Tissue levels of prostaglandins and what do they mean? Studies on the guinea-pig uterus

The ratios of the concentrations of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha in guinea-pig uterine horns, which were removed and placed in ethanol in 1.5 to 2 min, were 0.3:1.0:0.6 on day 7 and 13.8:1.0:0.8 on day 15 of the oestrous cycle. Adding indomethacin (10 micrograms/ml) to the ethanol had no significant effect on the tissue levels observed. These ratios were similar to the ratios of the outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha from the guinea-pig uterus (0.6:1.0:0.9 on day 7 and 7.6:1.0:1.5 on day 15), but were different (particularly on day 7, but only for 6-keto-PGF1 alpha on day 15) to the ratios of the amounts of the three PGs synthesized by homogenates of the guinea-pig uterus (7.2:1.0:2.4 on day 7 and 11.7:1.0:3.3 on day 15). Consequently, the measurement of tissue levels of PGs in the guinea-pig uterus reflects PG synthesis by intact tissue and changes in this synthesis, rather than PG synthesis by homogenates (broken cell preparations). Therefore, it appears meaningful to measure levels of PGs in the guinea-pig uterus since they reflect uterine PG output. Separation of the endometrium from the myometrium, which involved handling and mild trauma, stimulated uterine PG levels, but the ratio of the levels of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha in the endometrium was still similar to that found in the non-separated uterus.     

Effect of melittin on prostaglandin production by guinea-pig uterus.

Melittin, an activator of phospholipase (PL) A-2, increased the outputs of prostaglandin (PG) F-2 alpha and 6-keto-PGF-1 alpha, but not of PGE-2, from Day-7 guinea-pig uterus superfused in vitro. Reducing the extracellular calcium concentration (by omitting calcium chloride from the superfusing fluid) partially inhibited the stimulatory effect of melittin on uterine PG production. TMB-8 (an intracellular calcium antagonist) completely prevented the stimulation of PGF-2 alpha and 6-keto-PGF-1 alpha output by melittin, although the production of both PGs tended to increase after stopping the melittin and TMB-8 treatments. TMB-8 also inhibited the increases in outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 and prevented contraction of the uterus induced by exogenous PLA-2. Trifluoperazine (a calmodulin antagonist) had no inhibitory effect on the increases in outputs of PGF-2 alpha and 6-keto-PGF-1 alpha produced by melittin; it potentiated the stimulatory effect of melittin on 6-keto-PGF-1 alpha output and allowed melittin to increase PGE-2 output. When melittin was applied twice to the superfused uterus with an interval of 1 h between each treatment, partial refractoriness of the responses to melittin was seen: the magnitudes of the increases in PGF-2 alpha and 6-keto-PGF-1 alpha outputs were 40-50% less after the second treatment than after the first treatment. These results show that melittin stimulates the synthesis of PGF-2 alpha and PGI-2 (measured as 6-keto-PGF-1 alpha) in guinea-pig uterus by mechanisms which are calcium dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin output from the early pregnant rat uterus superfused in vitro, and the effect of A23187 and trifluoperazine

The outputs of prostaglandin (PG) E-2 and 6-oxo-PGF-1 alpha from the early pregnant rat uterus superfused in vitro were significantly higher (P less than 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-2 alpha output was significantly higher (P less than 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-2 alpha and/or 6-oxo-PGF-1 alpha 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-1 alpha output and, except on Day 4 (09:00-10:00 h), PGF-2 alpha output at all times studied. A23187 had little effect on PGE-2 output. The greatest stimulatory effect of A23187 on 6-oxo-PGF-1 alpha and PGF-2 alpha 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 microM), 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.     

Effects of TMB-8, an intracellular calcium antagonist, and W-7, a calmodulin antagonist, on prostaglandin output from the guinea-pig uterus

TMB-8, an intracellular Ca2+ antagonist, inhibited the A23187-induced increase in outputs of prostaglandin (PG) F-2 alpha and 6-keto-PGF-1 alpha from the guinea-pig uterus superfused in vitro. The high basal output of PGF-2 alpha 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-2 alpha. W-7, a calmodulin antagonist, had similar actions except that PGF-2 alpha 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-2 alpha synthesis during the last third of the oestrous cycle.     

Investigations into the mechanism by which sodium fluoride stimulates prostaglandin production in guinea-pig uterus

Sodium fluoride (10 mM) caused a slow increase in the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and, to a lesser extent, PGE-2 from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. This stimulatory action of sodium fluoride was not prevented by using calcium-free Krebs' solution. There was also a faster stimulation of 6-keto-PGF-1 alpha output from the Day-7 guinea-pig uterus produced by sodium fluoride, and this quicker response was abolished by using calcium-free Krebs' solution. TMB-8 (an intracellular calcium antagonist) inhibited the stimulatory action of sodium fluoride on the outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus. W-7 and trifluoperazine (calmodulin antagonists) and neomycin (an inhibitor of phospholipase C) had no inhibitory effect on the increases in outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus produced by sodium fluoride. These results indicate that sodium fluoride slowly stimulates uterine PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha synthesis in the guinea-pig uterus by mobilizing intracellular calcium by a mechanism which apparently does not involve the activation of phospholipase C or the participation of calmodulin (or a related compound). The initial, faster stimulation of 6-keto-PGF-1 alpha synthesis in the Day-7 guinea-pig uterus by sodium fluoride is dependent upon extracellular calcium.     

The identification of trihydroxyeicosatrienoic acids as products from the incubation of arachidonic acid with washed blood platelets

Arachidonic acid is converted by washed platelets from man, horse and dog into a mixture of 8, 9, 12-trihydroxyeicosa-5, 10, 14-trienoic acid and 8, 11, 12-trihydroxyeicosa-5, 9, 14-trienoic acid (termed 8, 9, 12-THETA and 8, 11, 12-THETA respectively and THETA collectively). Gas chromatographic — mass spectrometric evidence of structure is discussed.     

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Investigations into the mechanisms controlling prostaglandin production by the guinea-pig placenta: roles of calcium and gonadotrophin-releasing hormone

The outputs of PGF(2 alpha), PGE(2) and 6-keto-PGF(1 alpha) were higher from the day 29 guinea-pig placenta than from the sub-placenta in culture, with PGF(2 alpha)being the major prostaglandin produced by the placenta. Lack of extracellular calcium reduced the production of all three prostaglandins by the sub-placenta and 6-keto-PGF(1 alpha) production by the placenta, but had no effect on the production of PGF(2 alpha) and PGE(2) by the placenta. EGTA (a calcium chelator) and a low concentration (30 microM) of TMB-8 (an intracellular calcium antagonist) generally inhibited prostaglandin output from the placenta and sub-placenta at various time points during culture, although EGTA had no effect on PGE(2) output from the placenta. Trifluoperazine and W-7 (calmodulin inhibitors) had no inhibitory effect on the outputs of PGF(2 alpha) and PGE(2) from the placenta, nor on the outputs of any prostaglandin from the sub-placenta. However, these two compounds inhibited the output of 6-keto-PGF(1 alpha) from the placenta. Nifedipine and verapamil (calcium channel blocking drugs) generally reduced the outputs of prostaglandins from the placenta and sub-placenta, except verapamil had no inhibitory effect on PGF(2 alpha) output from the sub-placenta. Gonadotrophin-releasing hormone (GnRH) did not stimulate the output of prostaglandins from the placenta, and tended to have a weak inhibitory action on this tissue. On the sub-placenta, GnRH had an initial inhibitory action on the outputs of PGF(2alpha) and 6-keto-PGF(1 alpha), which was then followed by a stimulation of the outputs of PGF(2 alpha) and, to a lesser extent, of PGE(2).