly from that observed for endothelial cells (Fi ure 1 nel). In this case, the radioactivity was almost MEequa y distr uted between neutral lipids and phospholipids. Additional TLC analysis showed however that the majority of the radioactivity associated with neutral lipids corresponded to triglycerides and not to unesterified arachidonate (results not shown). Distribution of r3H 1 -oleate among cell lipids The amounts of 13H 1 -oleic acid incorporated into cell lipids were very similar for both, endothelial and smooth muscle cells : after 24 hours in the presence of 2 uCi/ml, 56.4% and 54.6% of the initial radioactivity were respectively incorporated. In addition, the patterns of distribution of the incorporated radioactivity among cell lipids were comparable in the two cell types (Figure 2 and Table I).
Endothelial cells -n
Smooth muscle cells
n -
3H -Arachidonate
PI
NL
7.9 + 0.8
(6)
43.5 + 2.4
(6)
PC
18.2 + 1.8
(6)
15.7 + 0.6
(6)
PE
26.4 + 1.3
(6)
10.8 + 1.5
(6)
32.7 + 3.7
(6)
17.7 + 0.8
(6)
NL
20.2 + 2.40
(4)
25.5 t 6
(6)
PC
44.1 + 6.90
(4)
30
(6)
+
2s
3H -0leate
+ 3.1
Table I : Distribution of radioactive fatty acids among cellular lipids. n : number of experiments.
67
front
origin
+
+ PC
IL
“.: b
I
PIP
r’
!@t A ,1’ 5
--
IL
PS
PI
‘.
origin
front
t
ion of radioactivity among cellular lipids after I-oleic acid (mean + SD, n = 6). Analysis of prostaglandin production Several authors have recently suggested that the metabo ism of an exogenously added tracer may not constitute an accurate ref ection of the overall metabolism of endogenous arachidonate (4, 17 . In our cell cultures we have thus compared the nature of the pros aglandins produced after a 24 h prelabelling with [jH ] -arachidonate, with those obtained by radioimmunologic determinations. As shown in Table II, both methods gave very similar results. Endothelial cells ps ted essentially prostacyclin and PGF2a whereas smooth muscle cells produced almost exclusively PGE2.
68
In the experiments carried out in the presence of C3H]-arachidonate the percentage of the incorporated radtoactivity conWed into prostaglandins was very low and mpt?%m&ed 0.6 and 0.45% far e#dohelial and smooth muscle cells respectively. After prelabelling with 15HI -oleate there was no radioactive prostaglandins recovered in the medium during the 2 h incubation period.
Endothelial cells
% of labelled secreted prostaglandins
% of imnunoreactive secreted prostaglan-
6 Keto-PGFIa
41 + 2 (8)
35 + 7 (6)
PGF2a
37 + 5 (8)
48 -I8 (6)
PGE2
11 + 2 (8)
16 t 3 (6)
Smooth muscle cells
6 Keto-PGFIa
4 -I1 (6)
n.d::
PGF2cJ
17 + 2 (6)
13 + 1 (5)
PGE2
63 + 3 (6)
87 + I (5)
Table II : Patterns of prostaglandin secretion by endothelial and smooth muscle cells of piglet aorta. The separation of the radioactive prostaglandins was made by high pressure liquid chromatography (15). *n.d : not determined. DISCUSSION Using two different methods for the determination of PG production, we have shown that endothelial and smooth muscle cells in culture are characterized by typical secretion profiles. Both, radioimmunological measurements of tot 1 PG production and radiochemical analysis of labelled PG after [ & ] -arachidonate incorporation gave
69 PROS’L-
D
similar results, suggesting thus that, in this case, [3H]-arachidonate provides a satisfactory reflection of the overall arachidonate metabolism, as far as PGs are concerned. As already described by other authors in various species (18-lg.),we demonstrate that PG12 secretion is mainly restricted to endothelial cells and that this characteristic is maintained in cultured cells. The analyses of the distribution of the incorporated radioactivity among cellular lipids also revealed a striking difference between endothelial nd smooth muscle cells. In endothelial cells, the per3 centage of [HI - arachidonic acid incorporated into neutral lipids was very low ( ~8%) and similar to that observed in many other cell In contrast, this' percentage amounted to more types (11, 13, 20). than 40% in smooth muscle cells. This difference between the two cell types appears to be specific for arachidonic acid, since the distribution of oleic acid among cell lipids was comparable for both cell types.
In addition, our results showed a preferential incorporation of r3Hl -oleate into PC in both cell types, whereas the incorporation of r3Hl -arachidonate was more equally distributed among the various phospholipids (with the exception of PI). Spector and coworkers (21) work'ng on human endothelial cells, obtained an incorporation profile of [ sHI -arachidonate very different from what we obtained with piglet endothelial cells. Nevertheless, differences in experimental conditions (arachidonate concentration and duration of incubation) might be entirely responsible for these discrepancies. Experiments carried out in the presence of endoperoxide have previously suggested that their transformation into different prostaglandins by various cell types was dependent upon the nature of the enzymes prostaglandin synthetase present into these cells (22, 23). Our present demonstration that the differences in the patterns of prostaglandin production between endothelial and smooth muscle cells were associated with a striking change in the distribution of the incorporated arachidonate between cellular lipids may indicate a possible relationship between these two parameters. This sugge tion is reinforced by the demonstration that the distribution of [ 5 H ] -01eate among cellular lipids was very comparable in the two types of cells. Recent experiments by Beaudry et al (24) have similarly demonstrated a very high incorporation of radiolabelled fatty acids into triglycerides in MOCK cells but it remains to determine whether TG hydrolysis may provide arachidonic acid for PG synthesis.
70
REFERENCES
B, Goldyne M, Granstr&n E, Hamberg M, HaaAlarstr&nS & Malmsten C : Prostaglandins and thromboxanes. Ann. Rev. Biochem. 47 : 997, 1978.
1. Samuelsson
2. Moncada S. : Biological importance of prostacyclin. Brit. J. Pharmat. 76 : 3, 1982. 3. Sirois P & Borgeat P : From slow reacting substance of anaphyla;;s~(SRS-A) to leukotriene D4 (LTD4). J. Imnunopharmac. 2 : 281, . 4. Irvine RF : How is the level of free arachidonic acid controlled in mammalian cells ? Biochem. J. 204 : 3, 1982. 5. Schwartzman M, Liberman E & Raz A : Bradykinin and angiotensin II activation of arachidonic acid deacylation and prostaglandin E2 formation in rabbit kidney. Hormone-sensitive versus hormone-insensitive lipid pools of arachidonic acid. J. Biol. Chem. 256 : 2329, 1981
6. Needleman P, Wyche A, Bronson SD, Holmberg S & Morrison AR : Specific regulation of peptide-induced renal prostaglandin synthesis. J. Biol. Chem. 254 : 9772, 1979. 7. Humes JL, Sadowski S, Galavage M, Goldenberg M, Subers E, Bonney RJ & Kuehl FA : evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. 257 : 1591, 1982. 8. Pong SS, Hong SC & Levine L : Prostaslandin production by methvlcholanthrene transformed mouse balb 37 . Requirement for protein synthesis. J. Biol. Chem. 252 : 1408, 197s . 9. Rittenhouse-Simmons S. : Production of diglyceride from phosphatidyl inositol in activated human platelets. J. Clin. Invest. 63 : 580, 1979. 10. Bell RL, Kennerly DA, Stanford N & Majerus PW : Diglyceride Lipa_ _ : a pathway for arachidonate release from human platelets. Proc. iitl. Acad. Sci. USA 76 : 3238, 1979.
11. Hong SC & Deykin D : Specificity of phospholipases in methylcho-
lanthrene-transformed mouse fibroblasts activated by bradykinin, thrombin, serum and ionophore A23187. J. Biol. Chem. 254 :
11463, 1979. 12. Bonney RJ, Wightman PD, Davies P, Sadowski SJ, Kuehl FA & Humes JL : Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages. Biothem. J. 176 : 433, 1978.
71
13. Honma Y, Kasukabe T, Hozumi M & Koshihara Y : Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells. J. cell. Physiol. 104 : 349, 1980. 14. Blackwell GJ, Duncombe WG, Flower RG, Parsons MF & Vane JR : The distribution and metabolism of arachidonic acid in rabbit platelets during aggregation and its modification by drugs. Brit. J. Pharmac. 59 : 353, 1977. 15. Ody C, Seillan C & Russo-Marie F. 6 ketoprostaglandin FI a , Prostaglandins E2, F2 a and thromboxane B production by endothelial cells, smooth muscle cells and fibrob: asts cultured from piglet aorta. Biochim. Biophys. Acta 712 : 103, 1982. 16. Abdel Latif AA, Akhtar RA & Hawthorne JN : Acetylcholine increases the break wn of triphosphoinositide of rabbit iris muscle prelaP phosphate. Biochem. J. 162 : 61, 1977. belled with tl? 17. Coene MC, Van Hove C, Claeys M & Herman AG : Arachidonic acid metabolism by cultured mesothelial cells. Different transformations of exogenously added and endogenously released substrate. Biochim. Biophys. Acta 710 : 437, 1982. 18. MC Intyre DE, Pearson JD & Gordon JL : Localisation and stimulation of prostacyclin production in vascular cells. Nature 271 : 549, 1978. 19. Weksler BB, Marcus AJ & Jaffe EA : Synthesis of prostaglandin 12 (prostacyclin) by cultured human and bovine endothelial cells. Proc. Natl. Acad. Sci. USA 74 : 3922, 1977. 20. Jakschick BA, Lee LM, Shuffer G & Parker CW : Arachidonic acid metabolism in rat basophilic leukemia (RBLI) cells. Prostaglandins 16 : 733, 1978. 21. Spector AA, Kaduce TL, Hoak JC & Fry GL : Utilization of arachidonic and linoleic acids by cultured human endothelial cells. J. Clin. Invest. 68 : 1003, 1981. 22. Needleman P, Moncada S, Bunting S, Vane JR, Hamberg M & Samuelsonn B :Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxydes. Nature 261. 558, 1976. 23. Moncada S, Gryglewski R, Bunting S & Vane JR : An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibit platelet aggregation. Nature 263 : 663, 1976. 24. Beaudry GA, King L, Daniel LW & Waite M : Stimulation of deacylation in Radin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetra-decanoyl-phorbol-13-acetate-treated cells. J. Biol. Chem. 257 : 10973, 1982.
72
1983
C. Ody+ and D. Duval**. *Respiratory Diseases, Hcpital Cantonal Universitaire, 1211 Geneva 4, Switzerland. **Department of Physiology and Pharmacology, INSERT U7, HijpitalNecker, Paris, France. (reprint requests to D. Duval) SUMARY In order to investigate the utilization of arachidonic acid by vasth muscle cular cells, we have compared cultured endothelial and cells from p glet aorta, after a 24 hour incubation with [y M-arachidonit acid ( f 4H] -AA). Ue studied both the release of labeled cyclo-oxygenase products, and the distribution of the radioactive fatty acid among lipids as determined by thin layer chromatography. As already described by others the main prostaglandins (PG) released by endothelial -cells were PGF2a and 6 keto-PGFIa whereas smooth muscle cells mainly produced PGE2. These differences wre associated with marked modification of the radioactivity distributi n among the various lipid classes. In particular, the proportion of [ sH] -AA incorporated into neutral lipids was much more important in smooth muscle than in endothelial cells ( -40% of the total incorpor ted radioactivity versus 8%). On the other hand, the distribution of [ 9 H 1 -oleic acid into cell lipids after a 24 hour incorporation was very similar in both types of cells. These results suggest the existence of precise mechanisms controlling the incorporation and the availability of the PG precursor in the various types of cells. This might in part explain the differences observed for different cell types in the PG secretion pattern. INTRODUCTION The metabolic pathways leading to the synthesis of eicosanoids have been extensively investigated over the past few years. It is now generally accepted that arachidonate, the fatty acid precursor of eicosanoids, is essentially derived from membrane phospholipid stores and then converted into various derivatives under the action of cyclooxygenase or lipoxygenate enzymes (l-3). However, many aspects of the intracellular regulation of arachidonic acid metabolism remain unclear as recently reviewed by Irvine (4). First, in many cell types, only a minimal proportion of the liberated arachidonate is converted into prostaglandins and hydroxyacids whereas the major part of the precursor 63
is either released in the extracellular medium, as unmetabolized fatty acid, or reincorporated into phospholipids under the action of acyltransferases. Several authors have in addition presented evidence for the existence of different pools of esterified arachidonate with different sensitivities to hormonal stimuli (5-7). Second, there are still controversies concerning the nature of the phospholipid species providing arachidonate for prostaglandin synthesis as well as the mechanisms of membrane lipid deacylation. Since arachidonate is generally esterified in the 2 position of phospholipids it was assumed that phospholipase A2 represents the major enzyme leading to arachidonate liberation (8). Nevertheless, there is now some evidence that this precursor can also be released from membrane phospholipids, mainly from phosphatidyl-inositol (PI) through the combined action of phospholipase C and diglyceride-lipase (9, 10). On the other hand, the pattern of incorporation of labelled arachidonic acid into cellular lipids varies greatly from one cell type to another : rat peritonal macrophages and MI myeloid cells show a preferential incorporation into phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE), platelets show a preferential incorporation into PE, whereas 3T3 cells show an exclusive incorporation into PC (11-14). In order to investigate the possible relationship between the pattern of fatty acid incorporation and that of prostaglandin production we have studied in parallel these two parameters in cultured endothelial and smooth muscle cells from the same origin. Our results demonstrate that the different profiles of PG production, which are observed in endothelial and smooth muscle cells, are associated with different distributions of arachidonate incorporation among the various lipid classes. MATERIAL AND METHODS Young piglets (large white strain, l-3 weeks old) were obtained from an agriculture center. All culture media and antibiotics were obtained from Gibco Biocult (Glasgow, Scotland). [3H] -arachidonic acid (78 Ci/mmole) was obtained from New England Nuclear (Paris, France) and 3H ] -oleic acid (504 Ci/mmole) was purchased from the Radiochemicaf Center (Amersham, UK). Organic solvents were bought from Merck (Darmstadt, Germany). Silica gel plates were Whatman LK6DF plates. Cell cultures were carried out as described previously (15). For endothelial cells first passage cultures were used, whereas for smooth muscle cells, cultures were taken from the first to the fifth passage. Incorporation of T3H1 -arachidonic acid and [3H] -oleic acid Confluent cell cultures were incubate for one hour with either 2 uCi/ml of r3H ] -arac idonate (2.5 x lo-6 ) or 1 u Ci/ml of [3H j oleic acid (2 x lo-'4M) in Dulbecco's medium. Fetal bovine serum ’
64
was then added to achieve a 2% final concentration, and the cells were further incubated for 23 more hours in the presence of the precursor. At the end of this incubation, the cells were washed three times with Hanks balanced salt solution (HBSS) at 37-C and received fresh mediun for an additional 2 hours incubation period. At the end of this incubation period, we measured in parallel the appearance of labelled PG into the medium and the incorporation into cellular lipids. Analysis of cellular lipids The cells were washed three times with cold HBSS, detached in 1 ml H20 with a rubber policeman and sonicated twice for 10 set at 4'C using a Branson 812 sonicator. Aliquots of the suspension were then taken for determination of radioactivity and protein contents. The rest of the sonicate was extracted twice by 3 ml of chloroform-methanol 2:l v/v. Lipid extracts were evaporated under N and separated on silica gel plates using chloroform-methanol-H20 $5: 25:4 (v/v/v) as a solvent system. After exposure to iodine vapor the radioactive lipids identified by comparison with known standards run in parallel, and the spaces in between were scraped off and counted by liquid scintillation spectrometry. For some samples, the identity of the different phospholipids was verified with a two dimensional thin layer chromatography system according to Abd el latif et al. (16) Prostaglandins were determined by following the release in the culture medium of radio-labelled materials or by radioimmunoassay. Previous experiments (15) have shown that the major prostaglandins produced by our cell cultures were 6 keto-PGF1, PGE2 and PGF2a and therefore, radioimmunoassays were carried out 50 measure these three compounds.
RESULTS Distribution of L3Hl-arachidonate among cellular lipids (a) Endothelial cells Arachidonic acid was readily incorporated into cellular lipids during the 24 h incubation period. The mean incorporation was 8115 + 875 dpm/ug prot. (mean + SD, n = 6) and represented more than 50% 07 the initially added rad?oactivity. As shown on the upper panel of Figure 1 the major incorporation was associated with phospholipix 2,8Q%, whereas only a small proportion was recovered in neutral lipids (free arachidonate + glycerides). The two dimensional thin layer chromatography analysis allowing the separation between phosphadidyl-
65
origin
front
I
I 0
PS
s
PE
CI;; 25c 0 I-
?C
;; s
--
Is1
I
o-
5o
b
t
I
IL --
.=.
.
r-
Ot origin
Figure 1: Distribution of radioactivity among cellular lipids after prelabelling with [3H]-arachidonic acid. Each value (mean + SD, n= 6) is expressed as a percentage of the total radioactivity hyered on the silicagel plate. (a) endothelial cells, (b) smooth muscle cells. PC: phosphatidylcholine, PE: phosphatidylPS: phosphatidylserine, ethanolamine, NL: neutral lipids. serine (PS) and phosphatidylinositol (PI) indicated that PI did not contain more than 2% of radioactive arachidonate (results not shown).
66
b) Smooth muscle cells Under similar experimental conditions, 3Hl carachidonic acid incorporation was lower in smooth muscle cclc s and amounted to 4450 + 786 dpm/ g prot. (mean + SD, n = 6). In addition, the distribution 07 the incorporated radioaztivity among the various lipids differed markedly from that observed for endothelial cells (Fi ure 1 nel). In this case, the radioactivity was almost MEequa y distr uted between neutral lipids and phospholipids. Additional TLC analysis showed however that the majority of the radioactivity associated with neutral lipids corresponded to triglycerides and not to unesterified arachidonate (results not shown). Distribution of r3H 1 -oleate among cell lipids The amounts of 13H 1 -oleic acid incorporated into cell lipids were very similar for both, endothelial and smooth muscle cells : after 24 hours in the presence of 2 uCi/ml, 56.4% and 54.6% of the initial radioactivity were respectively incorporated. In addition, the patterns of distribution of the incorporated radioactivity among cell lipids were comparable in the two cell types (Figure 2 and Table I).
Endothelial cells -n
Smooth muscle cells
n -
3H -Arachidonate
PI
NL
7.9 + 0.8
(6)
43.5 + 2.4
(6)
PC
18.2 + 1.8
(6)
15.7 + 0.6
(6)
PE
26.4 + 1.3
(6)
10.8 + 1.5
(6)
32.7 + 3.7
(6)
17.7 + 0.8
(6)
NL
20.2 + 2.40
(4)
25.5 t 6
(6)
PC
44.1 + 6.90
(4)
30
(6)
+
2s
3H -0leate
+ 3.1
Table I : Distribution of radioactive fatty acids among cellular lipids. n : number of experiments.
67
front
origin
+
+ PC
IL
“.: b
I
PIP
r’
!@t A ,1’ 5
--
IL
PS
PI
‘.
origin
front
t
ion of radioactivity among cellular lipids after I-oleic acid (mean + SD, n = 6). Analysis of prostaglandin production Several authors have recently suggested that the metabo ism of an exogenously added tracer may not constitute an accurate ref ection of the overall metabolism of endogenous arachidonate (4, 17 . In our cell cultures we have thus compared the nature of the pros aglandins produced after a 24 h prelabelling with [jH ] -arachidonate, with those obtained by radioimmunologic determinations. As shown in Table II, both methods gave very similar results. Endothelial cells ps ted essentially prostacyclin and PGF2a whereas smooth muscle cells produced almost exclusively PGE2.
68
In the experiments carried out in the presence of C3H]-arachidonate the percentage of the incorporated radtoactivity conWed into prostaglandins was very low and mpt?%m&ed 0.6 and 0.45% far e#dohelial and smooth muscle cells respectively. After prelabelling with 15HI -oleate there was no radioactive prostaglandins recovered in the medium during the 2 h incubation period.
Endothelial cells
% of labelled secreted prostaglandins
% of imnunoreactive secreted prostaglan-
6 Keto-PGFIa
41 + 2 (8)
35 + 7 (6)
PGF2a
37 + 5 (8)
48 -I8 (6)
PGE2
11 + 2 (8)
16 t 3 (6)
Smooth muscle cells
6 Keto-PGFIa
4 -I1 (6)
n.d::
PGF2cJ
17 + 2 (6)
13 + 1 (5)
PGE2
63 + 3 (6)
87 + I (5)
Table II : Patterns of prostaglandin secretion by endothelial and smooth muscle cells of piglet aorta. The separation of the radioactive prostaglandins was made by high pressure liquid chromatography (15). *n.d : not determined. DISCUSSION Using two different methods for the determination of PG production, we have shown that endothelial and smooth muscle cells in culture are characterized by typical secretion profiles. Both, radioimmunological measurements of tot 1 PG production and radiochemical analysis of labelled PG after [ & ] -arachidonate incorporation gave
69 PROS’L-
D
similar results, suggesting thus that, in this case, [3H]-arachidonate provides a satisfactory reflection of the overall arachidonate metabolism, as far as PGs are concerned. As already described by other authors in various species (18-lg.),we demonstrate that PG12 secretion is mainly restricted to endothelial cells and that this characteristic is maintained in cultured cells. The analyses of the distribution of the incorporated radioactivity among cellular lipids also revealed a striking difference between endothelial nd smooth muscle cells. In endothelial cells, the per3 centage of [HI - arachidonic acid incorporated into neutral lipids was very low ( ~8%) and similar to that observed in many other cell In contrast, this' percentage amounted to more types (11, 13, 20). than 40% in smooth muscle cells. This difference between the two cell types appears to be specific for arachidonic acid, since the distribution of oleic acid among cell lipids was comparable for both cell types.
In addition, our results showed a preferential incorporation of r3Hl -oleate into PC in both cell types, whereas the incorporation of r3Hl -arachidonate was more equally distributed among the various phospholipids (with the exception of PI). Spector and coworkers (21) work'ng on human endothelial cells, obtained an incorporation profile of [ sHI -arachidonate very different from what we obtained with piglet endothelial cells. Nevertheless, differences in experimental conditions (arachidonate concentration and duration of incubation) might be entirely responsible for these discrepancies. Experiments carried out in the presence of endoperoxide have previously suggested that their transformation into different prostaglandins by various cell types was dependent upon the nature of the enzymes prostaglandin synthetase present into these cells (22, 23). Our present demonstration that the differences in the patterns of prostaglandin production between endothelial and smooth muscle cells were associated with a striking change in the distribution of the incorporated arachidonate between cellular lipids may indicate a possible relationship between these two parameters. This sugge tion is reinforced by the demonstration that the distribution of [ 5 H ] -01eate among cellular lipids was very comparable in the two types of cells. Recent experiments by Beaudry et al (24) have similarly demonstrated a very high incorporation of radiolabelled fatty acids into triglycerides in MOCK cells but it remains to determine whether TG hydrolysis may provide arachidonic acid for PG synthesis.
70
REFERENCES
B, Goldyne M, Granstr&n E, Hamberg M, HaaAlarstr&nS & Malmsten C : Prostaglandins and thromboxanes. Ann. Rev. Biochem. 47 : 997, 1978.
1. Samuelsson
2. Moncada S. : Biological importance of prostacyclin. Brit. J. Pharmat. 76 : 3, 1982. 3. Sirois P & Borgeat P : From slow reacting substance of anaphyla;;s~(SRS-A) to leukotriene D4 (LTD4). J. Imnunopharmac. 2 : 281, . 4. Irvine RF : How is the level of free arachidonic acid controlled in mammalian cells ? Biochem. J. 204 : 3, 1982. 5. Schwartzman M, Liberman E & Raz A : Bradykinin and angiotensin II activation of arachidonic acid deacylation and prostaglandin E2 formation in rabbit kidney. Hormone-sensitive versus hormone-insensitive lipid pools of arachidonic acid. J. Biol. Chem. 256 : 2329, 1981
6. Needleman P, Wyche A, Bronson SD, Holmberg S & Morrison AR : Specific regulation of peptide-induced renal prostaglandin synthesis. J. Biol. Chem. 254 : 9772, 1979. 7. Humes JL, Sadowski S, Galavage M, Goldenberg M, Subers E, Bonney RJ & Kuehl FA : evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. 257 : 1591, 1982. 8. Pong SS, Hong SC & Levine L : Prostaslandin production by methvlcholanthrene transformed mouse balb 37 . Requirement for protein synthesis. J. Biol. Chem. 252 : 1408, 197s . 9. Rittenhouse-Simmons S. : Production of diglyceride from phosphatidyl inositol in activated human platelets. J. Clin. Invest. 63 : 580, 1979. 10. Bell RL, Kennerly DA, Stanford N & Majerus PW : Diglyceride Lipa_ _ : a pathway for arachidonate release from human platelets. Proc. iitl. Acad. Sci. USA 76 : 3238, 1979.
11. Hong SC & Deykin D : Specificity of phospholipases in methylcho-
lanthrene-transformed mouse fibroblasts activated by bradykinin, thrombin, serum and ionophore A23187. J. Biol. Chem. 254 :
11463, 1979. 12. Bonney RJ, Wightman PD, Davies P, Sadowski SJ, Kuehl FA & Humes JL : Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages. Biothem. J. 176 : 433, 1978.
71
13. Honma Y, Kasukabe T, Hozumi M & Koshihara Y : Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells. J. cell. Physiol. 104 : 349, 1980. 14. Blackwell GJ, Duncombe WG, Flower RG, Parsons MF & Vane JR : The distribution and metabolism of arachidonic acid in rabbit platelets during aggregation and its modification by drugs. Brit. J. Pharmac. 59 : 353, 1977. 15. Ody C, Seillan C & Russo-Marie F. 6 ketoprostaglandin FI a , Prostaglandins E2, F2 a and thromboxane B production by endothelial cells, smooth muscle cells and fibrob: asts cultured from piglet aorta. Biochim. Biophys. Acta 712 : 103, 1982. 16. Abdel Latif AA, Akhtar RA & Hawthorne JN : Acetylcholine increases the break wn of triphosphoinositide of rabbit iris muscle prelaP phosphate. Biochem. J. 162 : 61, 1977. belled with tl? 17. Coene MC, Van Hove C, Claeys M & Herman AG : Arachidonic acid metabolism by cultured mesothelial cells. Different transformations of exogenously added and endogenously released substrate. Biochim. Biophys. Acta 710 : 437, 1982. 18. MC Intyre DE, Pearson JD & Gordon JL : Localisation and stimulation of prostacyclin production in vascular cells. Nature 271 : 549, 1978. 19. Weksler BB, Marcus AJ & Jaffe EA : Synthesis of prostaglandin 12 (prostacyclin) by cultured human and bovine endothelial cells. Proc. Natl. Acad. Sci. USA 74 : 3922, 1977. 20. Jakschick BA, Lee LM, Shuffer G & Parker CW : Arachidonic acid metabolism in rat basophilic leukemia (RBLI) cells. Prostaglandins 16 : 733, 1978. 21. Spector AA, Kaduce TL, Hoak JC & Fry GL : Utilization of arachidonic and linoleic acids by cultured human endothelial cells. J. Clin. Invest. 68 : 1003, 1981. 22. Needleman P, Moncada S, Bunting S, Vane JR, Hamberg M & Samuelsonn B :Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxydes. Nature 261. 558, 1976. 23. Moncada S, Gryglewski R, Bunting S & Vane JR : An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibit platelet aggregation. Nature 263 : 663, 1976. 24. Beaudry GA, King L, Daniel LW & Waite M : Stimulation of deacylation in Radin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetra-decanoyl-phorbol-13-acetate-treated cells. J. Biol. Chem. 257 : 10973, 1982.
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