603
Biochimica @ Elsevier
et Biophysics
Scientific
Acta,
Publishing
431
(1976)
Company,
603-614
Amsterdam
- Printed
in The Netherlands
BBA 56803
BIOSYNTHESIS OF PROSTAGLANDIN Fzcu FROM ARACHIDONIC AND PROSTAGLANDIN ENDOPEROXIDES IN THE UTERUS
PAULINA
WLODAWER,
Department
(Received
of Chemistry,
December
22nd,
HANS KINDAHL Karolinska
ACID
and MATS HAMBERG
Institutet,
S-104
01 Stockholm
(Sweden)
1975)
summary Formation of prostaglandin FZa in the cow and guinea pig uterus microsomes was studied using i4C-labeled arachidonic acid and prostaglandin Hz. The total conversion of arachidonic acid was of a low order and underwent fluctuations during the estrous cycle of the guinea pig, being highest towards the end of the cycle. Injections of P-estradiol-3-benzoate also resulted in higher activity of the uterine prostaglandin synthetase. The uterine prostaglandin synthesizing system appeared to differ in several respects from that present in seminal vesicles, with regard to the proportions of the products formed and the effects of various agents, e.g. reduced glutathione. An inhibiting factor which suppressed the fatty acid cycle-oxygenase was found to be present in uterine preparations. Prostaglandin endoperoxide (prostaglandin H,) was very efficiently reduced to prostaglandin Fzo, by cow and guinea-pig uterus microsomes. Prostaglandin Gz also gave rise to prostaglandin F?, . Prostaglandin Ez, on the other hand, was not reduced. Both the inhibiting factor and the endoperoxide reducing activity are likely to be parts of a highly specialized mechanism that modulates prostaglandin Fzcu formation in the uterus.
Introduction It has been generally accepted that a local relationship exists between the uterus and the life span of the corpus luteum in the ovary and that the uterus produces a luteolytic substance which is transported into the ovary. In a number of animal species prostaglandin Fzor has been implicated as the uterine luteolytic principle which leads to regression of the corpus luteum when pregnancy has not occurred (for a review see ref. 1). This conclusion is mostly based on the findings that the onset of corpus luteum regression coincides with an increase of prostaglandin F?, level in the uterus [2,3,4] and in the uteroovarian venous blood as well [5-S], It has also been shown that infusion of
604
physiological amounts of prostaglandin F,, into the uterine vein of sheep and cow causes premature corpus luteum regression [ lO,ll]. If the luteolytic effect of the uterus is due to release of prostaglandin Fzar, mechanisms should exist which control its production in the uterus so that a cyclic release is obtained. However, few attempts have been made, as yet, to study the prostaglandin synthesizing system of the uterus. Poyser [3] found that guinea-pig uterus homogenates possess the capability to synthesize prostaglandins from endogenous precursors on every day of the estrous cycle and that the amounts of prostaglandins, especially prostaglandin F,, , considerably increase towards the end of the cycle as well as after estradiol benzoate injections [ 121. Ham et al. [4] studied prostaglandin formation from arachidonic acid by rat uterus microsomes and concluded that the activity of the synthetase fluctuates during the estrous cycle, apparently under the influence of estrogens. However, our knowledge of the factors regulating prostaglandin synthesis in the uterus is still limited and this study was undertaken in order to gain more information on the production of prostaglandin Fzo, by uterine preparations. Materials and Methods Prost~l~din Ez and FZLYwere obtained from the Upjohn Company, Kalamazoo, Michigan, U.S.A., arachidonic acid from the Hormel Institute, Austin, Minn., U.S.A. and [ 1-‘4C]arachidonic acid (54 Ci/mol) from the Radiochemical Centre, Amersham, U.K., GSH, NADPH, L-epinephrine, p-hydroxymercuribenzoate and 17/3-estradiol-3-benzoate were products of Sigma Chem. Comp. Prostaglandin endoperoxides Hz and GZ, as well as 12L-hydroxy-5,8,10heptadecat~enoic acid were prepared according to published methods [13,14]. They were kept under argon in dry distilled acetone at -20°C and checked for purity by thin layer chromatography before use. [ 17,18-3H]prostaglandin Ez was a gift from Dr. Krister Green. Sheep vesicular gland microsomes were prepared from glands stored at -80°C for several months and the procedure was identical to that described in detail elsewhere [ 151, Cow uteri were obtained from the slaughterhouse and immediately frozen in dry ice. The animals were supposedly at various stages of the estrous cycle, but no attempts were made to correlate the results with the stage of the cycle. The separated endometrium was homogenized in 3 vol. of Tris-buffered 0.25 M sucrose pH 7.4 and cellular fractions were obtained by differential ~entrifugation at the same conditions as for the sheep vesicular glands [ 151. Guinea pigs were laboratory adult females weighing 600-800 g each, fed commercial diet and given drinking water ad libitum. Estrous cycles were recorded by daily examinations of the vagina and day 1 was taken as the day when the vulva was completely open. Injected animals obtained subcu~eously 10 gg of 17~~stradiol-3-benzoate dissolved in propylene glycol(O.1 ml). The animals were killed by cervical dislocation and the uteri were immediately removed. They were weighed, disintegrated with scissors and homogenized in 3 vol. of Tris-buffered 0.25 M sucrose. Subcellular fractions were obtained as indicated above.
605
Portions of subcellular fractions were routinely incubated in 1 ml of 0.1 M potassium phosphate buffer pH 7.8 and details of incubation are given in the appropriate figures and tables. Incubations were terminated with 7 vol. chloroform/methanol (1 : 1) and lipids were extracted as elsewhere described [ 151. Products formed during incubation were separated by thin layer chromatography on pre-coated silica gel 20 X 20 cm plates, activated before use at 120°C for 0.5 h. Two solvent systems were used: Solvent I, benzene/dioxane/acetic acid 80 : 20 : 2 and Solvent II which consisted of the organic phase of the following components: ethyl acetate/2,2,4-trimethylpentane/benzene/water/acetic acid 50 : 15 : 10 : 50 : 5. Radioactivity on the plates was determined using a Berthold Diinnschichtscanner II. The radioactive zones were scraped off and counted in a Packard Tri-Carb liquid scintillation spectrometer model 3375. Authentic samples of prostaglandin E,, F,, and arachidonic acid were used as references and visualized by iodine vapours. For mass-spectrometric analysis the appropriate zone was scraped off from the plate and eluted with methanol. The methanol extract was concentrated to a small volume, acidified with 0.2 M HCl and extracted with diethyl ether. The residue of the ether extract was converted into the methyl ester with diazomethane, derivatized and analyzed using a LKB 9000 instrument equipped with a 1% SE-30 column on Gas-chrom Q. Protein was estimated by Lowry’s method [ 161 with bovine serum albumin as standard. Results I. Conversion
of arachidonic
acid by uterus preparations
Homogenates of cow endometrium were prepared and portions of cell-free homogenate, cell particles (including mitochondria and microsomes), isolated microsomes, and particle-free 105 000 Xg supematant, were incubated with [1-14C]arachidonate in order to determine the localization of the synthetase. Prostaglandin-synthesizing activity was found in all fractions except the highspeed supernatant and it was highest in the microsomes. In most experiments microsomes were used as the enzyme source. Products appeared which on thin-layer plates had the motilities of prostaglandin F,, , E, (and thromboxane B,) and Dz, respectively. In view of the small amounts of the products formed their structures could not be confirmed by mass-spectrometric analysis. The effects of several agents on the conversion of arachidonic acid were also investigated (Table I). The following conclusions could be drawn from the experiments: 1. The conversion of arachidonic acid into prostaglandins was of a very low order and substrate of rather high specific activity had to be used in order to enable accurate determination of the amounts of products formed. It should be noted that the yields of prostaglandins differed in various preparations (possibly i.a. as a result of the stage in the estrous cycle at which the animal had been killed) and values between 5 and 14% conversion were obtained. 2. The product corresponding to prostaglandin Fzo! constituted a large part
606
TABLE
I
EFFECTS OF CERTAIN ADDITIONS COW UTERUS MICROSOMES
ON THE
CONVERSION
OF [1-‘4C]ARACHIDONIC
ACID
BY
The animal was supposedly in the mid-cycle, as estimated by gross examination of the endometrium and the ovaries. Microsomes corresponding to 1 g endometrial tissues (3.5 mg protein) were incubated with 3 pg (10 PM) [l-14C1arachidonic acid in the form of ammonium salt (60 000 cpm) for 20 min in 1 ml of 0.1 M potassium phosphate buffer pH ‘1.8 at 37’C, and additions were made as indicated. Reaction was started by addition of substrate after 2 min preincubation and terminated with 7 ml chloroform/methanol 1 : 1. Lipids were extracted and products separated by thin-layer chromatography in solvent systems I and II, as described elsewhere [151. Additions
Product formation
a
Product formation
Prostaglandin b
Fza ’
(%)
(%) None GSH 0.5 mM GSH + hydroquinone (0.5 mM each) L-Epincphrine 10 mM High-speed supernatant (0.2 g tissue) High-speed supematant (0.4 g tissue) Boiled supernatant (0.2 g tissue) Supernatant (0.2 g tissue) + GSH 0.5 mM a Products b Products was taken c % of total
13.0 8.8 9.2 13.8
100 67 70 105
2.8 2.5 2.9 5.8
8.9
70
1.4
7.3
55
1.2
8.1
67
1.5
5.0
37
0.9
not including monohydroxyacids; % of total radioactivity recovered from the plate. not including monohydroxyacids; the percentage obtained in the absence of any addition as 100. radioactivity recovered from the plate.
of the total and in,various preparations it contained between 20 and 40% of the radioactivity recovered from the plate. In the presence of L-epinephrine the conversion was shifted towards higher production of prostaglandin Fzcu, without any significant change in the yield of total prostaglandins. 3. GSH and hydroquinone were found to inhibit the uterine prostaglandin synthetase. 4. Addition of high-speed supernatant to the incubation medium also resulted in a dose-dependent inhibition of prostaglandin formation. The possibility was considered that the poor conversion of the labeled arachidonic acid could be due to its dilution by endogenous (unlabeled) substrate. However, using a Clark electrode, extemely small O2 uptake by uterus microsomes was observed after addition of increasing amounts of arachidonic acid. Increasing the concentration of microsomes did not improve O2 consumption which consistently was too low to be accurately measured. When the relationship between the concentration of microsomes in the incubation mixture and the yield of prostaglandins formed from arachidonic acid was systematically determined, it appeared (Table II) that by increasing the amounts of microsomes no significant increase in the percentage conversion of arachidonic acid could be achieved. This finding as well as the observation that increasing the concentration of
607
TABLE
II
EFFECT
OF
CONCENTRATION
[1-14ClARACHIDONIC The animal
OF
COW
UTERUS
MICROSOMES
ON
THE
was supposedly
on day 3 or 4 of the estrous cycle.
Conditions
of incubation
in Table I, except that the concentration of microsomes was varied as indicated made. Microsomes corresponding to 1 g tissue contained 3.5 mg protein. Microsomes (g of tissue)
(S)
0.1 0.2 0.4 0.8 1.0 1.5 2.0
4.4 5.4 4.8 5.1 5.7 5.2 5.8 a Conversion
CONVERSION
OF
ACID
Conversion
into products
were the same as
and no additions
were
a
not including
monohydroxy
acids.
microsomes had practically no effect on O2 uptake suggested that an inhibitor was present in the endometrial tissue and that by increasing the amounts of microsomes the concentration of inhibitor was also elevated. In order to check this supposition, sheep vesicular gland microsomes were incubated with [ l-14C]arachidonic acid and varying amounts of microsomes from either cow or guinea pig uterus were included in the incubation mixture. A radiochromatogram of the products formed from [l-‘4C]arachidonate by vesicular gland microsomes in the absence and in the presence of cow uterus microsomes is shown in Fig. 1. Table III shows the effect of concentration of uterus microsomes on prostaglandin formation. It can be seen that uterus microsomes effectively inhibited the production of prostaglandins by vesicular gland microsomes, in a dose-dependent fashion, and that the inhibition was somewhat lowered but not abolished by heating. Table IV shows that O2 consumption by vesicular gland microsomes after addition of arachidonic acid was also inhibited in a very similar way. Those facts indicated that inhibition of the conversion of labeled arachidonate was not due to its dilution by endogenous fatty acids and that the inhibition appeared to be at the level of the fatty acid cycle-oxygenase, the enzyme responsible for the conversion of arachidonic acid into prostaglandin G2. II. Reduction of prostaglandin endoperoxides by uterus microsomes It can be seen in Fig. 2 that when cow uterus microsomes were incubated with [l-‘4C]prostaglandin Hz, a considerable part of the substrate was reduced and appeared as prostaglandin Fzar. Microsomes were also prepared from several organs of the guinea-pig and incubated with prostaglandin Hz in the presence or absence of certain additions (Table V). It can be seen that in the presence of microsomes from lung, liver, and kidney, prostaglandin F,, was never formed in any significant amounts and addition of either GSH or NADPH was without effect. Practically identical results were obtained in several separate experi-
608
Distance
from
origin
(cml
Fig. 1. Thin layer radiochromatogram showing the products of arachidonic acid conversion by sheep vesicular gland microsomes alone (upper panel) and in the presence of cow uterus microsomes corresponding to 0.4 g tissue (lower panel). Vesicular gland microsomes corresponding to 50 mg tissue were incubated with 0.1 mM [l-14C]arachidonic acid (30 000 cpm) for 10 min in 1 ml potassium phosphate buffer pH 7.8 and products were isolated as indicated in Table I. The designations AA, PGE2 and PGFzo, refer to the positions on the plate of arachidonic acid, prostaglandin Ez, and prostaglandin Fz~, respectively. Fig. 2. Thin layer chromatography of the products formed during incubation of uterus microsomes with prostaglandin Hz. (b) Cow uterus microsomes corresponding to 0.8 g tissue were incubated with [1-14C] prostaglandin H2 (1 pg. 44 000 cpm) for 10 min in 1 ml 0.1 M phosphate buffer pH 7.8. Lipids were extracted as indicated in Table I and products were separated by thin-layer chromatography in solvent system II. (a) Authentic sample of [3Hlhydroxyheptadecatrienoic acid (25 000 cpm) applied immediately before development of the plate. (c) [l-14Clprostaglandin H2 (15 000 cpm) applied immediately,before development. Designations PGFzo, and PGE2 refer to authentic prostaglandins Fzol and E2.
ments. On the other hand, uterus microsomes possessed a very high capacity to form prostaglandin Fzo, from prostaglandin Hz (Table V). The reducing activity did not require any cofactors (GSH or NADPH), but was completely abolished in the presence of 1 mM p-hydroxymercuribenzoate. Fig. 3 shows that a positive relationship existed between the concentration of uterus microsomes and formation of prostaglandin F,, from a given amount of prostaglandin Hz. As shown in Fig. 4, the amount of prostaglandin Fza formed was proportional to the concentration of prostaglandin H2 (up to 4 pg/ml) but it leveled off with higher concentrations (more than 7 pg/ml). A large scale incubation was performed using microsomes corresponding to 5 g of cow uterus endometrium and 25 pg prostaglandin Hz. The zone corresponding to prostaglandin FZa was scraped off from the plate and mass spectra were taken of the trimethylsilyl (Me,Si) derivative of the methyl ester of the product (carbon value 24.0 on 1% SE-30 column). The mass spectrum showed ions of high intensity at m/e 584 (M, molecular ion), 569 (M-15; loss of CHJ),
609
TABLE
III
EFFECT OF UTERUS MICROSOMES VESICULAR GLAND MICROSOMES
ON THE CONVERSION
OF ARACHIDONIC
ACID
BY SHEEP
Sheep vesicular gland microsomes corresponding to 50 mg tissue were incubated with [1-14Clarachidonate (0.1 mM, 30 000 cpm) in the absence or presence of varying amounts of uterus microsomes. Details of incubation are shown in Table I. L._____,_~___ __.-._._~ ~._. ..-. ~~.-..--~___ Additions __
Conversion _.________.___
~_.
None Cow uterus microsomes
(96) __ _.__.._...
.. . .
Inhibition
a ._~_._~.
(%) ~_ -
g g g g g
74 66 49 36 21 27
Boiled cow uterus microsomes:
0.2 g 0.4 g
52 45
30 39
Guinea-pig
0.2 g
43
42
0.4 g
33
56
corresponding
to:
0.1 0.2 0.4 0.8 1.0
uterus microsomes:
.~_.______.._~~~
----.
a Conve&on into covered
products
11 34 51 64 64
----not
including
monohydroxyacids;
.___ .__.~ ~~-
percentage
__._
._.____
of total radioactivity
re-
from the plate.
513 (M-71; loss of (CH2)4CH,), 494 (M-90; loss of Me&OH), 443 (M-141; cleavage between C-7 and C-8), 423 (M-(90+71)), 353 (M-(90+141)), 333 (M(2 X 90+71)), 199 (side chain attached to C12). This evidence confirmed the identity of the product formed from pros~gl~din H, as pros~landin Fzcr. The reducing activity of cow uterus microsomes was also checked using prostaglandin Gz as substrate. In incubations containing 2 /_tg [l-14C]prostaglandin Gz (88 000 cpm) and microsomes corresponding to either 0.4 or 0.8 g the substrate was found to produce 20% and 25%, respectively, of prostaglandin Fz,. However, no reduction of pros~gl~din E, occurred in incubations of uterus microsomes with ‘H-labeled I!& for 20 or 30 min.
TABLE EFFECT SOMES
IV OF
UTERUS
MICROSOMES
ON 02
UPTAKE
BY SHEEP
VESICULAR
GLAND
MICRO-
Sheep vesicular gland microsomes corresponding to 50 mg tissue were incubated in 1 ml of 0.1 M potassium phosphate buffer pH 7.8 in the absence or presence of varying amounts of cow uterus microsomes. Incubations were performed in an oxygraph cell equipped with a Clark electrode and maintained at 37’C. After addition of araehidonate (0.1 mM final concentration) total 02 uptake during 3 min was measured. --.~~_._.____~~ Addition
02 uptake (arbitrary units)
Inhibition
None
68
-
56 50 34 30
18 26 50 56
Uterus microsomes
0.2 0.4 0.6 0.8
g g g g
(W)
610
o-7iTx-z-r g (tissue
1’6
1.6
PGHZ
1
Fig. 3. Relationship between concentration tions identical to those shown in the legend somes was varied as indicated.
1 pg
)
of microsomes and formation of prostaglandin Fzo. Condito Fig. 2 except that the concentration of cow uterus micro-
Fig. 4. Effect of increasing concentrations of prostaglandin H2 on formation of prostagiandin Fzo. Cow uterus microsomes corresponding to 0.8 g tissue were incubated with [1-14Clprostaglandin Hz in varying concentrations. Other details are identical to those indicated in the legend to Fig. 2.
TABLE
V
FORMATION CROSOMES
OF PROSTAGLAN~IN
FzOi FROM
~l-i4ClPROSTAGLANDIN
H2 BY VARIOUS
MI-
Microsomes prepared from various tissues were incubated with [l- 14C]prostaglandin H2 (50 000 cpm. spec. act. 44 000 cpm/ng) for 10 min in 0.1 M potassium phosphate buffer pH 7.8 at 37’C in the presence and absence of certain additions. Total volume was 1 ml. GSH and hydroquinone (HQ) were 0.5 mM each, NADPH was 0.1 mM, and p-hydroxymercuribenlooate @HMB) was 1 mM. The reaction was terminated with 7 ml chIoroform/meth~ol 1 : 1, &ids were extracted as described 1151 and products were separated by thin layer chromatography in solvent system II. _.._____-. -.-. -..-...- .Incubation
Additions
(%I
-_-_--____
none
lung micrOSOmeS
Guinea pig liver microsomes
Guinea pig kidney
F2o
_. ._ __._~.____ -_-...-..___._-.___.-.._-^_.-..--.- ..-.
Buffer
Guinea-pig
Prostaglandin
microsomes
Guinea pig uterus microsomes Cow uterus microsomes
(0.2 8) (0.4 (0.8 (0.4 (0.4 (0.1 (0.2 (0.4 (0.2 (0.2 (0.2 (0.4 (0.8 (0.4 (0.4 (0.2 (0.4 (0.2 (0.4 (0.4 (0.4 (0.2
g) g) g) 9) g> g> gf g) 9) gl g) g) gf g) g) 9) 9) 9) g) g> g)
(0.4 gf
ncme NADPH none none none GSH + HQ NADPH mme none none GSH + HQ NADPH none n0ne none GSH + HQ N ADPH none none none nime GSH + HQ NADPH pHMB pHMB
3 4 4 4 4 3 4 4 9 10 11 11 9 9 8 9 10 9 35 46 51 55 56 56 5 3
____.“..__ .._.._ -- ..-
611
Microsomes solubilized with 1% Cutscum still retained high reducing activity, but it was completely lost in microsomes solubilized with 1.5% Tween 20. When 100 000 X g supe~at~t of Cu~cum-solubilized microsomes was applied to a Sephadex G-75 column, the reducing activity appeared in the void volume, together with the bulk of protein. The reducing activity was found to be highly resistant to heating and a microsomal suspension kept in a boiling water bath for 10 min still retained up to 80% activity. When the boiled suspension of microsomes was centrifuged at 100 000 Xg for 60 min, the activity appeared in the sediment and was absent from the supernatant. When uterus microsomes were extracted with either ethyl acetate or 10% aqueous acetone, the reducing activity was present in the lipid-depleted particles and absent from the lipid extract.
Guinea pigs were used to study the relationship between the estrous cycle and prostaglandin formation by uterus microsomes. Some animals received injections of estradiol benzoate as indicated (Table VI). It can be seen that the percentage conversion of [l-‘4C]arachidonic acid was even lower than with cow uterus microsomes. The conversion into both total products and into prostaglandin FZ, fluctuated in the course of the estrous cycle and was highest to-
TABLE VI CONVERSION OF [l-14ClARACHIDON~~
ACID BY GUINEA PIG UTERUS
Microsomes were prepared from uteri of animals on various days of the estrous cycle and portions containing about 1 mg of protein were incubated with 7 yM (2 @g) [l- 14Clarachidordc acid (120 000 cpm) in the absence of any additions. Details of incubation and determination of products are given in Table I. Animal and treatment
Weight of uterus (9)
Conversion a (%>
Prostaglandin Fzol b (%)
day 6 5 between 4 and 10 12 15 16 16 1 day 4 : 3 injections of estradiol benzoate (10 pg each) starting on day 2 day 8 : 5 injections of e&radio1 benzoate starting on day 4 day 12: 5 injections of estradiol benzoate starting on day 8
1.2 1.2 1.0 1.0 1.3 2.0 1.9 1.9
2.1 2.6 2.4 3.4 4.0 6.3 6.8 6.3
0.9 0.7 1.1 1.2 1.5 1.8 2.7 2.4
2.0
5.5
1.8
2.1
4.8
1.7
2.4
3.2
1.4
a Conversion into products not including monobydroxy acids; percentage of total radioactivity recovered from the thin-layer plate. b Percentage of total radioactivity recovered from the plate.
612
wards the end of the cycle. Since the uterus considerably increased in size at the end of the estrous cycle, the total production of prostaglandins from the radioactive precursor can be estimated to be 6-8 times higher at the end than on other days of the cycle. Uterus microsomes obtained from animals injected with estradiol benzoate also possessed higher capacity to convert the labeled arachidonic acid (Table VI). Injection of the vehicle alone (0.1 ml of propylene glycol) has been found to have no effect. Microsomal preparations of uteri from guinea pigs at different stages of the estrous cycle were also assayed with regard to their ability to inhibit prostaglandin formation by sheep vesicular gland microsomes. All preparations were found to possess high inhibiting activity. Production of prostaglandins from 20 lug [l-‘4C]arachidonic acid by vesicular gland microsomes (50 mg tissue) was inhibited by 40-50% in the presence of uterus microsomes (0.2 g tissue). Some differences in inhibiting activity existed, but they were small and could not be accurately enough evaluated by the method used. Thus, the degree of inhibition could not unequivocally be related to the estrous cycle. Discussion While much work has already been done on the release of prostaglandins from the uterus and on the role of prostaglandin F,, as the luteolyzing factor, very few reports have appeared as yet dealing with the uterine prostaglandin synthesizing system in vitro. This could, partially at least, be due to the low capacity of the uterine tissue to produce prostaglandins either from endogenous [3,12] or exogenous [4] precursors. In order to measure the small amounts of prostaglandin Ez and prostaglandin F,, formed in guinea pig homogenates, Poyser [3] used a bioassay on the rat fundus strip, Ham et al. [ 41 determined prostaglandins in rat uterus microsomes by radioimmunoassays, and Naylor and Poyser [12] measured the amounts of prostaglandin F,, by radioimmunoassay or combined gas chromatography-mass-spectrometry. In the present study, it has been possible to measure the conversion into products of radioactive arachidonic acid by microsomes of either cow endometrium or guinea pig uterus. It appeared that the synthetase system in the uterus differed in several respects from the highly efficient systems in the ovine and bovine seminal vesicles. In accordance with the authors cited above [ 3,4], the capacity to form prostaglandins from arachidonic acid was always very low, although it underwent fluctuations during the estrous cycle, as discussed later. Prostaglandin Fzc, comprised up to 40% of the total products of conversion while it is only a minor component of the products formed by sheep vesicular gland microsomes (Fig. 1 and ref. 17). GSH and hydroquinone that are known to promote prostaglandin biosynthesis in sheep [18] and bull [ 191 seminal vesicles and have been routinely included in incubation media of other tissues as well (e.g. refs. 20-22) were found in this study to persistently inhibit the already poor conversion of arachidonic acid into prostaglandins. The same effect was observed with the particle-free supernatant that also promotes prostaglandin formation by sheep vesicular gland microsomes [ 181. Substantial differences have been observed between the prostaglandin-synthesizing systems in rabbit or dog brain [22] and rabbit kidney [21] as compared to the one from bovine
613
seminal vesicles [23], and it is quite conceivable that prostaglandin synthetase from different tissues may possess differing properties which could be of physioIogic~ importance. In accordance with the findings by Poyser 131, the prostaglandin synthesizing activity in the guinea-pig uterus has been found to be highest at the end of the estrous cycle. Injections of estradiol benzoate also resulted in higher percentage conversion of arachidonic acid by guinea-pig microsomes as compared to the controls (see also ref. 12) and these findings seem to confirm the suggestion made by Kuehl et al. [24] that prostaglandin synthesizing enzyme systems are subject to fine hormonal control. Not only more prostaglandin Fzor was formed from the labeled arachidonic acid at the end of the cycle. The same was also true for other products of conversion which on thin-layer plates had the motilities of prostaglandin E, (and thrombox~e B,) and prostaglandin D2. However, the actual formation of those products is difficult to evaluate, since the possibility cannot be excluded that at least a part of them may represent that portion of endoperoxides which had not been converted into prostaglandin F Zoland underwent non-enzymatic rearrangement in the aqueous medium [ 13,251. The total conversion of arachidonic acid into products not including monohydroxyacids actually reflects the production of endoperoxides by uterus microsomes. Even at the height of activity the synthetic capacity of the uterus is of a very low order, The possibility exists that this may be due to the presence in the tissue of an inhibiting factor which suppresses the early steps in prostaglandin biosynthesis and is even able to counteract the extremely efficient prostaglandin synthetase in sheep vesicular glands. The nature of the inhibiting factor has not been elucidated so far. It is present in uterus homogenate and microsomes and, to a lesser extent, in the particle-free supernatant as well (this could possibly account for the inhibiting effect of the supernatant on prostaglandin formation by microsomes), it is resistant to heating and its effect is lowered but not abolished by boiling for several minutes. It seems conceivable that its activity also fluctuates during the estrous cycle thus leading to modulations in the amounts of endoperoxide intermediates formed, although our method of estimation was not sensitive enough to measure the small fluctuations in inhibition with the desired degree of precision. In the present study, uterus tissue was found to possess a very high capacity to convert exogenous endoperoxide, prostaglandin HZ, into prostaglandin Fzo,. This feature seems to be unique for uterus microsomes, since microsomes from guinea-pig kidney, lung and liver gave only a very small yield of prostaglandin F,, from prostaglandin HZ. Enzymes are known to exist which catalyze reduction of prostaglandin E compounds to prostaglandin F compounds [ 26-281. However, no transformation of tritium-labeled prostaglandin E2 incubated with uterus microsomes was detected in the present study. The capability to reduce prostaglandin H2 to prostagl~din F,, was found to be completely abolished by p-hydroxymercuribenzoate, an SH-group agent, but any attempts to isolate and purify the reducing factor have been as yet unsuccessful, and the elucidation of its real nature must await further studies. In conclusion, the results of the present study suggest that a highly specialized mechanism exists which modulates prostaglandin Fza: formation in the
614
uterus. Inhibition of the cycle-oxygenase reaction results in keeping prostaglandm F,, well below the level which could induce luteolysis of the corpus luteum during most of the estrous cycle. At some defined stage in the cycle, when the inhibition is likely to be somewhat less pronounced, more endoperoxide can be formed and, in the presence of an active reducing factor, efficiently converted into prostaglandin F?, which, after being discharged into the blood, can affect its target in the ovary. Acknowledgements This work was supported by grants from the World Health Organisation the Swedish Medical Research Council (Project no. 03X-217).
and
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2
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3
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4
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S.A.
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RR.
Butcher,
(1972)
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M.E.
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R.L.
and
Rec.
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(1972).
28,
51-73
J. Animal
Sci.
34.9349
147-159 and
Kuehl,
Jr.,
F.A.
(1975)
Proc.
Natl.
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Sci.
U.S.
72,
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BBA 56803
BIOSYNTHESIS OF PROSTAGLANDIN Fzcu FROM ARACHIDONIC AND PROSTAGLANDIN ENDOPEROXIDES IN THE UTERUS
PAULINA
WLODAWER,
Department
(Received
of Chemistry,
December
22nd,
HANS KINDAHL Karolinska
ACID
and MATS HAMBERG
Institutet,
S-104
01 Stockholm
(Sweden)
1975)
summary Formation of prostaglandin FZa in the cow and guinea pig uterus microsomes was studied using i4C-labeled arachidonic acid and prostaglandin Hz. The total conversion of arachidonic acid was of a low order and underwent fluctuations during the estrous cycle of the guinea pig, being highest towards the end of the cycle. Injections of P-estradiol-3-benzoate also resulted in higher activity of the uterine prostaglandin synthetase. The uterine prostaglandin synthesizing system appeared to differ in several respects from that present in seminal vesicles, with regard to the proportions of the products formed and the effects of various agents, e.g. reduced glutathione. An inhibiting factor which suppressed the fatty acid cycle-oxygenase was found to be present in uterine preparations. Prostaglandin endoperoxide (prostaglandin H,) was very efficiently reduced to prostaglandin Fzo, by cow and guinea-pig uterus microsomes. Prostaglandin Gz also gave rise to prostaglandin F?, . Prostaglandin Ez, on the other hand, was not reduced. Both the inhibiting factor and the endoperoxide reducing activity are likely to be parts of a highly specialized mechanism that modulates prostaglandin Fzcu formation in the uterus.
Introduction It has been generally accepted that a local relationship exists between the uterus and the life span of the corpus luteum in the ovary and that the uterus produces a luteolytic substance which is transported into the ovary. In a number of animal species prostaglandin Fzor has been implicated as the uterine luteolytic principle which leads to regression of the corpus luteum when pregnancy has not occurred (for a review see ref. 1). This conclusion is mostly based on the findings that the onset of corpus luteum regression coincides with an increase of prostaglandin F?, level in the uterus [2,3,4] and in the uteroovarian venous blood as well [5-S], It has also been shown that infusion of
604
physiological amounts of prostaglandin F,, into the uterine vein of sheep and cow causes premature corpus luteum regression [ lO,ll]. If the luteolytic effect of the uterus is due to release of prostaglandin Fzar, mechanisms should exist which control its production in the uterus so that a cyclic release is obtained. However, few attempts have been made, as yet, to study the prostaglandin synthesizing system of the uterus. Poyser [3] found that guinea-pig uterus homogenates possess the capability to synthesize prostaglandins from endogenous precursors on every day of the estrous cycle and that the amounts of prostaglandins, especially prostaglandin F,, , considerably increase towards the end of the cycle as well as after estradiol benzoate injections [ 121. Ham et al. [4] studied prostaglandin formation from arachidonic acid by rat uterus microsomes and concluded that the activity of the synthetase fluctuates during the estrous cycle, apparently under the influence of estrogens. However, our knowledge of the factors regulating prostaglandin synthesis in the uterus is still limited and this study was undertaken in order to gain more information on the production of prostaglandin Fzo, by uterine preparations. Materials and Methods Prost~l~din Ez and FZLYwere obtained from the Upjohn Company, Kalamazoo, Michigan, U.S.A., arachidonic acid from the Hormel Institute, Austin, Minn., U.S.A. and [ 1-‘4C]arachidonic acid (54 Ci/mol) from the Radiochemical Centre, Amersham, U.K., GSH, NADPH, L-epinephrine, p-hydroxymercuribenzoate and 17/3-estradiol-3-benzoate were products of Sigma Chem. Comp. Prostaglandin endoperoxides Hz and GZ, as well as 12L-hydroxy-5,8,10heptadecat~enoic acid were prepared according to published methods [13,14]. They were kept under argon in dry distilled acetone at -20°C and checked for purity by thin layer chromatography before use. [ 17,18-3H]prostaglandin Ez was a gift from Dr. Krister Green. Sheep vesicular gland microsomes were prepared from glands stored at -80°C for several months and the procedure was identical to that described in detail elsewhere [ 151, Cow uteri were obtained from the slaughterhouse and immediately frozen in dry ice. The animals were supposedly at various stages of the estrous cycle, but no attempts were made to correlate the results with the stage of the cycle. The separated endometrium was homogenized in 3 vol. of Tris-buffered 0.25 M sucrose pH 7.4 and cellular fractions were obtained by differential ~entrifugation at the same conditions as for the sheep vesicular glands [ 151. Guinea pigs were laboratory adult females weighing 600-800 g each, fed commercial diet and given drinking water ad libitum. Estrous cycles were recorded by daily examinations of the vagina and day 1 was taken as the day when the vulva was completely open. Injected animals obtained subcu~eously 10 gg of 17~~stradiol-3-benzoate dissolved in propylene glycol(O.1 ml). The animals were killed by cervical dislocation and the uteri were immediately removed. They were weighed, disintegrated with scissors and homogenized in 3 vol. of Tris-buffered 0.25 M sucrose. Subcellular fractions were obtained as indicated above.
605
Portions of subcellular fractions were routinely incubated in 1 ml of 0.1 M potassium phosphate buffer pH 7.8 and details of incubation are given in the appropriate figures and tables. Incubations were terminated with 7 vol. chloroform/methanol (1 : 1) and lipids were extracted as elsewhere described [ 151. Products formed during incubation were separated by thin layer chromatography on pre-coated silica gel 20 X 20 cm plates, activated before use at 120°C for 0.5 h. Two solvent systems were used: Solvent I, benzene/dioxane/acetic acid 80 : 20 : 2 and Solvent II which consisted of the organic phase of the following components: ethyl acetate/2,2,4-trimethylpentane/benzene/water/acetic acid 50 : 15 : 10 : 50 : 5. Radioactivity on the plates was determined using a Berthold Diinnschichtscanner II. The radioactive zones were scraped off and counted in a Packard Tri-Carb liquid scintillation spectrometer model 3375. Authentic samples of prostaglandin E,, F,, and arachidonic acid were used as references and visualized by iodine vapours. For mass-spectrometric analysis the appropriate zone was scraped off from the plate and eluted with methanol. The methanol extract was concentrated to a small volume, acidified with 0.2 M HCl and extracted with diethyl ether. The residue of the ether extract was converted into the methyl ester with diazomethane, derivatized and analyzed using a LKB 9000 instrument equipped with a 1% SE-30 column on Gas-chrom Q. Protein was estimated by Lowry’s method [ 161 with bovine serum albumin as standard. Results I. Conversion
of arachidonic
acid by uterus preparations
Homogenates of cow endometrium were prepared and portions of cell-free homogenate, cell particles (including mitochondria and microsomes), isolated microsomes, and particle-free 105 000 Xg supematant, were incubated with [1-14C]arachidonate in order to determine the localization of the synthetase. Prostaglandin-synthesizing activity was found in all fractions except the highspeed supernatant and it was highest in the microsomes. In most experiments microsomes were used as the enzyme source. Products appeared which on thin-layer plates had the motilities of prostaglandin F,, , E, (and thromboxane B,) and Dz, respectively. In view of the small amounts of the products formed their structures could not be confirmed by mass-spectrometric analysis. The effects of several agents on the conversion of arachidonic acid were also investigated (Table I). The following conclusions could be drawn from the experiments: 1. The conversion of arachidonic acid into prostaglandins was of a very low order and substrate of rather high specific activity had to be used in order to enable accurate determination of the amounts of products formed. It should be noted that the yields of prostaglandins differed in various preparations (possibly i.a. as a result of the stage in the estrous cycle at which the animal had been killed) and values between 5 and 14% conversion were obtained. 2. The product corresponding to prostaglandin Fzo! constituted a large part
606
TABLE
I
EFFECTS OF CERTAIN ADDITIONS COW UTERUS MICROSOMES
ON THE
CONVERSION
OF [1-‘4C]ARACHIDONIC
ACID
BY
The animal was supposedly in the mid-cycle, as estimated by gross examination of the endometrium and the ovaries. Microsomes corresponding to 1 g endometrial tissues (3.5 mg protein) were incubated with 3 pg (10 PM) [l-14C1arachidonic acid in the form of ammonium salt (60 000 cpm) for 20 min in 1 ml of 0.1 M potassium phosphate buffer pH ‘1.8 at 37’C, and additions were made as indicated. Reaction was started by addition of substrate after 2 min preincubation and terminated with 7 ml chloroform/methanol 1 : 1. Lipids were extracted and products separated by thin-layer chromatography in solvent systems I and II, as described elsewhere [151. Additions
Product formation
a
Product formation
Prostaglandin b
Fza ’
(%)
(%) None GSH 0.5 mM GSH + hydroquinone (0.5 mM each) L-Epincphrine 10 mM High-speed supernatant (0.2 g tissue) High-speed supematant (0.4 g tissue) Boiled supernatant (0.2 g tissue) Supernatant (0.2 g tissue) + GSH 0.5 mM a Products b Products was taken c % of total
13.0 8.8 9.2 13.8
100 67 70 105
2.8 2.5 2.9 5.8
8.9
70
1.4
7.3
55
1.2
8.1
67
1.5
5.0
37
0.9
not including monohydroxyacids; % of total radioactivity recovered from the plate. not including monohydroxyacids; the percentage obtained in the absence of any addition as 100. radioactivity recovered from the plate.
of the total and in,various preparations it contained between 20 and 40% of the radioactivity recovered from the plate. In the presence of L-epinephrine the conversion was shifted towards higher production of prostaglandin Fzcu, without any significant change in the yield of total prostaglandins. 3. GSH and hydroquinone were found to inhibit the uterine prostaglandin synthetase. 4. Addition of high-speed supernatant to the incubation medium also resulted in a dose-dependent inhibition of prostaglandin formation. The possibility was considered that the poor conversion of the labeled arachidonic acid could be due to its dilution by endogenous (unlabeled) substrate. However, using a Clark electrode, extemely small O2 uptake by uterus microsomes was observed after addition of increasing amounts of arachidonic acid. Increasing the concentration of microsomes did not improve O2 consumption which consistently was too low to be accurately measured. When the relationship between the concentration of microsomes in the incubation mixture and the yield of prostaglandins formed from arachidonic acid was systematically determined, it appeared (Table II) that by increasing the amounts of microsomes no significant increase in the percentage conversion of arachidonic acid could be achieved. This finding as well as the observation that increasing the concentration of
607
TABLE
II
EFFECT
OF
CONCENTRATION
[1-14ClARACHIDONIC The animal
OF
COW
UTERUS
MICROSOMES
ON
THE
was supposedly
on day 3 or 4 of the estrous cycle.
Conditions
of incubation
in Table I, except that the concentration of microsomes was varied as indicated made. Microsomes corresponding to 1 g tissue contained 3.5 mg protein. Microsomes (g of tissue)
(S)
0.1 0.2 0.4 0.8 1.0 1.5 2.0
4.4 5.4 4.8 5.1 5.7 5.2 5.8 a Conversion
CONVERSION
OF
ACID
Conversion
into products
were the same as
and no additions
were
a
not including
monohydroxy
acids.
microsomes had practically no effect on O2 uptake suggested that an inhibitor was present in the endometrial tissue and that by increasing the amounts of microsomes the concentration of inhibitor was also elevated. In order to check this supposition, sheep vesicular gland microsomes were incubated with [ l-14C]arachidonic acid and varying amounts of microsomes from either cow or guinea pig uterus were included in the incubation mixture. A radiochromatogram of the products formed from [l-‘4C]arachidonate by vesicular gland microsomes in the absence and in the presence of cow uterus microsomes is shown in Fig. 1. Table III shows the effect of concentration of uterus microsomes on prostaglandin formation. It can be seen that uterus microsomes effectively inhibited the production of prostaglandins by vesicular gland microsomes, in a dose-dependent fashion, and that the inhibition was somewhat lowered but not abolished by heating. Table IV shows that O2 consumption by vesicular gland microsomes after addition of arachidonic acid was also inhibited in a very similar way. Those facts indicated that inhibition of the conversion of labeled arachidonate was not due to its dilution by endogenous fatty acids and that the inhibition appeared to be at the level of the fatty acid cycle-oxygenase, the enzyme responsible for the conversion of arachidonic acid into prostaglandin G2. II. Reduction of prostaglandin endoperoxides by uterus microsomes It can be seen in Fig. 2 that when cow uterus microsomes were incubated with [l-‘4C]prostaglandin Hz, a considerable part of the substrate was reduced and appeared as prostaglandin Fzar. Microsomes were also prepared from several organs of the guinea-pig and incubated with prostaglandin Hz in the presence or absence of certain additions (Table V). It can be seen that in the presence of microsomes from lung, liver, and kidney, prostaglandin F,, was never formed in any significant amounts and addition of either GSH or NADPH was without effect. Practically identical results were obtained in several separate experi-
608
Distance
from
origin
(cml
Fig. 1. Thin layer radiochromatogram showing the products of arachidonic acid conversion by sheep vesicular gland microsomes alone (upper panel) and in the presence of cow uterus microsomes corresponding to 0.4 g tissue (lower panel). Vesicular gland microsomes corresponding to 50 mg tissue were incubated with 0.1 mM [l-14C]arachidonic acid (30 000 cpm) for 10 min in 1 ml potassium phosphate buffer pH 7.8 and products were isolated as indicated in Table I. The designations AA, PGE2 and PGFzo, refer to the positions on the plate of arachidonic acid, prostaglandin Ez, and prostaglandin Fz~, respectively. Fig. 2. Thin layer chromatography of the products formed during incubation of uterus microsomes with prostaglandin Hz. (b) Cow uterus microsomes corresponding to 0.8 g tissue were incubated with [1-14C] prostaglandin H2 (1 pg. 44 000 cpm) for 10 min in 1 ml 0.1 M phosphate buffer pH 7.8. Lipids were extracted as indicated in Table I and products were separated by thin-layer chromatography in solvent system II. (a) Authentic sample of [3Hlhydroxyheptadecatrienoic acid (25 000 cpm) applied immediately before development of the plate. (c) [l-14Clprostaglandin H2 (15 000 cpm) applied immediately,before development. Designations PGFzo, and PGE2 refer to authentic prostaglandins Fzol and E2.
ments. On the other hand, uterus microsomes possessed a very high capacity to form prostaglandin Fzo, from prostaglandin Hz (Table V). The reducing activity did not require any cofactors (GSH or NADPH), but was completely abolished in the presence of 1 mM p-hydroxymercuribenzoate. Fig. 3 shows that a positive relationship existed between the concentration of uterus microsomes and formation of prostaglandin F,, from a given amount of prostaglandin Hz. As shown in Fig. 4, the amount of prostaglandin Fza formed was proportional to the concentration of prostaglandin H2 (up to 4 pg/ml) but it leveled off with higher concentrations (more than 7 pg/ml). A large scale incubation was performed using microsomes corresponding to 5 g of cow uterus endometrium and 25 pg prostaglandin Hz. The zone corresponding to prostaglandin FZa was scraped off from the plate and mass spectra were taken of the trimethylsilyl (Me,Si) derivative of the methyl ester of the product (carbon value 24.0 on 1% SE-30 column). The mass spectrum showed ions of high intensity at m/e 584 (M, molecular ion), 569 (M-15; loss of CHJ),
609
TABLE
III
EFFECT OF UTERUS MICROSOMES VESICULAR GLAND MICROSOMES
ON THE CONVERSION
OF ARACHIDONIC
ACID
BY SHEEP
Sheep vesicular gland microsomes corresponding to 50 mg tissue were incubated with [1-14Clarachidonate (0.1 mM, 30 000 cpm) in the absence or presence of varying amounts of uterus microsomes. Details of incubation are shown in Table I. L._____,_~___ __.-._._~ ~._. ..-. ~~.-..--~___ Additions __
Conversion _.________.___
~_.
None Cow uterus microsomes
(96) __ _.__.._...
.. . .
Inhibition
a ._~_._~.
(%) ~_ -
g g g g g
74 66 49 36 21 27
Boiled cow uterus microsomes:
0.2 g 0.4 g
52 45
30 39
Guinea-pig
0.2 g
43
42
0.4 g
33
56
corresponding
to:
0.1 0.2 0.4 0.8 1.0
uterus microsomes:
.~_.______.._~~~
----.
a Conve&on into covered
products
11 34 51 64 64
----not
including
monohydroxyacids;
.___ .__.~ ~~-
percentage
__._
._.____
of total radioactivity
re-
from the plate.
513 (M-71; loss of (CH2)4CH,), 494 (M-90; loss of Me&OH), 443 (M-141; cleavage between C-7 and C-8), 423 (M-(90+71)), 353 (M-(90+141)), 333 (M(2 X 90+71)), 199 (side chain attached to C12). This evidence confirmed the identity of the product formed from pros~gl~din H, as pros~landin Fzcr. The reducing activity of cow uterus microsomes was also checked using prostaglandin Gz as substrate. In incubations containing 2 /_tg [l-14C]prostaglandin Gz (88 000 cpm) and microsomes corresponding to either 0.4 or 0.8 g the substrate was found to produce 20% and 25%, respectively, of prostaglandin Fz,. However, no reduction of pros~gl~din E, occurred in incubations of uterus microsomes with ‘H-labeled I!& for 20 or 30 min.
TABLE EFFECT SOMES
IV OF
UTERUS
MICROSOMES
ON 02
UPTAKE
BY SHEEP
VESICULAR
GLAND
MICRO-
Sheep vesicular gland microsomes corresponding to 50 mg tissue were incubated in 1 ml of 0.1 M potassium phosphate buffer pH 7.8 in the absence or presence of varying amounts of cow uterus microsomes. Incubations were performed in an oxygraph cell equipped with a Clark electrode and maintained at 37’C. After addition of araehidonate (0.1 mM final concentration) total 02 uptake during 3 min was measured. --.~~_._.____~~ Addition
02 uptake (arbitrary units)
Inhibition
None
68
-
56 50 34 30
18 26 50 56
Uterus microsomes
0.2 0.4 0.6 0.8
g g g g
(W)
610
o-7iTx-z-r g (tissue
1’6
1.6
PGHZ
1
Fig. 3. Relationship between concentration tions identical to those shown in the legend somes was varied as indicated.
1 pg
)
of microsomes and formation of prostaglandin Fzo. Condito Fig. 2 except that the concentration of cow uterus micro-
Fig. 4. Effect of increasing concentrations of prostaglandin H2 on formation of prostagiandin Fzo. Cow uterus microsomes corresponding to 0.8 g tissue were incubated with [1-14Clprostaglandin Hz in varying concentrations. Other details are identical to those indicated in the legend to Fig. 2.
TABLE
V
FORMATION CROSOMES
OF PROSTAGLAN~IN
FzOi FROM
~l-i4ClPROSTAGLANDIN
H2 BY VARIOUS
MI-
Microsomes prepared from various tissues were incubated with [l- 14C]prostaglandin H2 (50 000 cpm. spec. act. 44 000 cpm/ng) for 10 min in 0.1 M potassium phosphate buffer pH 7.8 at 37’C in the presence and absence of certain additions. Total volume was 1 ml. GSH and hydroquinone (HQ) were 0.5 mM each, NADPH was 0.1 mM, and p-hydroxymercuribenlooate @HMB) was 1 mM. The reaction was terminated with 7 ml chIoroform/meth~ol 1 : 1, &ids were extracted as described 1151 and products were separated by thin layer chromatography in solvent system II. _.._____-. -.-. -..-...- .Incubation
Additions
(%I
-_-_--____
none
lung micrOSOmeS
Guinea pig liver microsomes
Guinea pig kidney
F2o
_. ._ __._~.____ -_-...-..___._-.___.-.._-^_.-..--.- ..-.
Buffer
Guinea-pig
Prostaglandin
microsomes
Guinea pig uterus microsomes Cow uterus microsomes
(0.2 8) (0.4 (0.8 (0.4 (0.4 (0.1 (0.2 (0.4 (0.2 (0.2 (0.2 (0.4 (0.8 (0.4 (0.4 (0.2 (0.4 (0.2 (0.4 (0.4 (0.4 (0.2
g) g) g) 9) g> g> gf g) 9) gl g) g) gf g) g) 9) 9) 9) g) g> g)
(0.4 gf
ncme NADPH none none none GSH + HQ NADPH mme none none GSH + HQ NADPH none n0ne none GSH + HQ N ADPH none none none nime GSH + HQ NADPH pHMB pHMB
3 4 4 4 4 3 4 4 9 10 11 11 9 9 8 9 10 9 35 46 51 55 56 56 5 3
____.“..__ .._.._ -- ..-
611
Microsomes solubilized with 1% Cutscum still retained high reducing activity, but it was completely lost in microsomes solubilized with 1.5% Tween 20. When 100 000 X g supe~at~t of Cu~cum-solubilized microsomes was applied to a Sephadex G-75 column, the reducing activity appeared in the void volume, together with the bulk of protein. The reducing activity was found to be highly resistant to heating and a microsomal suspension kept in a boiling water bath for 10 min still retained up to 80% activity. When the boiled suspension of microsomes was centrifuged at 100 000 Xg for 60 min, the activity appeared in the sediment and was absent from the supernatant. When uterus microsomes were extracted with either ethyl acetate or 10% aqueous acetone, the reducing activity was present in the lipid-depleted particles and absent from the lipid extract.
Guinea pigs were used to study the relationship between the estrous cycle and prostaglandin formation by uterus microsomes. Some animals received injections of estradiol benzoate as indicated (Table VI). It can be seen that the percentage conversion of [l-‘4C]arachidonic acid was even lower than with cow uterus microsomes. The conversion into both total products and into prostaglandin FZ, fluctuated in the course of the estrous cycle and was highest to-
TABLE VI CONVERSION OF [l-14ClARACHIDON~~
ACID BY GUINEA PIG UTERUS
Microsomes were prepared from uteri of animals on various days of the estrous cycle and portions containing about 1 mg of protein were incubated with 7 yM (2 @g) [l- 14Clarachidordc acid (120 000 cpm) in the absence of any additions. Details of incubation and determination of products are given in Table I. Animal and treatment
Weight of uterus (9)
Conversion a (%>
Prostaglandin Fzol b (%)
day 6 5 between 4 and 10 12 15 16 16 1 day 4 : 3 injections of estradiol benzoate (10 pg each) starting on day 2 day 8 : 5 injections of e&radio1 benzoate starting on day 4 day 12: 5 injections of estradiol benzoate starting on day 8
1.2 1.2 1.0 1.0 1.3 2.0 1.9 1.9
2.1 2.6 2.4 3.4 4.0 6.3 6.8 6.3
0.9 0.7 1.1 1.2 1.5 1.8 2.7 2.4
2.0
5.5
1.8
2.1
4.8
1.7
2.4
3.2
1.4
a Conversion into products not including monobydroxy acids; percentage of total radioactivity recovered from the thin-layer plate. b Percentage of total radioactivity recovered from the plate.
612
wards the end of the cycle. Since the uterus considerably increased in size at the end of the estrous cycle, the total production of prostaglandins from the radioactive precursor can be estimated to be 6-8 times higher at the end than on other days of the cycle. Uterus microsomes obtained from animals injected with estradiol benzoate also possessed higher capacity to convert the labeled arachidonic acid (Table VI). Injection of the vehicle alone (0.1 ml of propylene glycol) has been found to have no effect. Microsomal preparations of uteri from guinea pigs at different stages of the estrous cycle were also assayed with regard to their ability to inhibit prostaglandin formation by sheep vesicular gland microsomes. All preparations were found to possess high inhibiting activity. Production of prostaglandins from 20 lug [l-‘4C]arachidonic acid by vesicular gland microsomes (50 mg tissue) was inhibited by 40-50% in the presence of uterus microsomes (0.2 g tissue). Some differences in inhibiting activity existed, but they were small and could not be accurately enough evaluated by the method used. Thus, the degree of inhibition could not unequivocally be related to the estrous cycle. Discussion While much work has already been done on the release of prostaglandins from the uterus and on the role of prostaglandin F,, as the luteolyzing factor, very few reports have appeared as yet dealing with the uterine prostaglandin synthesizing system in vitro. This could, partially at least, be due to the low capacity of the uterine tissue to produce prostaglandins either from endogenous [3,12] or exogenous [4] precursors. In order to measure the small amounts of prostaglandin Ez and prostaglandin F,, formed in guinea pig homogenates, Poyser [3] used a bioassay on the rat fundus strip, Ham et al. [ 41 determined prostaglandins in rat uterus microsomes by radioimmunoassays, and Naylor and Poyser [12] measured the amounts of prostaglandin F,, by radioimmunoassay or combined gas chromatography-mass-spectrometry. In the present study, it has been possible to measure the conversion into products of radioactive arachidonic acid by microsomes of either cow endometrium or guinea pig uterus. It appeared that the synthetase system in the uterus differed in several respects from the highly efficient systems in the ovine and bovine seminal vesicles. In accordance with the authors cited above [ 3,4], the capacity to form prostaglandins from arachidonic acid was always very low, although it underwent fluctuations during the estrous cycle, as discussed later. Prostaglandin Fzc, comprised up to 40% of the total products of conversion while it is only a minor component of the products formed by sheep vesicular gland microsomes (Fig. 1 and ref. 17). GSH and hydroquinone that are known to promote prostaglandin biosynthesis in sheep [18] and bull [ 191 seminal vesicles and have been routinely included in incubation media of other tissues as well (e.g. refs. 20-22) were found in this study to persistently inhibit the already poor conversion of arachidonic acid into prostaglandins. The same effect was observed with the particle-free supernatant that also promotes prostaglandin formation by sheep vesicular gland microsomes [ 181. Substantial differences have been observed between the prostaglandin-synthesizing systems in rabbit or dog brain [22] and rabbit kidney [21] as compared to the one from bovine
613
seminal vesicles [23], and it is quite conceivable that prostaglandin synthetase from different tissues may possess differing properties which could be of physioIogic~ importance. In accordance with the findings by Poyser 131, the prostaglandin synthesizing activity in the guinea-pig uterus has been found to be highest at the end of the estrous cycle. Injections of estradiol benzoate also resulted in higher percentage conversion of arachidonic acid by guinea-pig microsomes as compared to the controls (see also ref. 12) and these findings seem to confirm the suggestion made by Kuehl et al. [24] that prostaglandin synthesizing enzyme systems are subject to fine hormonal control. Not only more prostaglandin Fzor was formed from the labeled arachidonic acid at the end of the cycle. The same was also true for other products of conversion which on thin-layer plates had the motilities of prostaglandin E, (and thrombox~e B,) and prostaglandin D2. However, the actual formation of those products is difficult to evaluate, since the possibility cannot be excluded that at least a part of them may represent that portion of endoperoxides which had not been converted into prostaglandin F Zoland underwent non-enzymatic rearrangement in the aqueous medium [ 13,251. The total conversion of arachidonic acid into products not including monohydroxyacids actually reflects the production of endoperoxides by uterus microsomes. Even at the height of activity the synthetic capacity of the uterus is of a very low order, The possibility exists that this may be due to the presence in the tissue of an inhibiting factor which suppresses the early steps in prostaglandin biosynthesis and is even able to counteract the extremely efficient prostaglandin synthetase in sheep vesicular glands. The nature of the inhibiting factor has not been elucidated so far. It is present in uterus homogenate and microsomes and, to a lesser extent, in the particle-free supernatant as well (this could possibly account for the inhibiting effect of the supernatant on prostaglandin formation by microsomes), it is resistant to heating and its effect is lowered but not abolished by boiling for several minutes. It seems conceivable that its activity also fluctuates during the estrous cycle thus leading to modulations in the amounts of endoperoxide intermediates formed, although our method of estimation was not sensitive enough to measure the small fluctuations in inhibition with the desired degree of precision. In the present study, uterus tissue was found to possess a very high capacity to convert exogenous endoperoxide, prostaglandin HZ, into prostaglandin Fzo,. This feature seems to be unique for uterus microsomes, since microsomes from guinea-pig kidney, lung and liver gave only a very small yield of prostaglandin F,, from prostaglandin HZ. Enzymes are known to exist which catalyze reduction of prostaglandin E compounds to prostaglandin F compounds [ 26-281. However, no transformation of tritium-labeled prostaglandin E2 incubated with uterus microsomes was detected in the present study. The capability to reduce prostaglandin H2 to prostagl~din F,, was found to be completely abolished by p-hydroxymercuribenzoate, an SH-group agent, but any attempts to isolate and purify the reducing factor have been as yet unsuccessful, and the elucidation of its real nature must await further studies. In conclusion, the results of the present study suggest that a highly specialized mechanism exists which modulates prostaglandin Fza: formation in the
614
uterus. Inhibition of the cycle-oxygenase reaction results in keeping prostaglandm F,, well below the level which could induce luteolysis of the corpus luteum during most of the estrous cycle. At some defined stage in the cycle, when the inhibition is likely to be somewhat less pronounced, more endoperoxide can be formed and, in the presence of an active reducing factor, efficiently converted into prostaglandin F?, which, after being discharged into the blood, can affect its target in the ovary. Acknowledgements This work was supported by grants from the World Health Organisation the Swedish Medical Research Council (Project no. 03X-217).
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