The effect of estradiol-17β and actinomycin D on the release of PGF and PGE from separated cells of human endometrium

Estradiol-17β selestively stimulated the release of PGF from separated glandular but not stromal cells of human secretory endometrium (p<0.025) but had no effect on PGF release from either type of cells obtained from proliferative endometrium. PGE release was not affected by estradiol-17β. Actinomycin D did not antagonise the effect of estradiol-17β on PGF release from secretory, glandular cells. Basal release of PGF from these cells was stimulated by actinomycin D alone (100 ng/ml) (p<0.025) and PGE release stimulated in the presence of estradiol-17β. Actinomycin D had no effect on PGF or PGE release from proliferative endometrium. These findings suggest that estradiol-17β stimulates PGF release by a mechanism that does not affect PGE release and which is not dependent on the synthesis of new protein. The basal release of PGF and PGE by glandular cells of secretory endometrium in vitro is regulated by protein/proteins which reduce PG release.     

Effects of 17ß-estradiol and progesterone on the levels of prostaglandins F2α and E in human endometrium

17ß-estradiol and progesterone were administered to post-menopausal women to determine their effects on the capacity of human endometrium to synthesize prostaglandins (PGs) F₂α and E. Basal amounts of PGF₂α and PGE synthesized by endometrium exposed to 17ß-estradiol and progesterone were significantly higher than the levels produced by endometrium exposed to 17ß-estradiol alone (p < 0.02 for both PGs). Levels found in the former endometrium were broadly comparable to levels in secretory endometrium and in the latter to amounts found in proliferative endometrium of spontaneous, ovulatory cycles.     

NAD-dependent 15-hydroxyprostaglandin dehydrogenase activity in human endometrium

The specific activity of NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase was measured in human endometrial tissue obtained from ovulatory and anovulatory women. Employing PGE₂ as substrate, the specific activity of this enzyme was found to be highest in endometrial tissue during the secretory phase of the cycle (ovarian cycle days 15–25) and lowest in menstrual (days 1–5) and premenstrual (days 26–28) endometrium. The specific activity of prostaglandin dehydrogenase in endometrium of anovulatory women was low, being similar to that found in proliferative endometrium (days 6–14) of ovulatory women. Prostaglandin dehydrogenase activity was found in the cytosolic fraction prepared from endometrial tissue, and was found principally in the glandular epithelium following separation of endometrial glands and stromal cells.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [¹⁴C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF₂α and TXA₂, as monitored from the formation of its stable degradation product TXB₂ (PGF_2α > TXB₂ > 6-keto-PGF_1α, the stable degradation product of PGI₂=PGD₂=PGE₂=13,14-dihydro-15-keto-PGF_2α). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE₂. PGE₂ and its degradation product 13,14-dihydro-15-keto-PGE₂ accounted for 63% of the PG products formed by submucosal structures (PGE₂ PGD₂ > 13,14-dihydro-15-keto-PGE₂ > PGF_2α=TXB₂=6-keto-PGF_1α > 13,14-dihydro- 15-keto-PGF_2α). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE₂ or PGF_2α. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE₂ degradative capacity but only 23% of total colonic protein. Approximately 15% of [³H] PGE₂ added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE₂ formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Oxytocin-stimulated production of prostaglandin F2α by isolated endometrium of rabbit: Modulation by ovarian steroids

To test the hypothesis that ovarian steroid hormones modulate oxytocin-induced release of prostaglandin F_2α (PGF_2α) from uterine endometrium, 2 ovariectomized rabbits were pretreated with progesterone (5 mg/day for 10 days), 2 with estradiol-17β (25 μg/day for 10 days), 2 with both steroids, and one with sesame oil only. On the last day of treatment, endometrial fragments were excised and incubated in vitro with or without oxytocin (100 μU/ml). Although endometrium from rabbits pretreated with combined steroids released more PGF_2α immediately after excision than did tissue from animals pretreated with either steroid by itself, endometrium from animals pretreated with estrabdiol-17β alone released the most PGF_2α during sustained incubation in vitro. Moreover, only this tissue exhibited significant oxytocin-dependent release of PGF_2α. At the dosages used, progesterone completely antagonized both of these effects of estradiol-17β. The results support the hypothesis that ovarian steroid hormones regulate oxytocindependent release of PGF_2α from endometrial cells. A possible mechanism of action is suggested.     

Analysis of Telomerase Expression and Proliferative Activity in the Different Layers of Cyclic Endometrium

To gain better insights into cell kinetics under physiological conditions, telomerase activity in the functional and basal layers of cyclic endometrium (n= 33) was compared with the immunostaining of glandular and stromal cells within these layers (n= 25). Two immunohistochemical proliferation markers were used to demarcate cells in the G₁phase of the cell cycle. In contrast to previous expectations, telomerase activity and both glandular and stromal proliferative activities were all significantly higher in the functional than in the basal endometrium (P< 0.002). The course of telomerase activity in the endometrial layers during the ovarian cycle was significantly associated with the proliferative scores for the functional and basal endometrial glands and the functional stroma but not the stromal compartment of the basal layer. Our findings indicate that the telomerase activity in cyclic endometrium is associated with the total number of proliferating glandular and stromal cells in the functional layer. Proliferating daughter cells of telomerase-competent stem cells may account for the lower levels of telomerase detected in normal basal endometrium.     

The effect of locally administered PGF2α on the contractility of the nonpregnant human uterus in vivo

The effect of locally administered prostaglandin F_2α on the sensitivity and reactivity of the nonpregnant human uterus during the menstrual cycle was studied. An increase in uterine contractility in response to as little as 1.0 μg PGF_2α could be observed in all patients during both the early and late portions of the menstrual cycle, but at the time of ovulation a marked decrease in sensitivity was noted. Endogenous prostaglandin normally occurs in the secretory endometrium in levels compatible with the amount of exogenous prostaglandin which elicited increased uterine activity. These findings support the hypothesis that PGF_2α plays an important physiological role in the cyclical regulation of uterine motility during the human menstrual cycle.     

The release of PGF2 alpha and PGE2 from separated cells of human endometrium and decidua

The capacity of separated glandular and stromal cells from endometrium and first trimester decidua to release prostaglandins (PGs) was studied over 48 hours in culture. Glandular preparations released more PGs than stromal preparations in all tissues. Stromal release of PGs did not alter throughout the cycle or in early pregnancy but the capacity of glandular preparations to release PGs varied considerably. Proliferative glands released most PGF2 alpha and PGE2 followed by secretory glands and decidua. Histamine (10(-5)) stimulated PG release from endometrial and decidual glands but the response of proliferative glands was greatest. Actinomycin D stimulated release of PGF2 alpha and PGE2 from glandular cells of secretory endometrium and decidua. These results suggest that in vitro release of PGs is suppressed after ovulation and is in part due to inhibition of PG release by a protein or proteins synthesized in the glandular fraction of secretory endometrium or decidua.     

Prostaglandin E2 enhances uterine stromal cell alkaline phosphatase activity

Prostaglandins have been implicated in the process of uterine decidualization , but sites of action are uncertain. Since one of the earliest changes in endometrial stroma following induction of decidualization is an increase in alkaline phosphataseactivity, we have investigated the effects of PGs on stromal cell alkaline phosphatase activity . Immature rats were pretreated with hormones to sensitize their uteri for the decidual cell reaction. Endometrial stromal cells were isolated and cultured for up to 4 days with PGE₂ (0–10 μg/ml) or PGF₂ (0–10 μg/ml) Analysis of variance revealed a highly significant interaction between day of culture and concentration of PGE₂ in medium (P<0.01). Stromal cell alkaline phosphatase activity decreased significantly with increasing culture duration (P<0.01). In the presence of PGE₂, alkaline phosphatase activity was significantly higher (P<0.01) regardless of day of culture. In contrast, PGF_2α had only a small and inconsistent effect. These data indicate that PGs, and in particular PGE₂, can act directly upon stromal cells.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE₁ or PGE₂, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva: PGF_2α was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 μg/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE₁, PGE₂, PGF_2α or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF_2α, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion. Endogenous prostaglandins themselves may not play a role in a secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, an inhibitor of prostaglandins biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion. The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD₄, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE_2α and PGI₂ (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD₄, methacholine and histamine. LTD₄ (3–100 nM), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE₂ and PGF_2α but not PGI₂, was decreased in tissues lacking epithelium. Indomethacin (1 μM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI₂ and is unlikely to be PGF_2α or PGE₂. However, the possibility remains that the basal release of PGE₂ and/or PGF_2α derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE₂ and PGF_2α which may be involved in the maintenance of baseline tone.     

Prostaglandin release from the guinea-pig uterus superfused in vitro. Effect of stage of estrous cycle, progesterone, estradiol, oxytocin and A23187

Prostaglandin (PG) E₂ was the major PG released from the superfused guinea-pig uterus on Day 7, followed by in descending order 6-oxo-PGF_1α, thromboxane (TX) B₂ and PGF_2α. However, the outputs of all four substances were low and were very similar. By Day 15, PGF_2α output from the superfused uterus had increased 21.9-fold, whereas the outputs of PGE₂, 6-oxo-PGF_1α and TXB₂ had increased only 1.8-, 2.9- and 1.2-fold, respectively. A mechanism is apparently “switched on” between Days 7 and 15 which causes a fairly specific increase in the release of PGF_2α from the uterus.Progesterone and/or estradiol had no effect on PG or TX release when superfused over the uterus on Day 7, nor did they have any effect on PG and TX release from the Day 15 uterus when administered separately. When administered together, however, they significantly inhibited PGF_2α, PGE₂ and 6-oxo-PGF_1α, but not TXB₂, release from the Day 15 uterus. Oxytocin had no effect on PG release from the Day 7 or Day 15 uterus, while A23187 stimulated PGF_2α, 6-oxo-PGF_1α and, to a lesser extent, PGE₂ release from the uterus on both Days 7 and 15 Oxytocin is apparently not important for stimulating PGF_2α release from the guinea-pig uterus in relation to luteolysis, whereas increasing intracellular free Ca⁺⁺ levels may be part of the mechanism for “switching on” uterine PG synthesis. Furthermore, changes in intracellular free Ca⁺⁺ levels in the endometrium may be responsible for the pulsatile nature of PGF_2α release from the uterus.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI₂; PGD₂; PGF₂ and PGE₂); BaCl₂ or prostaglandin metabolites (15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α; 6-keto-PGF_1α and 6-keto-PGE₁ in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl₂, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF_2α was shifted to the right of that for PGF_2α itself; the curve for 6-keto-PGF_1α was displaced to the right of the curve for PGI₂ and that for 6-keto-PGE₁ to the left.It was also demonstrated that the uterine motility elicited by 10⁻⁵ M PGF_2α and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI₂; 6-keto-PGF_1α and BaCl₂, but not the contractions following 6-keto-PGE₁, which disappeared much earlier. The contractile tension after PGF_2α; 15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α and PGI₂, increased as time progressed whilst that evoked by 6-keto-PGF_2α or BaCl₂ fluctuated during the same period around more constant levels.The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI₂, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF_1α, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

The effect of estradiol-17 beta and actinomycin D on the release of PGF and PGE from separated cells of human endometrium

Estradiol-17 beta selectively stimulated the release of PGF from separated glandular but not stromal cells of human secretory endometrium (p less than 0.025) but had no effect on PGF release from either type of cells obtained from proliferative endometrium. PGE release was not affected by estradiol-17 beta. Actinomycin D did not antagonise the effect of estradiol-17 beta on PGF release from secretory, glandular cells. Basal release of PGF from these cells was stimulated by actinomycin D alone (100 ng/ml) (p less than 0.025) and PGE release stimulated in the presence of estradiol-17 beta. Actinomycin D had no effect on PGF or PGE release from proliferative endometrium. These findings suggest that estradiol-17 beta stimulates PGF release by a mechanism that does not affect PGE release and which is not dependent on the synthesis of new protein. The basal release of PGF and PGE by glandular cells of secretory endometrium in vitro is regulated by protein/proteins which reduce PG release.     

Prostanoid synthesis in whole blood cells from fish of the Arabian Gulf

The ability to synthesise prostaglandins and thromboxane from ¹⁴C-labelled arachidonic acid was investigated in 11 species of fish from the Arabian Gulf. Cyclooxygenase activity was assessed in washed whole blood cells. Arachidonic acid and its metabolites were extracted and separated on silicic acid columns and thin layer chromatography (silica gel G). Total capacity to convert [¹⁴C]arachidonic acid to prostanoids varied from 1 to 35% among the 11 fish species studied. Gray shark (Chiloscyllium griseum) blood cells had the highest capacity (37±0.4%) to convert arachidonate into prostanoids and two species of catfish (Arius bilineatus and A. thalassinus) exhibited greater than 10% capacity to convert [¹⁴C]arachidonate into prostanoids. The major prostanoid synthesised by the two catfish (A. bilineatus and A thalassinus) was 6-keto PGF_1α, a stable metabolite of prostacyclin, PGI₂. In contrast, A. teunispinis synthesised thromboxane B₂, a stable metabolite of thromboxane A₂. Thromboxane B₂ (TXB₂) was the major product synthesised by all three species of shark studied (Chil. griseum, Carcharhinus plumbeus, Carch. melanopterus), with 6-keto PGF_1α a minor product. Other fish studied showed a varied pattern of prostanoid synthesis. The synthesis of these prostanoids was almost completely blocked by preincubation of the whole blood cells from catfish and shark with indomethacin (0.5 μM) suggesting the involvement of cyclooxygenase-mediated prostanoid synthesis.     

The effect of intrauterine administration of prostacyclin on the contractility of the non-pregnant uterus in vivo

Prostacyclin was infused into the uterus of non-pregnant women either during early menstruation or in the secretory phase of the cycle. A dose of 5 μ/min produced little systemic effect and caused a significant decrease in uterine activity on Days 1 and 2 of the menses whereas 0.5 μg/min did not. However, when dysmenorrhoeic pain and increased uterine activity were produced by infusion of PGF_2α during the secretory phase, PGI₂ neither decreased the activity nor the pain. This indicates that PGI₂ does not cause uterine relaxation by blocking the action of PGF_2α.     

Radioimmunoassays of prostaglandin F2α and prostaglandin F2α-main urinary metabolite with prostaglandin-I-tyrosine methylester amide

Radioimmunoassays for measuring prostaglandin F_2α (PGF_2α) and 5α, 7α-dihydroxy-11-keto tetranorprosta-1,16-dioic acid, PGF_2α-main urinary metabolite (PGF_2α-MUM), with ¹²⁵I-tyrosine methylester amide (TMA) of PGF_2α and PGF_2α-MUM were developed.Antibody to PGF_2α was produced in rabbits immunized with conjugates of PGF_2α coupled to bovine serum albumine. Antibody to PGF_2α-MUM was also produced in rabbits immunized with conjugates of PGF_2α-MUM coupled to bovine serum albumin.PGF_2α-¹²⁵I-TMA had an affinity to antiserum to PGF_2α. PGF_2α-MUM-¹²⁵I-TMA also responded to antiserum to PGF_2α-MUM.     

Effects of PGD2 and PGF2α on longitudinal and circular muscles of guinea-pig isolated proximal colon

The mechanical effects of PGD₂ and PGF_2α on longitudinal and circular muscles of the guinea-pig isolated proximal colon were investigated. PGD₂ and PGF_2α (1 nM – 10 μM) produced a dose-dependent contraction in longitudinal and circular muscles. The contractile action of PGD₂ was more potent than that of PGF_2α in circular muscle and was less potent in longitudinal muscle.Contractions induced by PGD₂ or PGF_2α(1 μM) were unaffected by atropine (1 μM) in both muscles, but tetrodotoxin (1 μM) slightly inhibited these contractions in longitudinal muscle.The results suggest that in longitudinal muscle PGD₂ and PGF_2α have largely a direct action on the muscle cells and a partial neuronal action on the non-cholinergic intrinsic nerves, whereas in circular muscle these PGs have only a direct action on the muscle cells.