Biosynthesis and biological actions of prostaglandins and thromboxanes

ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 214, No. 2, April 1, pp. 431-445, 1982

INVITED PAPER Biosynthesis

and Biological Actions of Prostaglandins and Thromboxanes’ SVEN HAMMARSTRijM

Department of Physiological Chemistry, Karolinska Institutet, P.O. Box SO&Xl,S-104 01 Stockholm, Sweden Received September 29, 1981, and in revised form November 30. 1981

Prostaglandins are oxygenated derivatives of polyunsaturated fatty acids with a wide range of biological effects (1). For instance, prostaglandins of D, E, and I types stimulate cyclic AMP formation in a variety of tissues. These prostaglandins mimic or modulate hormonal and neurohormonal responses (1). Prostaglandin Fz,, on the other hand, is a luteolytic hormone (2) in several mammalian species. Receptors for these prostaglandins have been demonstrated in target tissues. The first part of this review will deal with a receptor for prostaglandin Fti in corpora lutea. Prostaglandins also modulate cell growth and immune responses (3-6). The second part of the review describes work on endogenous synthesis of prostaglandins in cultured fibroblasts, and changes in the regulation of prostaglandin synthesis that occur following transformation of the cells by polyoma virus. Effects of the altered prostaglandin production on cyclic AMP formation and cell division are also described. Thromboxane AZ, a labile bicyclic compound formed from prostaglandin endoperoxides (7), is a physiological mediator of platelet function. The last part of the paper will review work on an enzyme from platelets and lung which catalyzes the transformation of prostaglandin endoperoxides to thromboxanes. ’ This work was supported by grants from the Swedish Medical Research Council (03X-5914) and the Swedish Cancer Society (1503-03X).

ROLE OF RECEPTORS IN PROSTAGLANDIN ACTIONS

A prostaglandin receptor was first described in fat cells (8). This receptor is specific for prostaglandin El and appears to be involved in the stimulation of fat cell adenylate cyclase by prostaglandins. Receptors for E-type prostaglandins have subsequently been demonstrated in a number of tissues, e.g., adrenal gland, corpus luteum, fibroblasts, L cells, liver, lymphoma cells, neuroblastoma cells, platelets, stomach, thymocytes, thyroid gland, and uterus (1). Correlations between receptor binding and activation of adenylate cyclase has been reported for L cells, for neuroblastoma and lymphoma cells (9, lo), and for adrenal glands (11). Furthermore, cells unresponsive to prostaglandin E lacked prostaglandin E receptors (9,10). Some physicochemical data have been reported for a prostaglandin E receptor from rat liver (12). Triton X-lOO-solubilized material had a molecular weight (excluding bound detergent) of 105,000 daltons based on the sedimentation coefficient (5.6-5.7 X lo-r3 s) and Stoke’s radius (53 A). The frictional ratio for the detergent containing complex was 1.3 and approximately 110 molecules of Triton were bound per receptor molecule. The uterus is required for the cyclic regression of corpora lutea in the estrus cycle of many mammalian species (13). A soluble factor, produced by the uterus, mediates luteolysis (14). It is transported in blood and transferred from the utero431

0003-9861/82/040431-15$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

432

SVEN HAMMARSTROM

ovarian vein to the ovarian artery by a countercurrent mechanism (14). The factor has been identified as prostaglandin Fti (2), a previously known luteolytic agent (15). The mechanism of action of this hormone is known in part: A specific receptor protein in plasma membranes of corpus luteum cells has been demonstrated. Some properties of the receptor will be discussed below. Interaction between prostaglandin Fza and the receptor is an early event in luteolysis (16). Receptor occupancy antagonizes the effects of luteinizing hormone (LH) by lowering LH-stimulated production of cyclic AMP (17). This leads to reduced progesterone secretion and regression of the corpus luteum.

optimal around pH 6.3; at lower pH values there was an increase in nonspecific binding, primarily, whereas total binding decreased at higher pH values. The binding data (at 23°C) in Fig. 1A followed a second-order rate equation with rate constant (Ki) = 7.5 X lo3 liter - mol-’ - s-l. The same value for K1 was obtained at various concentrations of prostaglandin Faa. The dissociation data in Fig. 1B followed a first-order rate equation with rate constant (k-,) = 2.1 X 10m4s-‘. The ratio of k-i to k, (2.8 - lo-’ liter - mol-‘) is the dissociation constant (&) for the interaction between prostaglandin Fza and the corpus luteum receptor. A similar value for the Kd (5 - 10e8liter. mol-‘) was obtained from Scatchard plot analysis of binding data at equilibrium. PROSTAGLANDIN Fz, RECEPTOR IN The dissociation constants for various CORPORALUTEA unlabeled prostaglandins were calculated from the decreases in apparent dissociaAffinity and Speci&city tion constants induced by fixed amounts Using 3H-labeled prostaglandin Fza and of these compounds added to series of rea binding assay on small columns of Seph- action mixtures with varying concentraadex G-50, specific binding of the hormone tions of [3H]PGF2, (analyses by Scatchard plots). The results obtained using receptor was demonstrated to particulate fractions from ovine (18) and bovine corpora lutea preparations from either ovine or bovine (19). Figure 1 shows time courses of pros- corpora lutea are summarized in Table I. They indicated that the carboxyl group taglandin FZa binding to such a fraction sedimenting between 1000 and 35,000g and the hydroxyl group at C-15 in PGF2, from homogenates of bovine corpora lutea. were very important for the binding reThe binding reaction was carried out at action; conversion of these groups to hydroxy (1,9,11,15-tetrahydroxyprosta-5,1323 or 37°C and Sephadex chromatographies at 4°C. The nonspecific binding, de- diene) and keto groups (15-keto-PGF& termined in the presence of 250- to 2500- increased the dissociation constants ca. lOO- and 200-fold, respectively. The hyfold excesses of unlabeled prostaglandin droxyl group at C-9 and the double bond F aa, was subtracted from the binding curves just mentioned, and is shown by the at A5 were of intermediate importance; conversions to a keto group (PGEa) and a third curve in Fig. 1A. The specific binding at 37’C was more rapid but less extensive saturated bond (PGF1,) increased the disthan that at 23°C. This phenomenon, which sociation constant cit. 50- and 40-fold, rehas also been observed in similar prepa- spectively. Finally, the hydroxyl group at rations by others (20) may be due to rapid C-11 and the Ai3 double bond were reladestruction at 37°C of receptor molecules tively nonimportant for the binding reaction; 9- and 4-fold increases in dissociaby proteases present in the preparations. Figure 1B illustrates the reversibility of tion constants were observed upon oxidation to ketone (PGDa) and reduction the binding reaction: after preincubating tritium-labeled prostaglandin Fzo,and the to saturated bond (13,14-dihydro-PGF&, particulate fraction until equilibrium was respectively. reached (23”C, 60 min) a 400-fold excess of unlabeled PGFZawas added and the re- Subcellular Distribution (21) Corpora lutea were removed and placed sidual specific binding was measured as a function of time. The specific binding was in cold 0.3 or 0.5 M sucrose/l mM NaHC03

PROSTAGLANDINS

0

AND THROMBOXANES

433

1.11,....,....,,.. 100

52 Time

(m(n)

Time

(mm)

150

100

50

0

1 ,“,‘T”“I“.-l”’

0

50

100

150

FIG. 1. Time courses for the (A) binding of prostaglandin Fb to a particulate fraction from bovine corpora lutea at 23 (0) and 37°C (A) (0, nonspecific binding) and (B) the dissociation of the prostaglandin F&-receptor complex at 0 (0) and 23°C (0). From Ref. (19).

as soon as possible following slaughter of cows. The tissue was homogenized with a Dounce homogenizer. The homogenates in 0.3 M sucrose were fractionated by differential centrifugation (6000~; 35,000~; 80,000~; and 270,0009). The sediments and the final supernatant were assayed for contents of protein, marker enzymes (Nacetyl-P-glucosaminidase, /I-glucuronidase, NADPH-cytochrome c reductase, NADHcytochrome c reductase, 5’-nucleotidase, and succinate dehydrogenase), and prostaglandin Fza receptor. The distributions of receptor and 5’-nucleotidase were very similar. The highest concentrations were detected in the top layer of the 35,000~ pellet and in the 80,000~ pellet. The other enzymes had distinctly different distributions which indicated that the receptor was specifically localized in plasma membranes and that cytosol, mi-

tochondria, lysosomes, and nuclei were devoid of PGFaa binding activity. Further evidence for this localization was obtained by isolating plasma membranes from the homogenates in 0.5 M sucrose. This was done by a combination of differential and density gradient centrifugations: A purified top layer of the 10,OOOg pellet was layered on top of a 2’745% sucrose gradient with a cushion of 50% sucrose. After a centrifugation at 96,OOOgfor 10 h, fractions were collected and the absorbance at 650 nm and the refractive index were measured. The refractive indexes were converted to densities of sucrose solutions. Fractions were combined as indicated in Fig. 2 and the relative specific activities of NADPH-cytochrome c reductase, succinate dehydrogenase, and Ei’nucleotidase as well as the prostaglandin Fz, receptor activity were

434

SVEN HAMMARSTROM TABLE

I

DISSOCIATIONCONSTANTS(&) FORTHE BINDING OF PROSTAGLANDINS TO RECEPTOR PREPARATIONSFROMOVINE AND BOVINECORPORALUTJZA

Prostaglandin Prostaglandin Az Prostaglandin & Prostaglandin Da Prostaglandin El Prostaglandin Ez Prostaglandin E3 Prostaglandin F1, Prostaglandin F% Prostaglandin F% 13,14-Dihydroprostaglandin Fb l&Ketoprostaglandin Fti 13,14-Dihydro-15-ketoprostaglandin Fti 1,9~,lla,l5(S)-Tetrahydroxyprosta-5-ci-diene

Ovine corpora lutea

Bovine corpora lutea

>260 >260 34 2.7 2.0 0.10 0.23 11 38

140 150 0.46 38 2.4 16 2.0 0.05 0.17 0.20 9 14 5.4

Source. Refs. (18) and (19).

determined. The same analyses were performed on the fractions from the differential centrifugation which preceded the sucrose gradient centrifugations (Fig. 3). The results showed that the receptor and 5’-nucleotidase had the same distributions and that the other marker enzymes had different patterns of distribution (maximum specific activities of receptor and 5’nucleotidase were observed in fraction 7 from the gradient and in the top layer of the 10,OOOg sediment and the 80,OOOgsediment from the differential centrifugation). Physicochemical Characterization

haviors on Sepharose 6B with those of reference proteins of known Stoke’s radii (Fig. 4). These comparisons gave a value of 63 A for the prostaglandin Fzu receptor. The molecular weight of a protein (or other macromolecule) can be calculated

(22)

To determine physicochemical data for the prostaglandin Fzu receptor from bovine corpora lutea this molecule was solubilized from membranes using various detergents. It was observed that the binding reaction was inhibited by low concentrations of detergents whereas the performed hormone-receptor complex was quite stable under these conditions. By using tritium-labeled hormone, a convenient marker for the complex was obtained. The Stoke’s radius was determined by comparisons of chromatographic be-

c 0

FIG. 2. Density gradient centrifugation of bovine corpus luteum homogenates. (x) Sucrose density; (0) absorbance at 650 nm. The various l-ml fractions were pooled as indicated to give eight larger fractions. From Ref. (21).

PROSTAGLANDINS

435

AND THROMBOXANES

from the Stoke’s radius and the sedimentation coefficient. In the case of detergentsolubilized membrane proteins it is also necessary to know the extent of detergent binding to the protein. One way of estimating this is to determine the partial specific volume of the detergent-protein complex because the partial specific volume of certain detergents (e.g., Triton X100 = 0.903cm3/g) differs appreciably from that of typical nonglycosylated proteins (V = 0.736 cm3/g). The sedimentation coefficient and the partial specific volume can both be determined from density gradient centrifugations. Since two parameters have to be determined two analyses under dif-

1.0 =r-

0.83m * $

0.6

L

0.4.

-

Laglobin

01 0

I zoo

400

600

Stokes,

radius

300

1000

1200

(nm)

FIG. 4. Stoke’s radius determination for Triton XlOO-solubilized prostaglandin Fe,-receptor complex. From Ref. (22).

ferent conditions are necessary. This can be done by performing the centrifugations in sucrose/Hz0 and sucrose/D20 gradients. The sedimentation coefficient is calculated as functions of the partial specific volume (between 0.60 and 0.90 cm3/g) in the two sets of experiments, respectively (Fig. 5). The two curves obtained intercepted once. The point of interception defines the sedimentation coefficient (4.65) and the partial specific volume (0.78 cm3/g) of the detergent-receptor complex. The values thus determined indicated that the apparent molecular weight of the complex was 144,000 daltons and that the content of Triton X100 was 26% (w/w). Table II summarizes

5’ NucleotidaSe

10 -

2H,0

;; 3 4

p

0 0

100 Pmteln I” lractm (% total,

0 Profen

y1 100 .-recovered irom qrodlent (Sb total,

FIG. 3. Distribution patterns of constituents after fractionation of bovine corpus luteum homogenates by differential centrifugation (A) and gradient centrifugation (B). The hatched bars represent the fraction most enriched in plasma membranes. lp, 1OOOg pellet; lob, bottom layer of 10,OOOgpellet; lot, top layer of 10,OOOgpellet; f3Op,30,OOOgpellet; 8Os, 80,OOOg supernatant. From Ref. (21).

5

30

0. -52 --J

01’ 0.60

0.70

0.80

“e krn3/9)

FIG. 5. Calculation of equivalent sedimentation coefficient (+,,J and partial specific volume (V,) from sedimentation data for Triton X-lOO-solubilized prostaglandin F&-receptor on sucrose/He0 and sucrose/ ‘He0 density gradients. From Ref. (22).

436

SVEN HAMMARSTROM TABLE

II

PROPERTIESOFTRITONX-100~SOLUBILIZEDPROSTAGLANDIN FaY RECEPTOR

Mr Wmol)

63

spg,wx lOI (cm/s * dyn)

” (cm3/g)

4.6

0.78

Percentage Triton (w/w) 26

Triton bound (mol/mol complex)

Receptordetergent complex

Receptor

144,000

107,000

58

f/f0 1.6

Source. Ref. (22).

the physicochemical data of the prostaglandin Fz, receptor including the frictional ratio (f/fO) and the number of Triton molecules bound per molecule of receptor. PROSTAGLANDIN CULTURED

BIOSYNTHESIS FIBROBLASTS

BY

Certain prostaglandins, notably El, Ez, Dz, and Iz, stimulate the formation of

cyclic AMP in a number of tissues and cells (1). The stimulation involves binding to specific receptors (see above) and activation of adenylate cyclase. Cyclic AMP appears to have a role in the regulation of cell growth: Exogenous cyclic AMP derivatives inhibit cell division (23), confluent, contact-inhibited cells have higher levels of cyclic AMP than growing cells (24), and several transformed cells have lower levels of cyclic AMP than corre-

FIG. 6. Concentration of prostaglandin E2 (open bars) and prostaglandin cell culture media from 3T3 fibroblasts (A), and polyoma virus-transformed From Ref. (26).

Fz, (hatched bars) in 3T3 (py 3T3) cells (B).

PROSTAGLANDINS

AND THROMBOXANES

437

sponding nontransformed cells (24). Prostaglandins produced by tumor cells can inhibit cytotoxicity of lymphocytes involved in host-defense mechanisms against cancer cells (6). These observations led us to investigate endogenous prostaglandin production by normal and transformed fibroblasts and to determine its effects on cyclic AMP formation and cell growth. Effects of Polywma Virus Transformation on Prostaglandin Formation Baby hamster kidney fibroblasts transOY formed by polyoma virus synthesized and 20 25 0 10 15 5 released considerable amounts of prostakachldonlc acid (sgigimi) glandin E2 into the growth media (up to 8. Prostaglandin E2 production by 3T3 (0) and 4.1 pg PGEJlOO pg of cellular DNA in ‘72 py FIG. 3T3 (0) cells in the presence of exogenous arah). Significant amounts of prostaglandin chidonic acid. After rinsing with phosphate-buffered Fzo,(up to 0.11 pg/lOO pg of cellular DNA) saline (3 X 5 ml) the cells were incubated with 3 ml were also formed (25). On the other hand, buffer containing arachidonic acid at various conregular baby hamster kidney fibroblasts centrations (0.25-25 *g/ml). Prostaglandin Ea levels produced much smaller quantities of PGEz were determined after 20 min and corrected for DNA (10.06 pg/lOO pg of DNA) and PGFZa content. From Ref. (28). (co.01 pg/lOO pg of DNA). Cells transformed by a temperature-sensitive mutant of polyoma virus synthesized PGEz and 10.016 at 31°C and ~0.007 pg/lOO pg DNA PGFz, in amounts intermediate to those at 39°C). mentioned above. The quantities produced More detailed investigations were perwere two- to three-fold higher at the per- formed using Balb/c 3T3 fibroblasts transmissive (31°C) than at the nonpermissive formed by polyoma virus (26-28). Figure temperature (39°C) (PGE2: 10.51 at 31°C 6B shows the concentrations of PGEz and and 10.16 kg/100 pg DNA at 39°C; PGF2,: PGFz, in growth media from these cells at various times after initiation of the cultures. These concentrations increased with time throughout the experiment. Figure 6A shows corresponding values for media from regular Balb/c 3T3 fibroblasts. It is obvious that no net synthesis of PGEz occurred after 24 h in culture whereas the concentrations of PGFB, increased also after Day 3. This suggested that polyoma virus transformation changes the regulation of prostaglandin E2 and Fz, biosynthesis in fibroblasts. Other prostaglandins were also produced in increased amounts by polyoma virus-transformed 3T3 fibroblasts (27). The concentrations of PGEl and 6-keto-PGF1, (hydrolysis product of Time after medium change (hani PGIB) were ea. 10 times lower than those of PGEz in media from either polyoma FIG. ‘7. Prostagiandin Ez production by 3T3 (A) and virus-transformed or regular 3T3 fibropy 3T3 (B) cell cultures during l-h incubations in 5 blasts. Consequently, ca. 100 times greater ml phosphate-buffered saline at various times after medium change. From Ref. (28). amounts of PGEl and PG12were produced

SVEN HAMMARSTRGM

0 0

I

2 Time I” culture

3 (days)

4

5

FIG. 9. Arachidonic acid concentrations in the growth media from 3T3 fibroblasts (open bars) and polyoma virus-transformed 3T3 fibroblasts (hatched bars). The analyses were performed by quantitative mass spectrometry. From Ref. (26).

by the transformed compared to the regular cells. Mechanism of Altered Prostaglandin Biosynthesis in Polyoma VirusTransfmed Fibroblasts (2%) As mentioned above, regular 3T3 fibroblasts produced prostaglandin E2 for a period of less than 24 h following a medium change. The time course of stimulation of PGEz synthesis by serum was therefore determined by analyzing at various times after the medium change the amounts of PGEz released during 60 min into phosphate-buffered saline (Fig. 7). Maximal synthesis (0.2 pg PGE&OO pg DNA) was observed 30 min after medium change. After 3 h, less than 0.05 pg/lOO pg DNA was produced. For comparison, corresponding analyses were also performed using polyoma virus-transformed 3T3 fibroblasts. These cells produced ca. 1 pg PGEz/lOO pg DNA. 60 min at all times following medium change, suggesting that synthesis was independent of serum stimulation. Therefore, polyoma virus transformation appears to alter mechanisms which normally decrease prostaglandin Ez synthesis following a stimulus (e.g., by serum). Addition of arachidonic acid markedly stimulated prostaglandin Ez synthesis in both 3T3 and polyoma virus-transformed 3T3 cells (Fig. 8). This indicated that the

concentration of precursor fatty acid was rate limiting in PGEz biosynthesis in these cells and suggested that polyoma virus transformation might alter the rate of release of arachidonic acid from cellular lipids. In fact it had been previously observed (26) that the levels of free arachidonic acid were higher in growth media from polyoma virus-transformed compared to regular 3T3 fibroblasts (Fig. 9). This would be compatible with increased acyl hydrolase activity in the transformed cells with release of liberated acids into the medium. To determine the hydrolytic release of

L L

0

’ 0

I 48

24

I 72

Time (hours)

FIG. 10. Release of radioactivity from 3T3 (0) and py 3T3 (0) cells prelabeled with [1-‘%]arachidonic acid. Cultures were incubated for 16 h in media containing radiolabeled arachidonic acid (0.02 &i/ml, 50-60 Ci/mol). After rinsing the cells, fresh medium was added and samples were taken at intervals for radioactivity determinations. From Ref. (28).

PROSTAGLANDINS

1

a

AND THROMBOXANES

439

b

time

(days

after

planting)

FIG. 11. Effects of indomethacin and indomethacin plus PGEz on PGE2 concentrations in growth media (a), cyclic AMP levels in cells (b), and DNA contents/dish (c). Symbols: control without additions (m, 0), 10 nM indomethacin (0, A), 1 pM indomethacin (W, A), and 1 pM indomethacin plus 10, 75, ‘75, 25, 120, 30, 180, 85, 300, and 250 rig/ml of PGEB on Days l-10, respectively (0, 0). From Ref. (33).

arachidonic acid from regular and polyoma virus-transformed 3T3 fibroblasts, the cells were labeled during growth in medium containing [l-‘4C]arachidonic acid. After rinsing, the release of radioactivity from the cells into the medium was determined (Fig. 10). Chromatographic analyses indicated that 90% of the radioactivity in the cells was bound in the phospholipid fraction and 10% in the neutral ester lipid fraction. Both cell types released about 10% of the radioactivity during the first 60 min after the medium change. During the next 71 h the transformed cells released an additional 22% of the labeled arachidonic acid from cellular lipids whereas the regular 3T3 cells released less than 4% during the same period of time. It has recently been demonstrated (2931) that corticosteroids are effective inhibitors of arachidonic acid release in various cells and that the mechanism of action is by induction of a phospholipase A2 inhibiting protein. When added to polyoma virus-transformed 3T3 fibroblasts, hydrocortisone totally prevented further prostaglandin Ez synthesis. No effect of

the steroid on PGEz levels in media from corresponding 3T3 fibroblasts was observed. These results strongly suggest that polyoma virus-transformation of 3T3 fibroblasts increases the basal acyl hydrolase activity in the cells. This leads to release of arachidonic acid from cellular lipids and to increased synthesis of PGE2, PGFz,, and PGIB. In addition, dihomo-ylinolenic acid is released since PGEl is synthesized by the transformed cells. The effects of polyoma virus transformation on the activities of the enzymes which convert arachidonic acid to PGEz (prostaglandin endoperoxide synthase and prostaglandin endoperoxide-E isomerase) and which metabolize prostaglandin Ez (probably 15-hydroxyprostaglandin dehydrogenase) in 3T3 cells were also determined. After addition of saturating concentrations of arachidonic acid, the initial rates of PGEe synthesis were greater (2.9fold) in regular compared with transformed cells. The rate of degradation of PGEz (determined in the presence of 1 PM indomethacin to inhibit endogenous synthesis) was the same in regular and

440

SVENHAMMARSTROM

transformed cells (tljz = 45 h). The decreased capacity to synthesize prostaglandin Ez in the transformed compared to the regular cells confirms that the activities of these enzymes is not rate limiting for PGEz synthesis in polyoma virus-transformed 3T3 fibroblasts. Stimulation of Cyclic AMP Synthesis and Inhibition of Cell Growth by Endogenous Prostaglandin E2 Form&cm in Polyoma VirusTran.@rmed 3T3 Fibroblasts Prostaglandin E2 stimulates or inhibits adenylate cyclase in various cells (1). It seemed of interest, therefore, to determine if endogenous prostaglandin production in polyoma virus-transformed 3T3 fibroblasts had effects on cyclic AMP synthesis and cell growth. Measurements of cyclic AMP showed that virus-transformed tibroblasts had higher levels than regular 3T3 cells, particularly when the concentrations of PGE2 were high in the growth medium (32,33; see Figs. lla and b, Days 7-11, fB: PGE2, 0: CAMP). More frequent growth medium changes decreased the concentrations of PGEz and also the levels of cyclic AMP (32). Conclusive evidence that PGEz synthesis was involved in the regulation of cyclic AMP concentrations was obtained using inhibitors of prostaglandin biosynthesis (aspirin, indomethacin, and 5,8,11,14-eicosatetraynoic acid) and exogenous additions of PGE2 (32,33). At minimal doses which inhibited PGE, synthesis completely the rise in cyclic AMP concentrations with time was abolished (results using 1 pM indomethacin are shown in Figs. lla and b. n . A: CAMP). At a lower concentration of indomethacin (10 nM), partial inhibition of PGEz synthesis (0) and a partial lowering of cyclic AMP levels (A) were observed (Figs. lla and b). Daily additions of PGEz to cells treated with 1 pM indomethacin (10,X, 75,25,120, 30,180,85,300, and 250 rig/ml on Days l10, respectively) gave similar values of PGEz in media (U) and cyclic AMP in cells (0) as observed in the control cultures which received neither indomethacin nor PGEp (l&l and 0, Figs. lla and b). Cell

growth was measured as amounts of DNA per dish in the same experiments (Fig. 11~). The results showed that 1 pM indomethacin significantly stimulated growth (30%; A), 10 nM indomethacin had no effect (A), and 1 pM indomethacin plus the daily PGEz additions mentioned above decreased growth by about 10% (0). Moreover, 10-20s reductions in DNA contents were observed on Day 3 in all experiments with indomethacin. This initial inhibitory effect may be due to inhibition of PGFzo, synthesis since PGFz, can stimulate cell proliferation (34). THROMBOXANESYNTHASE

Thromboxane AZ is formed from the prostaglandin endoperoxide PGHz by cleavages of the cyclopentane ring between C-11 and C-12 and of the endoperoxide bridge between the two oxygen atoms. Two new bonds are also formed: the oxygen atom at C9 becomes attached to C-11 and the oxygen atom at C-11 becomes attached to C-12. The resulting bicyclic oxane, oxetane compound (thromboxane A,) is rapidly hydrolyzed in aqueous solution to a hemiacetal derivative, thromboxane B2 (tllz: 30 s at pH 7.4 and 37°C (7). The transformation of PGHz to thromboxane A2 is catalyzed by a microsomal enzyme which has been isolated from platelets and lung (35-37). In addition to forming thromboxane Az, this enzyme also catalyzes a fragmentation of prostaglandin endoperoxides to Cl,-hydroxy acids and a three-carbon fragment, malondialdehyde (38). The latter reaction predominates when the endoperoxide substrate lacks a cis double between C-5 and C-6 (PGH1, A4-cti PGHl (38, 39). Thromboxane AZ has similar biological effects as its precursors, PGGz and PGHz, but is more potent. These compounds induce platelet aggregation and contraction of arterial smooth muscles (7). Synthesis of PGG2, PGHz, and thromboxane AZ is required for normal platelet function since a genetic lack of cyclooxygenase in platelets results in defective release and a prolonged bleeding time (40).

PROSTAGLANDINS

441

AND THROMBOXANES

Assay and PurQkaticm [l-14C]PGHz (PGGz) was prepared by incubating [l-‘4C]arachidonic acid with ovine vesicular gland microsomes. The product was isolated by rapid ether extraction and drying since it is labile in aqueous media (tl,2 = 5 min). After purification by silicic acid chromatography the endoperoxides were stored at -80°C dissolved in acetone. These methods are described in (41, 42). [l-14C]PGGz was incubated with subcellular fractions from human platelets. The conversion to thromboxane Bz (the primary product, thromboxane Az, is hydrolyzed (tl12 = 0.5 min) in aqueous media to thromboxane Bz) was determined by thinlayer chromatography. In larger-scale experiments, the thromboxane Bz formed was isolated and the structure verified by gas-liquid chromatographic-mass spectrometric analyses. Table III shows the specific activity of thromboxane synthase in fractions obtained from disrupted platelets by differential centrifugation or according to published procedures (plasma menbranes, a granules, dense bodies). The results showed that thromboxane synthase (and also prostaglandin endoperoxide synthase) was TABLE

III

SUBCELLULAR DISTRIBUTIONS OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASE AND THROMBOXANE SYNTHASE IN PLATELETS sp act

Platelet fraction Disrupted platelets 1,WOgSediment 12,OOOg Sediment 100,600gSediment 160,000gSupernatant Plasma membranes (I Granules Dense bodies

Prostaglandin endoperoxide synthase” 11.6 5.9 12.0 20.4 0 0 -

Thromboxane synthas$ 17.2 14.8 47.6 34.4 0 0 0 0

Source. Ref. (35). ‘Sum of nmol of HHT and thromboxane Rs formed from arachidonic acid/mg of protein in 45 s at 37°C. * nmol of thromboxane BI formed from PGGs/mg of protein in 20 8 at 37’C.

Y.

Distance

from

origin

tcm)

FIG. 12. Thin-layer chromatograms of products formed from [%]PGHs by purified thromboxane synthase after 10-s (upper) and 2-min incubations at 37°C (lower chromatogram). No PGHs remained after 10s and reactions were stopped by addition of methanol to convert thromboxane As to O-methyl thromboxane Bs (O-Me TXBs). From Ref. (38).

enriched in the 100,000~ sediment fraction (35). These enzymes, which convert arachidonic acid to PGH2 and PGH2 to thromboxane AZ, were released from the platelet microsomes by treatment with Triton X100 (0.5%, v/v). Chromatography on DEAE-cellulose separated the prostaglandin endoperoxide synthase and thromboxane synthase activities (35). The former was eluted with 0.01 M potassium phosphate buffer at pH 7.4 and the latter with the same buffer at 0.2 M concentration. Further purification of thromboxane synthase was achieved by chromatography on phenyl-Sepharose, isoelectric focusing (~1 = 5.2) and Sepharose CL 6B chromatography (K,, = 0.5).2 The purified enzyme was stable for several months at -80°C. Substrate Specificity and Formation of Cl,-Hydroxy Fatty Acids A number of 14C- or tritium-labeled prostaglandin endoperoxides were prepared from polyunsaturated fatty acids by procedures analogous to those described s Diczfalusy, U., Kylden, U., and Hammarstrom, unpublished results.

S.,

442

SVEN HAMMARSTRiiM

.;(ww 15

10 Distance

from

5 origin

0 Icm I

FIG. 13. Thin-layer chromatograms of products from PGG2 incubations with purified thromboxane synthase before (upper chromatogram), and after SnCI, reduction (lower chromatogram). The products were converted to methyl esters before analysis. From Ref. (42).

above for the preparation of PGHz. The endoperoxides were tried as substrates for purified preparations of thromboxane synthase. Products were separated by thinlayer chromatography and structures were determined by mass spectrometric analyses of suitable derivatives. Using PGHz as substrate it was originally observed (35) that chromatographic fractions containing thromboxane synthase also catalyzed the conversion of PGHz to a product identified as 12hydroxy-5,8,10-heptadecatrienoic acid (HHT). HHT had earlier been recognized as a by-product of prostaglandin biosynthesis, formed by elimination of C-9 to C-11 of the precursor fatty acids as malondialdehyde (43). Purifications of thromboxane synthase failed to separate thromboxane Bz- and HHT-forming activities and the ratio of HHT/thromboxane Bz formed remained constant (ca. 1.6) during the purifications.’ Several inhibitors of thromboxane synthase have been recognized (44, 45). These compounds inhibited not only the conversion of PGHz to thromboxane B2 but also the conversion of PGHz to HHT. Moreover, the degrees of inhibition of both reactions were the

same for various concentrations of all inhibitors tested. These results suggest that thromboxane synthase catalyzes the conversion of PGH2 both to thromboxane AZ and to HHT (38). Since thromboxane Az is very labile, it was conceivable that it was an intermediate in the formation of HHT. Thromboxane A2 was therefore trapped as the 12-O-methyl derivative of thromboxane Bz (7) after different times of incubation of PGHz with purified enzyme. The results (Fig. 12) showed that thromboxane Az and HHT were formed simultaneously and that thromboxane AX was degraded to thromboxane Bz but not to HHT in aqueous solution under the con-, ditions used (38). This excluded that thromboxane A2 was an intermediate in the formation of HHT. Analogous conversions have subsequently been demonstrated using other endoperoxide substrates. The 15-hydroperoxy analog of PGHz, PGGz, was thus transformed into two products, each one less polar than thromboxane B, (see Fig. 13) and HHT, respectively (42). These products were converted by stannous chloride reduction to thromboxane Bz and HHT and by lead tetraacetate dehydration to 15-ketothromboxane Bz and 12-keto5,8,10-heptadecatrienoic acid (the reduced and dehydrated products were identified by mass spectrometry). Based on these transformations the initial products were 15-hydroperoxy thromboxane Bz (AJ and 12- hydroperoxy - 5,8,10- heptadecatrienoic acid. The ratio of C17-acid/thromboxane was 2.3 when PGGz was used as substrate. PGHl is the endoperoxide intermediate in the conversion of dihomo-y-linolenic acid to prostaglandins of the one-series. Incubations of PGHr with purified preparations of thromboxane synthase unexpectedly gave no (or very little) formation of thromboxane B1. Instead, the endoperoxide was nearly quantitatively transformed into a product identified as 12-hydroxy-8,10-heptadecadienoic acid (HHD; ratio of C17-hydroxy acid/thromboxane > 40). This indicated that the A5-cis double bond in PGHz and PGGz was important for conversion to thromboxanes. Additional studies were performed to investi-

PROSTAGLANDINS

443

AND THROMBOXANES

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SO-

I

X5 OTMS

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;

453

155

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173

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100

150

200

250

300

FIG. 14. Mass spectra of thromboxanes mass spectrum; methyl ester, trimethylsilyl

‘-2’

IHPHT 1 a/o-tlomo HHT

400

450

500

,.,,! 550

formed from w-homo- (upper) or ol-homo-PGHa ether derivatives). From Ref. (46).

gate the effect of changing the position of the double bond in the carboxyl side chain of prostaglandin endoperoxides. 4,8,11,14-Eicosatetraynoic acid was converted to the corresponding &#HJpolyenoic acid by reduction with tritium gas and Lindlar catalyst and further to a/rhomo PGH2 PGH*

350

524

599 ,,, 600

(lower

3H-labeled A4-cis PGHl as described above. The latter was characterized by chemical transformations into A4-PGF1, and A4PGE1. This A4-isomer of PGHz was converted efficiently into a Cl,-hydroxy acid (12-hydroxy-4,8,10-heptadecatrienoic acid; A4-HHD (39)). No conversion to A4-throm-

A4 -PGH,

PGH,

'X4

(HPTXA,~ a/w-horn0

TXA2

FIG. 15. Transformations of prostaglandin endoperoxides by purified thromboxane synthase. HPHT, 12-hydroperoxy-5,8,10-heptadecatrienoic acid; HPTXAa, 15hydroxyperoxy thromboxane Aa; R, = (CHa)&OOH except in a-homo compounds in which R1 = (CH&COOH, Ra = (CHa)&Hs except in w-homo compounds in which Re = (CH&CHa; R3 = (CH&COOH.

444

SVEN HAMMARSTRbM

boxane B1 was detected (ratio of A4-HHD/ A4-thromboxane B1 > 40). This suggested a strict requirement for a cia double bond at precisely the A5 position of the endoperoxide to make possible transformation to thromboxanes. Similar experiments were performed with 5,8,11,14and I 6,9,12,15-heneicosatetraenoic acids. The tritium-labeled endoperoxides (whomo- and Lu-homo-PGHz, respectively) were converted by purified preparations of thromboxane synthase into mixtures of C18-hydroxy acids and thromboxanes (12hydroxyoctadecatrienoic acid plus w-homothromboxane Bz and 13-hydroxyoctadecatrienoic acid plus a-homo thromboxane Bz, respectively). The mass spectra of methyl ester, trimethylsilyl ether derivatives of o- and cY-homo-thromboxane Bz are shown in Fig. 14. The ratios of hydroxy acid to thromboxane formed were 1.2 when o- or cy-homo PGHz was used. Under the same conditions PGHB gave a ratio of HHT to thromboxane B2 of 1.3 (46). a-Homo PGHz contains a A6 double bond at the same distance from the cyclopentane ring as the A5double bond in PGH2. The results obtained with this endoperoxide show that the distance between the carboxyl group and the cis double bond can be altered without interfering with its conversion to thromboxane. The inability to convert A4PGHl to A4-thromboxane B1 is therefore probably due to the altered distance between the double of the carboxyl side chain and the cyclopentane ring in this endoperoxide. Figure 15 summarizes the conversions of various endoperoxides to thromboxanes and hydroxy fatty acids by purified preparations of thromboxane synthase. REFERENCES

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