Onapristone (a progesterone antagonist) or ICI 182780 (an oestrogen antagonist) administered to guinea-pigs on days 11–14 of the cycle significantly reduced uterine PGF2α output on day 15. Concentrations of progesterone in plasma of onapristone-treated and ICI 182780-treated guinea-pigs were still high on day 15 indicating that luteal regression had been prevented. These findings indicate that progesterone and oestradiol are necessary for increased PGF2α production by the uterus towards the end of the cycle, and support the hypothesis that oestradiol acting on a progesterone-primed uterus is the physiological stimulus for increased uterine PGF2α synthesis and release in guinea-pigs. The capacity of the endometrium to synthesize PGF2α on day 15 was reduced by treatment with ICI 182780 and, unexpectedly, by treatment with onapristone, indicating that onapristone may also be antagonizing the release or action of oestradiol in some way. Tamoxifen was an agonist in guinea-pigs since it induced vaginal opening. It had no inhibitory effect on uterine PGF2α output and did not delay luteal regression when administered between days 11 and 14 of the cycle. However, it redirected PG synthesis in homogenates of endometrium and myometrium from PGI2 (as indicated by 6-keto-PGF1α) to PGF2α. The output of 6-keto-PGF1α from the uterus of day 15 guinea-pigs was reduced following tamoxifen treatment, but the high output of PGF2α from the uterus was not affected.
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N. L. Poyser
Summary. Arachidonic acid increased the outputs of prostaglandin (PG) F-2α, PGE-2 and 6-keto-PGF-1α from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. Similar increases in PG output were observed when the arachidonic acid treatment was repeated after an interval of 1, 3 or 5 h. Phospholipase (PL) A-2 increased the outputs of PGF-2α, PGE-2 and 6-keto-PGF-1α from the Day-7 guinea-pig uterus, but repeating the PLA-2 treatment 1 h later failed to stimulate PG output. The increase in outputs of PGF-2α and PGE-2 caused by PLA-2 were partly restored after 3 h and were fully restored after 5 h, whereas the increase in 6-keto-PGF-1α output produced by PLA-2 was only partly restored after 3 and 5 h. PLA-2 had little or no effect on PGF-2α and PGE-2 outputs from the Day-15 guinea-pig uterus initially, and when repeated after 1, 3 and 5 h. This was probably due to the output of these two PGs, particularly of PGF-2α, being stimulated in vivo before removal of the uterus. PLA-2 increased 6-keto-PGF-1α output from the Day-15 uterus initially, but failed to cause a response when administered again 1 h later. After 3 and 5 h, the increase in 6-keto-PGF-1α output from the Day-15 uterus caused by PLA-2 was partly restored. A23187 and PLC increased the outputs of PGF-2α, PGE-2 and 6-keto-PGF-1α from the Day-7 and Day-15 guinea-pig uterus. These responses to A23187 and PLC were reduced (but not abolished) when the two compounds were administered again 1 h later. After 3 and 5 h, the increases in output of PGF-2α and PGE-2 produced by A23187 and PLC had returned to the initial values. The increases in output of 6-keto-PGF-1α from the Day-7 and Day-15 guinea-pig uterus produced by A23187 and PLC were partly restored after 3 and 5 h, except for the response to PLC on Day 7 which was fully restored after 5 h.
The results show that there is no failure with time in the mechanism which converts arachidonic acid into PGF-2α in the guinea-pig uterus. The refractoriness of uterine PGF-2α production following repeated stimulation apparently occurs at the level of arachidonic acid release, particularly as regards the action of PLA-2. There appear to be one or several pools of bound arachidonic acid which are readily releasable but which take 3–5 h to refill completely. This phenomenon may explain, in part, the pulsatile nature of uterine PGF-2α release.
Keywords: guinea-pig; uterus; phospholipase A-2; phospholipase C; A23187; arachidonic acid; prostaglandins
N. L. Poyser
Onapristone (a progesterone receptor antagonist) administered to guinea-pigs on days 11–14 of pregnancy had no effect on uterine PGF2α output and endometrial PGF2α synthesizing capacity when measured on day 15. Peripheral plasma progesterone concentrations were still high on day 15, although the weight of the conceptuses was decreased by 50%. These findings indicate that the lack of increase in PGF2α production by the uterus during early pregnancy is not due to an inhibitory action of progesterone on uterine PGF2α synthesis and release. The output of PGF2α from the guinea-pig uterus remained low during early pregnancy, showing that the uterus is not the source of increased PGF2α secretion, as indicated previously by an increase in PGF2α metabolite concentrations in the urine, after day 24 of pregnancy. Of the conceptual tissues examined, the fetal placenta had the highest PGF2α synthesizing capacity, and it increased 2.3-fold between days 29 and 36 of pregnancy. The fetal placenta may therefore be the source of increased PGF2α production during pregnancy. Onapristone administered to guinea-pigs on days 27 and 28 or on days 34 and 35 of pregnancy resulted in the guinea-pigs being in the early, middle or late stages of abortion when examined on days 29 or 36, respectively. Increased PG production, particularly of PGF2α, by the uterus occurred in those guinea-pigs that were in the middle or late stages of abortion; uterine PG production in guinea-pigs that were in the early stages of abortion remained low. Peripheral concentrations of plasma progesterone remained high in guinea-pigs in the early and middle stages of abortion, but fell by 70% in those guinea-pigs in the late stages of abortion. These results indicate that increased uterine PG production following onapristone treatment in mid-pregnancy occurs as a result of the abortion process and is not responsible for the initiation of pregnancy termination.
N. L. Poyser
Summary. The outputs of prostaglandin (PG) F-2α and PGE-2, but not of 6-oxo-PGF-1α, from the guinea-pig uterus were significantly lower on Days 7 and 15 of pregnancy than on the corresponding days of the cycle. Uterine PGF-2α output increased 28-fold between Days 7 and 15 of the cycle but only 4- to 5-fold between these same days of pregnancy. Uterine PGE-2 and 6-oxo-PGF-1α outputs increased 2- to 3-fold between Days 7 and 15 of the cycle and of pregnancy. Endometrial PGF-2α synthesizing capacity was 60–70% lower on Days 7 and 15 of pregnancy than on the corresponding days of the cycle, although it increased 2-fold and 2·5-fold between these days of pregnancy and of the cycle, respectively. Endometrial PGE-2 and 6-oxo-PGF-1α synthesizing capacities showed no significant variation amongst Days 7 and 15 of the cycle and of pregnancy, except that endometrial PGE-2 synthesizing capacity was lower on Day 7 of the cycle.
Oestradiol treatment (10 μg s.c. daily from Days 10 to 14 of pregnancy) did not affect plasma progesterone concentrations, uterine 6-oxo-PGF-1α output, and endometrial PGF-2α, PGE-2 and 6-oxo-PGF-1α synthesizing capacities in 9/12 guinea-pigs when examined on Day 15. Uterine PGF-2α and PGE-2 outputs increased 3- and 1·5-fold, respectively, in these guinea-pigs, but were still much lower than the outputs from the Day-15 non-pregnant uterus. The pregnancies appeared unaffected in these oestradiol-treated guinea-pigs. In the other 3 oestradiol-treated animals, uterine PGF-2α output was 20- to 30-fold higher than in untreated, pregnant guinea-pigs on Day 15, and 2- to 3-fold higher than in Day-15 non-pregnant guinea-pigs. Uterine PGE-2 and 6-oxo-PGF-1α outputs also tended to be higher in these treated guinea-pigs. In these 3 guinea-pigs, endometrial PGF-2α, PGE-2 and 6-oxe-PGF-1α synthesizing capacities were 4·0-, 3·4- and 2·5-fold higher, respectively, than in untreated, pregnant guinea-pigs on Day 15, and tended to be higher than in Day-15 non-pregnant guinea-pigs. Plasma progesterone concentrations were much lower in these 3 animals than in the other 9 treated with oestradiol, and also much lower than in untreated, pregnant guinea-pigs on Day 15. However, only 1 of these 3 guinea-pigs was in the process of aborting, and this had the lowest progesterone concentration (0·5 ng/ml).
In the pregnant guinea-pig a large increase in output of PGF-2α from the uterus after Day 11 does not occur, uterine PGE-2 does not appear to be involved in luteal maintenance and general uterine PG production is not increased at the time of implantation.
N. L. Poyser
Summary. TMB-8, an intracellular Ca2+ antagonist, inhibited the A23187-induced increase in outputs of prostaglandin (PG) F-2α and 6-keto-PGF-1α from the guinea-pig uterus superfused in vitro. The high basal output of PGF-2α from the Day-15 guinea-pig uterus was not inhibited by TMB-8, indicating that a maintained high intracellular free Ca2+ concentration is not necessary for maintaining this high output of PGF-2α. W-7, a calmodulin antagonist, had similar actions except that PGF-2α output from the Day-15 uterus was reduced 20–30 min after the W-7 treatment had stopped. Overall, these findings suggest that, in the guinea-pig, oestradiol acting on a progesterone-primed uterus causes a prolonged stimulation of endometrial phospholipase A-2 in the absence of a maintained high Ca2+ concentration, thus providing a continuous release of arachidonic acid for increased endometrial PGF-2α synthesis during the last third of the oestrous cycle.
N. L. Poyser
Summary. The outputs of prostaglandin (PG) E-2 and 6-oxo-PGF-1α from the early pregnant rat uterus superfused in vitro were significantly higher (P < 0·05) on Day 4 (09:00–10:00 h) and Day 5 (14:00–15:00 h) than on Day 2 (09:00–10:00 h) and Day 5 (14:00–15:00 h). PGF-2α output was significantly higher (P < 0·05) only on Day 5 (09:00–10:00 h). PGE-2 was the major PG released at all times, although the amounts of PGF-2α and/or 6-oxo-PGF-1α released were often only slightly less. These findings are consistent with uterine PGs having a role in implantation in the rat.
A23187 stimulated 6-oxo-PGF-1α output and, except on Day 4 (09:00–10:00 h), PGF-2α output at all times studied. A23187 had little effect on PGE-2 output. The greatest stimulatory effect of A23187 on 6-oxo-PGF-1α and PGF-2α outputs occurred on Day 5 (09:00–10:00 h), which is the day of highest uterine PGH-2 synthetase activity. These increases in response to A23187 were prevented by trifluoperazine (100 μm), a calmodulin antagonist. Trifluoperazine had no inhibitory effect on the high basal output of PGs on Day 5 (09:00–10:00 h), but caused a small increase in uterine PG output.
N. L. Poyser
Summary. Trifluoperazine, a calmodulin antagonist, inhibited the A23187-induced increase in outputs of prostaglandin (PG) F-2α and 6-oxo-PGF-1α from the Day 7 and Day 15 guinea-pig uterus superfused in vitro. The basal outputs of, and the arachidonic acid-induced increase in outputs of PGF-2α, PGE-2 and 6-oxo-PGF-1α from the guinea-pig uterus were not inhibited by trifluoperazine. In contrast, indomethacin inhibited A23187-stimulated, arachidonic acid-stimulated and the basal outputs of PGs from the guinea-pig uterus, indicating that trifluoperazine was not inhibiting cyclo-oxygenase. Since the action of A23187 is dependent upon extracellular Ca2+, the present findings provide evidence that calmodulin is involved in Ca2+-induced increases in uterine PG output from the guinea-pig uterus.
Trifluoperazine, but not indomethacin, inhibited A23187-induced contraction of the guinea-pig uterus, which is consistent with calmodulin being involved in smooth muscle contraction. Arachidonic acid treatment did not contract the guinea-pig uterus. These findings indicate that PGs are not involved in the contraction induced by A23187. Other findings of interest were (i) trifluoperazine caused a small, sometimes significant (P < 0·05), increase in uterine PG output, (ii) exogenous arachidonic acid failed to increase PGF-2α output from the Day 15 uterus in contrast to the stimulant action of A23187, and (iii) exogenous arachidonic acid caused a fairly large increase in uterine PGE-2 output in contrast to the small effect with A23187.
N. L. Poyser
Summary. Phospholipases (PL) A-2 and C stimulated the outputs of prostaglandin (PG) F-2α, PGE-2 and 6-keto-PGF-1α from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. PLC had a more pronounced effect than PLA-2, particularly on the output of PGE-2. The ratios of the outputs of PGF-2α and PGE-2 were similar after stimulation by A23187 and PLA-2, but this ratio was lower after stimulation by PLC. It appears that the stimulation of endometrial PGF-2α synthesis by Ca2+ is via activation of PLA-2 rather than via activation of PLC, although the PLC used was of bacterial origin (which uses phosphatidylcholine as substrate) rather than of mammalian origin (which uses phosphatidylinositol as substrate).
Forskolin (which increased endometrial and myometrial cyclic AMP levels) and phorbol 12-myristate-13-acetate had no effect on uterine PG output, indicating that cyclic AMP and protein kinase C are not involved in the stimulation of endometrial PGF-2α synthesis in the guinea-pig. Uterine PG output was not stimulated by 54 mm-KC1, which shows that the pulsatile nature of endometrial PGF-2α synthesis and release is not due to an intermittent, synchronous depolarization of the endometrial cells.
N. L. Poyser
Summary. Intrauterine, but not systemic, administration of actinomycin D on Day 10 increased oestrous cycle length in guinea-pigs. Peripheral plasma progesterone levels remained elevated during these lengthened cycles presumably because luteal life-span had been extended. Prostaglandin (PG) F-2α production in vitro, on Day 15, by the uterus of guinea-pigs which had received intrauterine actinomycin D was much lower than control values. This decrease in PG production was not due to lack of precursor, increased metabolism, re-direction of synthesis towards PGE-2, or a direct inhibition by actinomycin D of the conversion of arachidonic acid to PGs. The effects of actinomycin D treatment were not reversed by oestradiol.
It is proposed that actinomycin D prevents the increase in uterine PG synthetase levels that normally takes place after Day 11, thereby reducing uterine PGF-2α synthesis and output in vivo, and resulting in luteal maintenance and longer oestrous cycles.
E. K. Naderali and N. L. Poyser
Using guinea-pig uterine tissues, indomethacin (a non-selective inhibitor of prostaglandin H synthase) inhibited prostaglandin (PG) synthesis by homogenates of the endometrium, by cultured endometrium and myometrium, and by cultured epithelial glandular cells and stromal cells derived from the endometrium. NS-398, a selective inhibitor of prostaglandin H synthase-2 (PGHS-2), also inhibited PG synthesis by endometrial homogenates, by cultured endometrium and myometrium, and by cultured epithelial glandular cells and stromal cells. Indomethacin and NS-398 inhibited PG production to similar extents, except for 6-keto-PGF1α production by the myometrium where indomethacin was more effective. In particular, indomethacin and NS-398 produced over 90% inhibition of PGF2α output from the epithelial glandular cells, the main source of PGF2α in the endometrium. These functional studies indicate that prostaglandin H synthase-2 is the predominant PG-forming enzyme in the guinea-pig uterus.