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Sex difference in the inhibitory effect of aspirin on prostacyclin production of rat aortae.

The effect of aspirin on the prostacyclin (PGI2) production of rat aorta was investigated, and the influence of sex hormones on the effect of aspirin was studied by the treatments of hormone administration, ovariectomy and castration. There was no significant sex difference in the arterial production of PGI2 between male and female rats. However, the PGI2 production was decreased with aspirin treatment, and the effect of aspirin was more efficient in male rats. The inhibitory effect of aspirin was reduced in the rats treated with estradiol and the castrated male rats, but it was potentiated in the rats treated with testosterone and the ovariectomized female rats. These results suggest that sex hormones may regulate the effect of aspirin on the PGI2 production in the aorta.

Abstract-The effect of aspirin on the prostacyclin (PG12) production of rat aorta was investigated, and the influence of sex hormones on the effect of aspirin was studied by the treatments of hormone administration, ovariectomy and castration. There was no significant sex difference in the arterial production of PG12 between male and female rats. However, the PG12 production was decreased with aspirin treatment, and the effect of aspirin was more efficient in male rats. The inhibitory effect of aspirin was reduced in the rats treated with estradiol and the castrated male rats, but it was potentiated in the rats treated with testosterone and the ovari ectomized female rats. These results suggest that sex hormones may regulate the effect of aspirin on the PG12 production in the aorta.
Aspirin has been reported to be an effective antithrombotic agent in clinical trials (1-3) and in animal experiments (4). However, the antithrombotic effect of aspirin was found to be more effective in males than in females (2,4).
That effect of aspirin is attributed to its inhibitory effect on PG12 synthesis; aspirin is considered to acetylate cyclooxygenase, an enzyme involved in PGs synthesis (5, 6). PG12 is the main product of the PGs in aorta (7). Chang et al. (8,9) reported that estradiol stimulated the PG12 production in cultured smooth muscle cells of rat aorta and promoted the PG12 production in intact rats. Nakao et al. (10) showed that testosterone suppressed the PG12 production in cultured rat aortic smooth muscle cells.
Animals: Wistar strain rats of both sexes were used for this study. At 6 weeks of age, the female rats were anesthetized with ether and then bilaterally ovariectomized via the back route. The male rats were anesthetized with ether and then bilaterally castrated via the ventral route. All rats were housed in a temperature-controlled room with a fixed lighting schedule (a.m. 7:00-p.m. 7:00) and allowed free access to standard laboratory food (Oriental Yeast Co. Ltd.) and water.
Administration of sex hormones: Testos terone or estradiol was dissolved in corn oil. Three days after the operation, the rats were treated with either 5 mg/kg testosterone or 10 /-tg/kg estradiol at intervals of three days from 6 weeks to 10 weeks. Control rats were given 1.0 ml/kg corn oil at the same interval from 6 weeks to 10 weeks.
Drug: Aspirin was suspended in 0.1% carboxymethylcellulose sodium salt solution for the p.o. administration.
The dose levels were 20, 50 and 100 mg/kg. Preparation of aortic tissues: At an hour after administration of aspirin, the animals were sacrified by bleeding from common carotid arteries, and the thoracic aortae were quickly isolated and kept in Krebs-Ringer Bicarbonate buffer (pH 7.4).
Measurement of PGI2 production: The aortae were incubated in 50 mM Tris HCI buffer (pH 7.4) at 22'C for 2, 5, 10, 15, 20 and 30 min. The production of endogenous PG12 was determined by the modified method of Harada et al. (11,12). The aorta was mechanically stimulated before each incu bation by holding the strip with two forceps and gently stretching the strip ten times within 20 sec. The amounts of PGI2 released in the medium were immediately bioassayed as the inhibitory activity against ADP induced human platelet aggregation (13). The amount of PGI2 increased time dependently for the first 10 min and then decreased, so the aortae were incubated for 10 min. The content of PGI2 was expressed as ng/mg of wet tissue, using synthetic PGI2 as the standard.
Measurement of PGI2 production from exogenous arachidonic acid: The aortae were incubated in Tris HCI buffer containing 1.0 dig/ml arachidonic acid at 37'C for 2 min. The PGI2 production was immediately determined as described under "Measurement of PGI2 production". Measurement of PG 12 production with addition of PGI2 as a substrate: The aortae were incubated in Tris HCI buffer containing 0.5 /cg/ml PGH2 at 37°C for 2 min. The PGI2 production was determined by the above described method of bioassay.
Measurement of PG 12 production after addition of aspirin in vitro: The aortae were preincubated with aspirin (2.0, 4.0, 6.0 and 10.0 /,,g/ml) in Tris HCI buffer (1.0 ml/2.5 mg tissue) at 22'C for 10 min and rinsed with Tris HCI buffer. Then they were incubated in Tris HCI buffer at 22°C for 10 min, and this incubation medium was measured for the PG12 production as described under "Measurement of PG1 2 production". In the study of interaction of aspirin with sex hormones, 1.0 /cg/ml testosterone and 1.0 ng/ml estradiol were used. The aortae were preincubated with aspirin (6 ,ig/ml) and testosterone or estradiol in Tris HCI buffer (1.0 ml/2.5 mg tissue) at 22°C for 10 min.

Results
Effect of aspirin on PGI2 production from endogenous arachidonic acid Figure 1 shows the inhibitory effect of aspirin on the PGI2 production. In the control group, no sex difference was found in the PGI2 production. In aspirin-treated group, the PGI2 production was decreased in a dose dependent manner. Aspirin at a dose 20 mg/ kg did not efficiently decrease the PG 12 production in female rats. Aspirin at a dose 50 mg/kg significantly decreased the PGI2 production and more significantly decreased it in male rats. Furthermore, aspirin at 100 mg/ kg completely inhibited the PGI2 production in both sexes of rats (data not shown). Effect of aspirin on PG I2 production from exogenous arachidonic acid Figure 2 shows that aspirin inhibited the PG12 production with the addition of arachidonic acid as a substrate. In the control group, there was no significant sex difference in the PG12 production, but the PG12 pro duction in females was slightly larger than in males. In the aspirin-treated group, aspirin dose-dependently decreased the PG 12 pro duction. These results agree with those on the PG12 production from endogenous arachi donic acid. The effect of aspirin was more efficient in males than in females. Figure 3 shows that aspirin inhibited the 6-keto-PGFta release from[ 3H]-arachidonic acid used as a substrate. The results cor respond with those from endogenous and exogenous arachidonic acid. Aspirin affected the 6-keto-PGF,a release in a dose dependent manner. This effect of aspirin was more effective in males than in females.  Effect of aspirin on PG I2 production with addition of PGH2 used as a substrate Table 1 shows the effect of aspirin on the PG12 production from exogenous PGH2. When arachidonic acid was used as the substrate, aspirin at a dose 50 mg/kg com pletely inhibited the PGI2 production of rat aorta in both sexes. However, when PGH2 was added as the substrate, aspirin had no influence on the PGI2 production. Effect of aspirin in vitro Figure 4 shows that the PGI2 production is inhibited by aspirin in vitro. In male rats, aspirin inhibited the PGI2 production at 4.0 ig/ml, 61.0±16.7%; at 6.0 /cg/ml, 32.6± 13.7%; and at 10.0 itg/ml, 19.4+10.5%.
There was a sex difference in the effect of aspirin at 4.0, 6.0 and 10.0 gig/ml on the PGI2 production in vitro. Influence of gonadectomy and sex hormones Table 2 shows the influence hormones and gonadectomy on PG 12 production and the effect of aspirin.
i) Change in PGI2 production by gon adectomy: In castrated male rats, aspirin showed a tendency to increase the PGI2 production. The inhibitory effect of aspirin on the PGI2 production was weaker in castrated male rats than in intact male rats. In ovari ectomized female rats, the effect of aspirin was enhanced, and the amount of the produced PGI2 in ovariectomized female rats was the same as that in intact male rats. Fig. 4. Effect of aspirin on prostacyclin production from male and female rat aortae in vitro. Thoracic aortae were preincubated with aspirin and then were incubated in Tris HCI buffer. PGI2 was measured as described under "Materials and Methods". Mean±S.D. (n=5). *': Significant difference from the control value for each sex (P<0.05), *2: Significant difference from female value (P<0.05). Each value is expressed as the relative rate with respect to the control for each sex. •: male, A: female. Table  2. Change in prostacyclin production by gonadectomy and sex hormone treatment ii) Change in PGI2 production by treat ment with estradiol: To study the influence of sex hormones on PG 12 production, estradiol was administered. Doses of 10, 50 and 100 peg/kg were chosen. The influence of estradiol was similar among these three doses.

Discussion
It is known that PGI2 is the main product in rat aorta and an important material in thrombosis. Some investigators have studied the PGI2 production in rat aorta. Pomerantz et al. (15) reported the sex hormonal modi fication of 6-keto-PGF1a release by intact and gonadectomized rats. Their results showed that more 6-keto-PGF,a was release by male rat aorta than by female rat aorta, estradiol decreased 6-keto-PGF1a, and tes tosterone had no influence on 6-keto-PGF1a release by rat aorta. On the other hand,  reported that estradiol increased the PGI2 production in rat aorta, and testosterone decreased the PGI2 production in the cultured smooth muscle cells of rat aorta. These two con clusions contradicted each other. However, sex hormones must have some influence on the PGI2 production in rat aorta. In this study, the effect of aspirin on the PGI2 production of rat aorta was examined in intact, gonadectomized and sex hormone treated rats.
In intact rats, there was no significant sex difference in the PGI2 production in rat aorta when endogenous and exogenous arachidonic acid were used as substates, but the PGI2 production in females was slightly more than in males. Aspirin decreased the PG 12 production dose-dependently, and the effect was more pronounced in males than in females.
Neither the significant sex difference nor the inhibitory effect of aspirin on PGI2 production was found in control rat aorta added with PGH2 as a substrate.
These results show that aspirin does not affect the PG 12 synthesis process from PGH2. The site for the effect of aspirin seems to be in the course of the conversion of arachidonic acid to PGH2.
is not due to a process involving the phar macokinetics of aspirin, but is a process involving the PG12 production.
In sex hormone treated rats, estradiol tended to increase the PG12 production in rat aorta, but testosterone tended to decrease it. In the male rats treated with estradiol, the effect of aspirin was weaker in castrated rats than in intact rats. It seems that in intact rats, endogenous testosterone may influence the action of exogenous estradiol in the process of PG12 production.
Accordingly, it seems that aspirin strongly inhibited the PG12 production in the intact male rats treated with estradiol. Estradiol tended to increase the PG12 production and suppress the effect of aspirin in intact and ovariectomized female rats. Moreover, it was proven by Chang et al. (8) that estradiol stimulated cyclooxygenase in the cultured smooth muscle of rat aorta. Their results agree with the results of this report. It is suggested from the results in this study that the low concentration of estradiol (10 cg/kg) must have affected the process of PG12 production and reduced the effect of aspirin. ment.
In female rats, it is likely that the capacity for PG 12 production is latently elevated by estradiol and the effect of aspirin consequently weakened. However, in male rats, it is likely that the capacity for PG12 production is reduced or not influenced by testosterone, and the effect of aspirin is strongly enhanced. It has not yet been reported whether testosterone suppresses cyclooxygenase.
These results suggest that the sex differ ence in the effect of aspirin is regulated by sex hormones.
This must be one of the main reasons that the effect of aspirin on the PG12 production in rat aorta is stronger in males than in females. Further studies on the enzyme level are to be carried out by the authors.