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The effects of somatostatin on the arachidonate cascade of platelets
PROSTAGLANDINS
THE EFFECTS
OF SOMATOSTATIN
ON THE ARACHIOCINATE CASCAOE
Zs. Mezei, Department
A. Geese,
OF PLATELETS
G. Telegdy
of Pathophysiology, Albert Szent-Gyorgyi Szeged, Hungary
Medical
University,
ABSTRACT Somatostatin (10e9 M) significantly elevated the synthesis of thromboxane B in rat platelets. The transformation of arachidonic acig to active lipsxygenase metabolites was suppressed by somatostatin (loand 10-O M). The ratio of the lipoxygenase/cyclooxygeRase products was signi-
ficantly reduced by the po+ypepQde (10 _5and 10 M) in rat platelets. Higher concentrations (10 , 10 and 10 M) of somatostatin did not modify the lipoxygenase pathway of the platelets. The synthesis of the vasoconstrictor - proa$gregatorN cyclooxygenase products was stimulated by the oolvoeotide (10 and 10 M). while the formation of vasodilatator antiaggrkgatory cyclooxygenaye mktaboiites was induced by higher concentrations of somatostatin (10 and 10 M). Somatostatin might act on the deacylation process of phospholipids, reducing the free arachidonic acid substrate level, resulting in a lower lipoxygenation rate in the platelets, which could be responsible for the increased formation of thromboxane. The contradictory results reported by others concerning the action of somatostatin on the platelet function might be explained by our results that the effect of somatostatin depends on the applied dose. INTROOUCTION
The cyclooxygenase and lipoxygenase pathways of the arachidonate cascade have been implicated in many physiologic and pathologic conditions (1). Arachidonate metabolites are not stored to any appreciable extent in any tissue (2), and their presence therefore reflects de novo synthesis and release. The precursor fatty acids are liberated by a receptor-mediated activation of phospholipase A phospholipase C and diacylglycerol kinase from phospholipids (3, 4), ?&lowed by the conversion of these precursor fatty acids to prostaglandins (PG-s), thromboxanes (TX-S) or peptide-containing lipids, leukotrienes (LT-s) via endoperoxide or peroxide formation (5, 6). The mechanisms by which various stimuli induce the deacylation and/or reacylation of arachidonic acid esterified in position 2 of phospholipids, and the synthesis of arachidonate metabolites, have not been unequivocally demonstrated. The evidence suggests that the structurally different neuropeptides may exert a regulatory role through activation of the calcium channel and/or phospholipid-arachidonate metabolism (1). Mielke et al. (7) found no clinically relevant changes in coagulation and platelet function after somatostatin treatment in diabetics. In vitro investigations have as yet provided no clear-cut evidence that somatostatin exerts a direct influence on human platelets (8, 9). In fact, while
SEPTEMBER
1988 VOL.
36 NO. 3
399
PROSTAGLANDINS
some investigators have detected a reduction in platelet number (10) and aggregation in man (8, 10) after the infusion of somatostatin, others have reported that the drug has some proaggregating effects in vivo (11). Gragnoli et al. (12) found that somatostatin treatment is accompained by an increased platelet activation and by a concurrent decrease in the level of the stable metabolite of TxA2. In vitro studies were undertaken to explore the possible role matostatin in the regulation of the cyclooxygenase and lipoxygenase ways of the arachidonate cascade in rat platelets.
of sopath-
MATERIALANDMETHODS Chemicals Arachidonic acid (grade I) was purchased from Sigma Chemical Co, St. Louis, MO (USA). l-14C-Arachidonic acid (2035 MBq/mMspec. act.) was obtained from Amersham (England). TC Medium 199 was purchased from DIFCO Laboratories, Detroit, Mich. (USA). Silica gel G thin layer plates (0.25 mm) were obtained from Merck AG, Oarmstadt (FRG). PGE2, PGD2, TxA2, TxB2, PGF2alpha and 6-oxo-PGFlalpha were generously provided by Or J.E. Pike, Upjonn Co, Kalamazoo (USA). Hydroxyeicosatetraenoic acid (HETE) standards were purchased from Calbiochem, La Jolla, Ca (USA). Somatostatin (Stilamin) was purchased from Serono, Italy. Chemicals were of analytical grade and obtained commercially, unless otherwise stated. Isolation
of platelets
Blood was drawn from the abdominal aorta of male rats of the CFY strain (body weight: 200+15 g) under light ether anaesthesia. Samples were diluted with phosphste buffer (pH 7.4) which contained 5.8 mMEIJTA and 5.55 mMglucose. The platelet-rich plasma was collected after the whole blood had been centrifugated at 200 g for 10 min at room temperature. The platelets were sedimented from the supernatant by means of centri$ugatfon at 20009 for 10 min. The pell@ cortained the platelets(2x 10 ml ) and the red blood cells (lx 10 ml ). Erythrocytes are capable to metabolize arachidonic acid by the lipoxygenase pathway and release 12-HETE, dihydroxyeicosatetraenoic acid and 6-sulfidopeptide-containing leukotrienes (13). Therefore erythrocytes were lysed with hyposmic ammonium chloride (O.B3%, 9 parts) containing EOTA (0.02%, 1 part) at 4 ‘C for 15 min. According to our preliminary experiments this treatment did not modify the arachidonate cascade of platelets. The platelets were washed 3 times with phosphate buffer (pH 7.4, containing 5.8 mMEDTA and 5.55 mMglucose). After centrifugation (at 2000 g, for 10 min, at room temperature), platelets were resuspended in TC Medium 199. TC Medium 199 was applied, because the arachidonate cascade of platel@ was the most ac ive in this medium and less active or inactive in Ca depleted or Ca” free incubation mixtures. Analysis
of eicosanoids:
The platelets (1OB ml-l_5in egch sample) were incubated at 37 ‘C for M) was then added to the incubation 5 min, and somatostatin (10 -10
400
SEPTEMBER
1988 VOL. 36 NO. 3
PROSTAGLANDINS
-14 C-amixture. The enzyme reaction was started by the introduction of 1 rachidonic acid (3.7 kBq) as tracer substrate in the incubation mixture. Ten minutes later, the enzyme reaction was quenched by bringing the pH to 3 with formic acid. According to Dahl and Uotila (14) and our unpublished results 5-10 min is an adaquate time period for labelling platelets. The samples were then extracted with ethyl acetate (2x3 ml). The organic phases were pooled and evaporated to dryness under nitrogen. The residues were reconstituted in 150 ul ethyl acetate and quantitatively applied to Silica-gel G thin layer plates. The plates were developed to a distance of 15 cm in an organic phase of ethyl acetate: acetic acid:2,2,4_trimethylpentane:water (110:20:30:100), a high pressure thin layer chromatograph being used. Each 3 mm band of the chromatograms was scraped off and the radioactivity was determined in a Beckman LS 1800 liquid scintillation counter, using 5 ml toluene containing 0.4% w/v PPO, 0.02% w/v POPOP and 10% v/v ethanol. The radiolabelled
products standards,
of arachidonic which were
High-performance Reversed-phase
acid were identified with unlabelled authentic detected with anisaldehyde reagent (15).
liquid
chromatography
high-performance
liquid
ChromatogrRphy
(HPLC) was per-
C formed on a column (4.6~250 mm) packed with LiChrosorb les). The solvent used for the detection of 12-HETE was a '#i~~u~~ ~~'~~~_ hanol:water (75~25) containing 0.01% of acetic acid and adjusted to pH 5.6 with NH40H and monitored at 235 nm with a UV detector 308 of Labor MIM,
Hungary. Statistical
analysis
was performed
via
Student’s
t test.
RESULTS
5
10 DISTANCE
15
IN CM
Fig. 1. Separation of arachidonate metabolites of rat platelets by high The plate was developed to a distance pressure thin layer chromatograph. of 15 cm - in 25 min - in an organic phase of ethyl acetate: acetic acid: 2,2,4_trimethylpentane:water (110:20:30:100). (1) 6-keto-PGFlalpha (2) PGF2alpha, (3) PGE2, (4) TxB2, (5) PGD2, (6) HHT, (7) 12-HETE, (8) 12-HPETE.
SEPTEMBER
1988 VOL.
36 NO. 3
401
PROSTAGLANDINS
A typical radiochromatogram of rat platelet arachidonate metabolites is shown on Fig. 1. All the major cyclooxygenase products of arachidonate cascade are synthesized by platelets and could be separated by high pressure thin layer chromatography (Fig. 1).
* 10
1
8 TxE$
_llll I_
6
% 4.
2.
C
ro-’
O-8 169 SOMATOSTATIN
10-6
w5 M
Fig. 2. In vitro effects of somatostatin on synthesis of TxB2 in rat platelets. TxB2 is expressed (on the ordinate) as a percentage of t$e total arachidonfte metapglites. C = control. Each sample contained 10 platelets ml and l- C-arachidonic acid (3.7 kBq). Each value is the mean L S.E. of 6 determinations. x p = 0.05 Table
I. In vitro effects of somatostatin on synthesis of proaggregatory and antiaggregatory prostanoids in rat platelets
c
1O-9 M
1O-B M
SOMATOEjTATIN lO-6 M lo- M
lO-5 M
Proaggregatory
13.77
17.;;
19.;:
13.57
14.79
14.02
PG-s
-t-o.54
+0.66 -
~1.41
50.96
~1.63
20.65
Antiagregatory
10.17
10.77
13.63
15.2!!
14.35
12.02
PG-s
-+0.58
_+0.38
_+1.66
-+1.73
-+1.47
LO.73
Each sample contained lo8 (3.7 kBq). The quantity @ percentage of the total prostanoids are PGFZalpha PG12, PGE2 and PG02. Each x p = 0.05; *X pcO.02. C
402
pQtelets ml-1 and 1-14C-arachidonic acid C-prostanoids (PG-s) is expressed as a C-arachidonate metabolites. Proaggregatory and TxB2. Antiaggregatory prostanoids are value is the mean -+ S.E. of 6 determinations. = control.
SEPTEMBER 1988 VOL. 36 NO. 3
PROSTAGLANDINS
The synthesis of Txl32 in rat platelet3 was modified by somatostatin. Onlv one concentration of somatostatin (10 M) induced a sionificant increase in the formation of TxB?, Ther& was a t$ndency to inhibition at certain somatostatin doses (10 , 10 and 10 M), but the changes were not significant (Fig. 2).
3
9
SOMATOSTATIN an Fig. 3. In vitro effects of somatostatin on ratio of proaggregatory g antiaggregatofy prostftoids in rat platelets. Each sample contained 10 platelets ml and l- C-arachidonic acid (3.7 kBq). C = control. Each value is the mean -+ S.E. of 6 determinations. x p-=0.05; x): ~~0.02. Table
II. In vitro effects of somatostatin on lipoxygenase oxygenase pathways of rat platelets.
and cyclo-
SOMATOSTATIN
C 1O-9 M
10-B M
lO-7 M
1O-6 M
lO-5 M
Lipoxygenase
75.97
ft* 71.97
66.4;
71.2:
70.83
73.90
products
_+0.32
+0.49 -
-+2.83
+2.75 _
_+3.10
-+1.37
Cyclooxygenase
24.03
X%X 28.03
33.5;
28.80
29.17
26.10
products
_+0.32
-+0.49
-+2.83
-i2.75
-+3.10
+1.37 -
acid Each sample contained 1OB platelets ml-l and l-14C-arachidonic (3.7 kBq). Lipoxygenase products are: HPETE-s and HETE-s. Cyclooxygenase products: PG12, PGF2alpha14PGE2, TxB2 and PGOZ. They are expressed as percentages of the total C-arachidonate metabolites. Each value is the mean) S.E. of 6 determinations. x ~~0.05; XX)! ~~0.01. C = control.
SEPTEMBER
1988 VOL. 36 NO. 3
403
PROSTAGLANDINS
Almost 1.5 times more proaggregatory - vasoconstrictor (TxA2 and PGF2aloha) then antiaaareoatorv - vasodilatator orostanoids (PGI2._PGE2 and6PGb2).were formed-in The platelets in vitro.’ Somatostatin (lOL’ and M) resulted in a significant decrease in the ratio of proaggregatory 10 - vasodilatator prostanoid synthesis (Fig. 3). This effect was the result of the elevated formation of antiaggregatory - vasodil,atator prostanoids (Table I). Lower concentrations of soM) resulted in an increased synthesis of proaggregatory matostatin (10 - vasoconstrictor cyclooxygenase products in rat platelets (Table I.).
In rat platelets, the lipoxygenase pathway of the arachidonate cascade predominates. In controls, three times more lipoxygegase metabolites and 10 M) sigwere formed than cyclooxygenase ones. Somatostatin (10 nificantly reduced the ratio of lipoxygenase and cyclooxygenase metabolites in the platelets (Fig. 4). Both the cyclooxygenase and lipoxygenaseq pathway8 of the arachidonate cascade were modified by somatostatin (10 and 10 M). Somatostatin resulted in an increased synthesis of cyclooxygenase products, while the formation of lipoxygenase metabolites was sign nificantly reduced. The optimal dose of somatostatin was found to be 10 M (Table II).
L/C
C
'
SOMATOSTATIN
il1 lo+
lO*M
Fig. 4. Effects of somatostatin on ratio of aipoxygenase a!f’ cycloo~xand lCgenase metabolites. Each sample contained 10 platelets ml arachidonic acid (3.7 kBq). L/C = ratio of lipoxygenase and cyclooxygenase metabolites of the arachidonate cascade. C = control. Each value is the mean 2 S.E. of 6 determinations. XX pcO.02; XXX p c 0.01.
404
SEPTEMBER
1988 VOL. 36 NO. 3
PROSTAGLANDINS
The formation of arachidonate metabolites in rat pla;$lets wapSsignificantly inhibited in the presence of somatostatin (10 and 10 M) (Fig. 5).
8 g
28
! l-
24
I
1U61O-5M SOMATOSTATIN
Fig. 5. In vitro effects of somatostatin on form8tion of arachifona$& metabolites in platelets. Each sample contained 10 platelets ml .l- -C-arachidonic acid transformed to cyclooxygenase and lipoxygenase products. C = control. Each value is the mean -+ S.E. of 6 determinations. x ~~0.05; 33% pco.01. DISCUSSION In accordance with the results of Srivastava and Tiwari (16) and Dahl and Uotila (14) obtained in human platelets all the major cyclooxygenase metabolites of arachidonate cascade are synthesized by rat platelets, as well. These in vitro data show that somatostatin in certain concentrations could modify the arachidonate cascade of rat platelets. The transformation of arachidgnic acidSto lipoxygenase metabolites was inhibited by somatostatin (10 and 10 M). This could be explained in terms of two mechanisms. First, this polypeptide might have a direct effect on the lipoxygenase pathway of the platelets, resulting in a reduced formation of 12HPETE and 12-HETE. Second, somatostatin might act on the deacylation process, reducing the free arachidonic acid substrate level, which might result in a lower lipoxygenation rate in the platelets. The second hypothesis could be supported by the results of Aharony et al. (17), who found a diminished platelet aggregability at higher concentrations of arachidonic acid, which was associated with marked increases in the ratio of lipoxygenase/cyclooxygenase products. The lipoxygenase product 12-HPETE has been
SEPTEMBER
1988 VOL.
36 NO. 3
405
PROSTAGLANDINS
proposed as an inhibitor of platelet aggregation and secretion induced by arachidonic acid or collagen (17, 18). This lipoxygenase metabolite could also inhibi-4 the formation of TX in activated platelets (17). The somatostatin (10 M) - induced low lipoxygenase activity and HPETE level might be responsible for the increased formation of proaggregatory and vasoconstrictor TX in rat platelets. Free arachidonic acid can be derived from inositol phospholipids through two consecutive reactions, catalysed by phospholipase C and diacylglycerol lipase (191, and in platelets it can also be derived from phosphatidic acid via its specific lipase (3, 20). However, in most tissues arachidonic acid is also released by phospholipase A2 from phosphatidylcholine and phosphatidylethanolamine. After receptor stimulation, the two phospholipase perhaps act in concert. It is important to note that diacylglycerol is produced in membranes only transiently, presumably due both to its conversion back to inositol phospholipids and to its further degradation to arachidonic acid for TX, HPETE and HETE synthesis. Somatostatin might shift the phospholipid breakdown to the phospholipase C and diacylglycerol lipase pathway, which results in a lower release of free arachidonic acid. The precise mechanism by which somatostatin affects cellular processes after binding to its receptors is not yet known conclusively. Several investigators have proposed that somatostatin binds to specific receptors, which results in the inhibition of adenylate cyclase activity, and this might reduce the phosphorylation of lipomodulin (21) by the CAMP -dependent protein kinase. Finally, the release of free arachidonic acid by phospholipases might be reduced.
The contradictory results reported concerning the action of somatostatin on the platelet function could be explained by our results that the effect of the polypeptide depends on the applied dose. 09r inves$gations demonstrated that low somatostatin concentrations (loand 10 M) increased the synthesis of proaggregatory cyclooxygenase rat platelets, while higher somatostatin concentrations induced the synthesis of antiaggregatory products.
meJybolites_$(10 and 10 M
ACKNOWLEDGEMEN This work was partly
Ministry
of Social
supported by the Scientific and Health, Hungary.
Research Council,
REFERENCES 1.
and G. Telegdy. Neuropeptides and arachidonate in the central nervous system. In: Front. Horm. Res. (G. Telegdy ed.) Karger, Easel, 1987. g:299.
Geese,
cascade
A., zs. Mezei
2.
Riper, p. and J. Vane. The release of prostaglandins other tissue. Ann. N.Y. Acad. Sci. 183:363, 1971.
3.
Billah,
406
from lung and
M.M., E.G. Lapetina and P. Cuatrecasas. Phospholipase A2 acspecific for phosphatidic acid. A possible mechanism for the production of arachidonic acid in platelets. J. biol. Chem. 256:5399, 1981.
tivity
SEPTEMBER
1988 VOL. 36 NO. 3
PROSTAGLANDINS
4.
Marcus, A.J., M.J. Broekman, 6.6. Neksler, E.A. Jaffe, L.B. Safier, H.L. Ullman, N. Islam and K. Tack-Goldman. Arachidonic acid metabolism in endothelial cells and platelets. Ann. N.Y. Acad. Sci. 401: 195, 1982.
5.
Hamberg, M. and B. Samuelsson. transformations of arachidonic Acad. Sci. USA -71:3400, 1974.
6.
Samuelsson B. Introduction: new trends in prostaglandin Adv. Prostaglandin Thromboxane Res. 1~1, 1976.
7.
Mielke, C.H., J.E. Gerich, M. Lorenzi, E. Tsalikian, R. Roviden and P.H. Forsham. The effect of somatostatin on coagulation and platelet function in man. N. Engl. J. Med. _293:4BO, 1975.
8.
Besser, G.M., A.M. Paxton and S.A.N. Johnson. function by growth hormone release-inhibiting 1975.
9.
Chiang,T.M., W.C. Duckworth, E.H. Beachey and A.H. Kang. The effect of somatostatin on platelet aggregation. Endocrinology -97:753, 1975.
Prostaglandin endoperoxides. Novel acid in human platelets. Proc. Natn.
research.
Impairment of platelet hormone. Lancet l-:1166,
10. Scheck, R., S. Raptis, H. Rasche and F. Escobar of somatostatin on platelets: in vitro studies. 219, 1977.
Jimenez. Diabetes
The effect Metab. -3:
11. Guigliano, IJ., I.Coppola, L. Misso, A. Tirelli, N. Passairello and F. O'Onofrio. Further studies on the significance of circulating platelet aggregates induced by somatostatin in man. Metabolism -30:172, 1981. 12. Gragnoli, G., A.M. Signorini and I. Tanganelli. Effects of somatostatin on the behaviour of thromboxane B and beta-thromboglobulin in &:104, 1985. type I diabetes.Acta Endocrinologica 13. Kobayashi, T. and L. Levine. Arachidonic cytes. J. Biol. Chem. _258:9116, 1983.
14.
~~~~;l,“~~~ma~~o~~n~~~i~~. ro. Prostaglandins
15. Kiefer,
tion 1975.
Verapamil C-arachidonic
Leukotrienes
acid metabolism
by erythro-
decreases the formation of thromacid in human platelets in vit-
and Medicine
-17:191,
1983.
H.C., C.R. Johnson and K.L. Arora. Calorimetric identificaof prostaglandins in subnanomole amounts. Anal. Biochem. -68:336,
16. Sriy$stava,
K.C. and K.P. Tiwari.
In vitro
platelet
utilization
of
humans of different age and sex and in acute myocardial infarction patients: A comparative study for possible diagnostic purposes. Prostaglandins and MedicineL:229, 1981.
(l-
C) arachidonic
SEPTEMBER
acid
1988 VOL.
in normal
36 NO. 3
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PROSTAGLANDINS
17. Aharony, O., J.B. Smith and M.J. Silver. Regulation of arachidonateinduced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid. Biochim. biophys. Acta 1718:193, 1982. 18. Vericel, E. and M. Layarde. 15-hydroperoxyeicosatetraenoic hibit human platelet aggregation. Lipids -15:472, 1980.
acid in-
19. Bell, R.L., D.A. Kennerley, N. Stanford and P.W. Majerus. Oiglyceride lipase: a pathway of arachidonate release from human platelets. Proc. natn. Acad. Sci. USA -76:3238, 1979. 20. Billah, M.M., E.G. Lapetina and P. Cuatrecasas. The initial action of thrombin on platelets conversion of phosphatidyl-inositol to phosphatidic acid preceding the production of arachidonic acid. J. biol. Chem. _256:5037, 1981. 21. Hirata, F. The regulation of lipomodulin, a phospholipase inhibitory protein in rabbit neurotrophils by phosphorylation. J. biol. them. -256:7730, 1981.
Editor:
408
P. Ramwell
Received:
3-28-88
Accepted:
7-8-88
SEPTEMBER 1988 VOL. 36 NO. 3
THE EFFECTS
OF SOMATOSTATIN
ON THE ARACHIOCINATE CASCAOE
Zs. Mezei, Department
A. Geese,
OF PLATELETS
G. Telegdy
of Pathophysiology, Albert Szent-Gyorgyi Szeged, Hungary
Medical
University,
ABSTRACT Somatostatin (10e9 M) significantly elevated the synthesis of thromboxane B in rat platelets. The transformation of arachidonic acig to active lipsxygenase metabolites was suppressed by somatostatin (loand 10-O M). The ratio of the lipoxygenase/cyclooxygeRase products was signi-
ficantly reduced by the po+ypepQde (10 _5and 10 M) in rat platelets. Higher concentrations (10 , 10 and 10 M) of somatostatin did not modify the lipoxygenase pathway of the platelets. The synthesis of the vasoconstrictor - proa$gregatorN cyclooxygenase products was stimulated by the oolvoeotide (10 and 10 M). while the formation of vasodilatator antiaggrkgatory cyclooxygenaye mktaboiites was induced by higher concentrations of somatostatin (10 and 10 M). Somatostatin might act on the deacylation process of phospholipids, reducing the free arachidonic acid substrate level, resulting in a lower lipoxygenation rate in the platelets, which could be responsible for the increased formation of thromboxane. The contradictory results reported by others concerning the action of somatostatin on the platelet function might be explained by our results that the effect of somatostatin depends on the applied dose. INTROOUCTION
The cyclooxygenase and lipoxygenase pathways of the arachidonate cascade have been implicated in many physiologic and pathologic conditions (1). Arachidonate metabolites are not stored to any appreciable extent in any tissue (2), and their presence therefore reflects de novo synthesis and release. The precursor fatty acids are liberated by a receptor-mediated activation of phospholipase A phospholipase C and diacylglycerol kinase from phospholipids (3, 4), ?&lowed by the conversion of these precursor fatty acids to prostaglandins (PG-s), thromboxanes (TX-S) or peptide-containing lipids, leukotrienes (LT-s) via endoperoxide or peroxide formation (5, 6). The mechanisms by which various stimuli induce the deacylation and/or reacylation of arachidonic acid esterified in position 2 of phospholipids, and the synthesis of arachidonate metabolites, have not been unequivocally demonstrated. The evidence suggests that the structurally different neuropeptides may exert a regulatory role through activation of the calcium channel and/or phospholipid-arachidonate metabolism (1). Mielke et al. (7) found no clinically relevant changes in coagulation and platelet function after somatostatin treatment in diabetics. In vitro investigations have as yet provided no clear-cut evidence that somatostatin exerts a direct influence on human platelets (8, 9). In fact, while
SEPTEMBER
1988 VOL.
36 NO. 3
399
PROSTAGLANDINS
some investigators have detected a reduction in platelet number (10) and aggregation in man (8, 10) after the infusion of somatostatin, others have reported that the drug has some proaggregating effects in vivo (11). Gragnoli et al. (12) found that somatostatin treatment is accompained by an increased platelet activation and by a concurrent decrease in the level of the stable metabolite of TxA2. In vitro studies were undertaken to explore the possible role matostatin in the regulation of the cyclooxygenase and lipoxygenase ways of the arachidonate cascade in rat platelets.
of sopath-
MATERIALANDMETHODS Chemicals Arachidonic acid (grade I) was purchased from Sigma Chemical Co, St. Louis, MO (USA). l-14C-Arachidonic acid (2035 MBq/mMspec. act.) was obtained from Amersham (England). TC Medium 199 was purchased from DIFCO Laboratories, Detroit, Mich. (USA). Silica gel G thin layer plates (0.25 mm) were obtained from Merck AG, Oarmstadt (FRG). PGE2, PGD2, TxA2, TxB2, PGF2alpha and 6-oxo-PGFlalpha were generously provided by Or J.E. Pike, Upjonn Co, Kalamazoo (USA). Hydroxyeicosatetraenoic acid (HETE) standards were purchased from Calbiochem, La Jolla, Ca (USA). Somatostatin (Stilamin) was purchased from Serono, Italy. Chemicals were of analytical grade and obtained commercially, unless otherwise stated. Isolation
of platelets
Blood was drawn from the abdominal aorta of male rats of the CFY strain (body weight: 200+15 g) under light ether anaesthesia. Samples were diluted with phosphste buffer (pH 7.4) which contained 5.8 mMEIJTA and 5.55 mMglucose. The platelet-rich plasma was collected after the whole blood had been centrifugated at 200 g for 10 min at room temperature. The platelets were sedimented from the supernatant by means of centri$ugatfon at 20009 for 10 min. The pell@ cortained the platelets(2x 10 ml ) and the red blood cells (lx 10 ml ). Erythrocytes are capable to metabolize arachidonic acid by the lipoxygenase pathway and release 12-HETE, dihydroxyeicosatetraenoic acid and 6-sulfidopeptide-containing leukotrienes (13). Therefore erythrocytes were lysed with hyposmic ammonium chloride (O.B3%, 9 parts) containing EOTA (0.02%, 1 part) at 4 ‘C for 15 min. According to our preliminary experiments this treatment did not modify the arachidonate cascade of platelets. The platelets were washed 3 times with phosphate buffer (pH 7.4, containing 5.8 mMEDTA and 5.55 mMglucose). After centrifugation (at 2000 g, for 10 min, at room temperature), platelets were resuspended in TC Medium 199. TC Medium 199 was applied, because the arachidonate cascade of platel@ was the most ac ive in this medium and less active or inactive in Ca depleted or Ca” free incubation mixtures. Analysis
of eicosanoids:
The platelets (1OB ml-l_5in egch sample) were incubated at 37 ‘C for M) was then added to the incubation 5 min, and somatostatin (10 -10
400
SEPTEMBER
1988 VOL. 36 NO. 3
PROSTAGLANDINS
-14 C-amixture. The enzyme reaction was started by the introduction of 1 rachidonic acid (3.7 kBq) as tracer substrate in the incubation mixture. Ten minutes later, the enzyme reaction was quenched by bringing the pH to 3 with formic acid. According to Dahl and Uotila (14) and our unpublished results 5-10 min is an adaquate time period for labelling platelets. The samples were then extracted with ethyl acetate (2x3 ml). The organic phases were pooled and evaporated to dryness under nitrogen. The residues were reconstituted in 150 ul ethyl acetate and quantitatively applied to Silica-gel G thin layer plates. The plates were developed to a distance of 15 cm in an organic phase of ethyl acetate: acetic acid:2,2,4_trimethylpentane:water (110:20:30:100), a high pressure thin layer chromatograph being used. Each 3 mm band of the chromatograms was scraped off and the radioactivity was determined in a Beckman LS 1800 liquid scintillation counter, using 5 ml toluene containing 0.4% w/v PPO, 0.02% w/v POPOP and 10% v/v ethanol. The radiolabelled
products standards,
of arachidonic which were
High-performance Reversed-phase
acid were identified with unlabelled authentic detected with anisaldehyde reagent (15).
liquid
chromatography
high-performance
liquid
ChromatogrRphy
(HPLC) was per-
C formed on a column (4.6~250 mm) packed with LiChrosorb les). The solvent used for the detection of 12-HETE was a '#i~~u~~ ~~'~~~_ hanol:water (75~25) containing 0.01% of acetic acid and adjusted to pH 5.6 with NH40H and monitored at 235 nm with a UV detector 308 of Labor MIM,
Hungary. Statistical
analysis
was performed
via
Student’s
t test.
RESULTS
5
10 DISTANCE
15
IN CM
Fig. 1. Separation of arachidonate metabolites of rat platelets by high The plate was developed to a distance pressure thin layer chromatograph. of 15 cm - in 25 min - in an organic phase of ethyl acetate: acetic acid: 2,2,4_trimethylpentane:water (110:20:30:100). (1) 6-keto-PGFlalpha (2) PGF2alpha, (3) PGE2, (4) TxB2, (5) PGD2, (6) HHT, (7) 12-HETE, (8) 12-HPETE.
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PROSTAGLANDINS
A typical radiochromatogram of rat platelet arachidonate metabolites is shown on Fig. 1. All the major cyclooxygenase products of arachidonate cascade are synthesized by platelets and could be separated by high pressure thin layer chromatography (Fig. 1).
* 10
1
8 TxE$
_llll I_
6
% 4.
2.
C
ro-’
O-8 169 SOMATOSTATIN
10-6
w5 M
Fig. 2. In vitro effects of somatostatin on synthesis of TxB2 in rat platelets. TxB2 is expressed (on the ordinate) as a percentage of t$e total arachidonfte metapglites. C = control. Each sample contained 10 platelets ml and l- C-arachidonic acid (3.7 kBq). Each value is the mean L S.E. of 6 determinations. x p = 0.05 Table
I. In vitro effects of somatostatin on synthesis of proaggregatory and antiaggregatory prostanoids in rat platelets
c
1O-9 M
1O-B M
SOMATOEjTATIN lO-6 M lo- M
lO-5 M
Proaggregatory
13.77
17.;;
19.;:
13.57
14.79
14.02
PG-s
-t-o.54
+0.66 -
~1.41
50.96
~1.63
20.65
Antiagregatory
10.17
10.77
13.63
15.2!!
14.35
12.02
PG-s
-+0.58
_+0.38
_+1.66
-+1.73
-+1.47
LO.73
Each sample contained lo8 (3.7 kBq). The quantity @ percentage of the total prostanoids are PGFZalpha PG12, PGE2 and PG02. Each x p = 0.05; *X pcO.02. C
402
pQtelets ml-1 and 1-14C-arachidonic acid C-prostanoids (PG-s) is expressed as a C-arachidonate metabolites. Proaggregatory and TxB2. Antiaggregatory prostanoids are value is the mean -+ S.E. of 6 determinations. = control.
SEPTEMBER 1988 VOL. 36 NO. 3
PROSTAGLANDINS
The synthesis of Txl32 in rat platelet3 was modified by somatostatin. Onlv one concentration of somatostatin (10 M) induced a sionificant increase in the formation of TxB?, Ther& was a t$ndency to inhibition at certain somatostatin doses (10 , 10 and 10 M), but the changes were not significant (Fig. 2).
3
9
SOMATOSTATIN an Fig. 3. In vitro effects of somatostatin on ratio of proaggregatory g antiaggregatofy prostftoids in rat platelets. Each sample contained 10 platelets ml and l- C-arachidonic acid (3.7 kBq). C = control. Each value is the mean -+ S.E. of 6 determinations. x p-=0.05; x): ~~0.02. Table
II. In vitro effects of somatostatin on lipoxygenase oxygenase pathways of rat platelets.
and cyclo-
SOMATOSTATIN
C 1O-9 M
10-B M
lO-7 M
1O-6 M
lO-5 M
Lipoxygenase
75.97
ft* 71.97
66.4;
71.2:
70.83
73.90
products
_+0.32
+0.49 -
-+2.83
+2.75 _
_+3.10
-+1.37
Cyclooxygenase
24.03
X%X 28.03
33.5;
28.80
29.17
26.10
products
_+0.32
-+0.49
-+2.83
-i2.75
-+3.10
+1.37 -
acid Each sample contained 1OB platelets ml-l and l-14C-arachidonic (3.7 kBq). Lipoxygenase products are: HPETE-s and HETE-s. Cyclooxygenase products: PG12, PGF2alpha14PGE2, TxB2 and PGOZ. They are expressed as percentages of the total C-arachidonate metabolites. Each value is the mean) S.E. of 6 determinations. x ~~0.05; XX)! ~~0.01. C = control.
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1988 VOL. 36 NO. 3
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PROSTAGLANDINS
Almost 1.5 times more proaggregatory - vasoconstrictor (TxA2 and PGF2aloha) then antiaaareoatorv - vasodilatator orostanoids (PGI2._PGE2 and6PGb2).were formed-in The platelets in vitro.’ Somatostatin (lOL’ and M) resulted in a significant decrease in the ratio of proaggregatory 10 - vasodilatator prostanoid synthesis (Fig. 3). This effect was the result of the elevated formation of antiaggregatory - vasodil,atator prostanoids (Table I). Lower concentrations of soM) resulted in an increased synthesis of proaggregatory matostatin (10 - vasoconstrictor cyclooxygenase products in rat platelets (Table I.).
In rat platelets, the lipoxygenase pathway of the arachidonate cascade predominates. In controls, three times more lipoxygegase metabolites and 10 M) sigwere formed than cyclooxygenase ones. Somatostatin (10 nificantly reduced the ratio of lipoxygenase and cyclooxygenase metabolites in the platelets (Fig. 4). Both the cyclooxygenase and lipoxygenaseq pathway8 of the arachidonate cascade were modified by somatostatin (10 and 10 M). Somatostatin resulted in an increased synthesis of cyclooxygenase products, while the formation of lipoxygenase metabolites was sign nificantly reduced. The optimal dose of somatostatin was found to be 10 M (Table II).
L/C
C
'
SOMATOSTATIN
il1 lo+
lO*M
Fig. 4. Effects of somatostatin on ratio of aipoxygenase a!f’ cycloo~xand lCgenase metabolites. Each sample contained 10 platelets ml arachidonic acid (3.7 kBq). L/C = ratio of lipoxygenase and cyclooxygenase metabolites of the arachidonate cascade. C = control. Each value is the mean 2 S.E. of 6 determinations. XX pcO.02; XXX p c 0.01.
404
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1988 VOL. 36 NO. 3
PROSTAGLANDINS
The formation of arachidonate metabolites in rat pla;$lets wapSsignificantly inhibited in the presence of somatostatin (10 and 10 M) (Fig. 5).
8 g
28
! l-
24
I
1U61O-5M SOMATOSTATIN
Fig. 5. In vitro effects of somatostatin on form8tion of arachifona$& metabolites in platelets. Each sample contained 10 platelets ml .l- -C-arachidonic acid transformed to cyclooxygenase and lipoxygenase products. C = control. Each value is the mean -+ S.E. of 6 determinations. x ~~0.05; 33% pco.01. DISCUSSION In accordance with the results of Srivastava and Tiwari (16) and Dahl and Uotila (14) obtained in human platelets all the major cyclooxygenase metabolites of arachidonate cascade are synthesized by rat platelets, as well. These in vitro data show that somatostatin in certain concentrations could modify the arachidonate cascade of rat platelets. The transformation of arachidgnic acidSto lipoxygenase metabolites was inhibited by somatostatin (10 and 10 M). This could be explained in terms of two mechanisms. First, this polypeptide might have a direct effect on the lipoxygenase pathway of the platelets, resulting in a reduced formation of 12HPETE and 12-HETE. Second, somatostatin might act on the deacylation process, reducing the free arachidonic acid substrate level, which might result in a lower lipoxygenation rate in the platelets. The second hypothesis could be supported by the results of Aharony et al. (17), who found a diminished platelet aggregability at higher concentrations of arachidonic acid, which was associated with marked increases in the ratio of lipoxygenase/cyclooxygenase products. The lipoxygenase product 12-HPETE has been
SEPTEMBER
1988 VOL.
36 NO. 3
405
PROSTAGLANDINS
proposed as an inhibitor of platelet aggregation and secretion induced by arachidonic acid or collagen (17, 18). This lipoxygenase metabolite could also inhibi-4 the formation of TX in activated platelets (17). The somatostatin (10 M) - induced low lipoxygenase activity and HPETE level might be responsible for the increased formation of proaggregatory and vasoconstrictor TX in rat platelets. Free arachidonic acid can be derived from inositol phospholipids through two consecutive reactions, catalysed by phospholipase C and diacylglycerol lipase (191, and in platelets it can also be derived from phosphatidic acid via its specific lipase (3, 20). However, in most tissues arachidonic acid is also released by phospholipase A2 from phosphatidylcholine and phosphatidylethanolamine. After receptor stimulation, the two phospholipase perhaps act in concert. It is important to note that diacylglycerol is produced in membranes only transiently, presumably due both to its conversion back to inositol phospholipids and to its further degradation to arachidonic acid for TX, HPETE and HETE synthesis. Somatostatin might shift the phospholipid breakdown to the phospholipase C and diacylglycerol lipase pathway, which results in a lower release of free arachidonic acid. The precise mechanism by which somatostatin affects cellular processes after binding to its receptors is not yet known conclusively. Several investigators have proposed that somatostatin binds to specific receptors, which results in the inhibition of adenylate cyclase activity, and this might reduce the phosphorylation of lipomodulin (21) by the CAMP -dependent protein kinase. Finally, the release of free arachidonic acid by phospholipases might be reduced.
The contradictory results reported concerning the action of somatostatin on the platelet function could be explained by our results that the effect of the polypeptide depends on the applied dose. 09r inves$gations demonstrated that low somatostatin concentrations (loand 10 M) increased the synthesis of proaggregatory cyclooxygenase rat platelets, while higher somatostatin concentrations induced the synthesis of antiaggregatory products.
meJybolites_$(10 and 10 M
ACKNOWLEDGEMEN This work was partly
Ministry
of Social
supported by the Scientific and Health, Hungary.
Research Council,
REFERENCES 1.
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Riper, p. and J. Vane. The release of prostaglandins other tissue. Ann. N.Y. Acad. Sci. 183:363, 1971.
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1988 VOL. 36 NO. 3
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Marcus, A.J., M.J. Broekman, 6.6. Neksler, E.A. Jaffe, L.B. Safier, H.L. Ullman, N. Islam and K. Tack-Goldman. Arachidonic acid metabolism in endothelial cells and platelets. Ann. N.Y. Acad. Sci. 401: 195, 1982.
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Hamberg, M. and B. Samuelsson. transformations of arachidonic Acad. Sci. USA -71:3400, 1974.
6.
Samuelsson B. Introduction: new trends in prostaglandin Adv. Prostaglandin Thromboxane Res. 1~1, 1976.
7.
Mielke, C.H., J.E. Gerich, M. Lorenzi, E. Tsalikian, R. Roviden and P.H. Forsham. The effect of somatostatin on coagulation and platelet function in man. N. Engl. J. Med. _293:4BO, 1975.
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Besser, G.M., A.M. Paxton and S.A.N. Johnson. function by growth hormone release-inhibiting 1975.
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Chiang,T.M., W.C. Duckworth, E.H. Beachey and A.H. Kang. The effect of somatostatin on platelet aggregation. Endocrinology -97:753, 1975.
Prostaglandin endoperoxides. Novel acid in human platelets. Proc. Natn.
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10. Scheck, R., S. Raptis, H. Rasche and F. Escobar of somatostatin on platelets: in vitro studies. 219, 1977.
Jimenez. Diabetes
The effect Metab. -3:
11. Guigliano, IJ., I.Coppola, L. Misso, A. Tirelli, N. Passairello and F. O'Onofrio. Further studies on the significance of circulating platelet aggregates induced by somatostatin in man. Metabolism -30:172, 1981. 12. Gragnoli, G., A.M. Signorini and I. Tanganelli. Effects of somatostatin on the behaviour of thromboxane B and beta-thromboglobulin in &:104, 1985. type I diabetes.Acta Endocrinologica 13. Kobayashi, T. and L. Levine. Arachidonic cytes. J. Biol. Chem. _258:9116, 1983.
14.
~~~~;l,“~~~ma~~o~~n~~~i~~. ro. Prostaglandins
15. Kiefer,
tion 1975.
Verapamil C-arachidonic
Leukotrienes
acid metabolism
by erythro-
decreases the formation of thromacid in human platelets in vit-
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H.C., C.R. Johnson and K.L. Arora. Calorimetric identificaof prostaglandins in subnanomole amounts. Anal. Biochem. -68:336,
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K.C. and K.P. Tiwari.
In vitro
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utilization
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(l-
C) arachidonic
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1988 VOL.
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PROSTAGLANDINS
17. Aharony, O., J.B. Smith and M.J. Silver. Regulation of arachidonateinduced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid. Biochim. biophys. Acta 1718:193, 1982. 18. Vericel, E. and M. Layarde. 15-hydroperoxyeicosatetraenoic hibit human platelet aggregation. Lipids -15:472, 1980.
acid in-
19. Bell, R.L., D.A. Kennerley, N. Stanford and P.W. Majerus. Oiglyceride lipase: a pathway of arachidonate release from human platelets. Proc. natn. Acad. Sci. USA -76:3238, 1979. 20. Billah, M.M., E.G. Lapetina and P. Cuatrecasas. The initial action of thrombin on platelets conversion of phosphatidyl-inositol to phosphatidic acid preceding the production of arachidonic acid. J. biol. Chem. _256:5037, 1981. 21. Hirata, F. The regulation of lipomodulin, a phospholipase inhibitory protein in rabbit neurotrophils by phosphorylation. J. biol. them. -256:7730, 1981.
Editor:
408
P. Ramwell
Received:
3-28-88
Accepted:
7-8-88
SEPTEMBER 1988 VOL. 36 NO. 3