Prostaglandin production by the pregnant and non-pregnant rabbit uterus

We have studied the prostaglandin synthesis of the pregnant and non-pregnant rabbit uterus in a microsomal membrane preparation, and in an perfused uterus preparation which retains agonist stimulated prostaglandin production. In both the microsomal and isolated perfused system, prostacyclin was the major arachidonic acid metabolite produced; PGE₂ was also produced in substantial quantities while TxB₂ and PGF_2α were not detectable. Moreover, oxytocin was a specific stimulus of PGE₂ release. the steroid hormone milieu influenced the level of agonist stimulated prostaglandin release; in general, ovariectomized, estrogen treated animals were more responsive to agonist stimulation than those treated with estrogen followed by progesterone. The microsomal studies indicated that the pregnant animal had a greatly enhanced capacity to metabolize arachidonic acid when compared with the non-pregnant animal. However, this was not reflected in the ability of agonists to stimulate prostaglandin release in the perfused preparation.     

Prostaglandin production by the pregnant and non-pregnant rabbit uterus

We have studied the prostaglandin synthesis of the pregnant and non-pregnant rabbit uterus in a microsomal membrane preparation, and in an ex vivo perfused uterus preparation which retains agonist stimulated prostaglandin production. In both the microsomal and isolated perfused system, prostacyclin was the major arachidonic acid metabolite produced; PGE2 was also produced in substantial quantities while TxB2 and PGF2 alpha were not detectable. Moreover, oxytocin was a specific stimulus of PGE2 release. The steroid hormone milieu influenced the level of agonist stimulated prostaglandin release; in general, ovariectomized, estrogen treated animals were more responsive to agonist stimulation than those treated with estrogen followed by progesterone. The microsomal studies indicated that the pregnant animal had a greatly enhanced capacity to metabolize arachidonic acid when compared with the non-pregnant animal. However, this was not reflected in the ability of agonists to stimulate prostaglandin release in the ex vivo perfused preparation.     

Arachidonic acid metabolites in pregnant rat uterus

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.     

Survival and recovery of cryopreserved rat platelets

Freezing studies were conducted using rat platelets to determine if electromagnetic thawing could be used to improve the survival time and recovery of platelets over that obtainable with conventional techniques. A total of 130 experiments were conducted. Seventy-four control experiments not involving freezing and 56 experimental freezings of rat platelets were performed. In most freezing experiments, ⁵¹Cr-labeled platelets were maintained in a mixture of RCD-20% plasma-6% Me₂SO. In this medium, the mean survival and recovery of unfrozen control platelets were 3.50 days and 67%, respectively. The results for frozen-thawed platelets were not encouraging. Using the same incubation mixture, the mean survival time was 1.18 days and the mean recovery was 7%, yielding a platelet viability index of 3.5%. Changing the thawing method (electromagnetic or waterbath), Me₂SO concentration (6 or 10%), buffer medium (RCD-20% plasma or plasma), or freezing or thawing rate did not improve the results. When these same methods were applied in humans, platelets could be successfully frozen and thawed. It would therefore appear that the rat model is an inappropriate one in which to develop improved techniques for freezing and thawing human platelets.     

Identification of a uterine elastase in the pregnant rat uterus

During development of the pregnant rat uterus there is a several fold increase in elastin content. Using Verhoeff's elastic fiber stain, we have shown that a significant proportion of these elastin fibers are in the extracellular matrix of the myometrium. They do not appear as an organized structure but rather in a variety of partially extended, random configurations. An elastase was identified in both the pregnant and the postpartum uterus. Partial characterization of the enzyme indicated that it is a serine protease with a molecular weight around 24,500 and a pH optimum of 8.5. In addition to the enzyme, relatively high levels on an elastase inhibitor were found in the uterine extracts. The inhibitor did not inhibit trypsin, indicating that it was not alpha-1-antiprotease. The data suggest that the elastase and inhibitor are uterine tissue derived and perhaps important in the normal remodeling process of uterine connective tissue.     

Prostacyclin formation by myometrial & decidual fractions of the pregnant rat uterus.

Chopped samples of myometrium, decidua and extrinsic blood vessels from the pregnant rat uterus when incubated at room temperature generated a prostacyclin-like substance. Activity in the incubation mixtures was compared against authentic prostacyclin in two assay systems: relaxation of strips of bovine coronary artery and inhibition of ADP-induced aggregation of rabbit platelet-rich plasma. Results estimated from inhibition of platelet aggregation showed that activity generated by all samples was low on day 12 of pregnancy (less than 0.25 ng/mg). However at the time of delivery (day 22) myometrial synthesis had increased 18.5 fold to over 3 ng/mg of prostacyclin whereas decidual production had only increased 5 times. As there was no increase in synthesis by the extrinsic uterine blood vessels over this period it is proposed that the myometrial muscle cells are the probable source of the prostacyclin-like material.     

Prostacyclin formation by myometrial & decidual fractions of the pregnant rat uterus

Chopped samples of myometrium, decidua and extrinsic blood vessels from the pregnant rat uterus when incubated at room temperature generated a prostacyclin-like substance. Activity in the incubation mixtures was compared against authentic prostacyclin in two assay systems: relaxation of strips of bovine coronary artery and inhibition of ADP-induced aggregation of rabbit platelet-rich plasma. Results estimated from inhibition of platelet aggregation showed that activity generated by all samples was low on day 12 of pregnancy (less than 0.25 ng/mg). However at the time of delivery (day 22) myometrial synthesis had increased 18.5 fold to over 3 ng/mg of prostacyclin whereas decidual production had only increased 5 times. As there was no increase in synthesis by the extrinsic uterine blood vessels over this period it is proposed that the myometrial muscle cells are the probable source of the prostacyclin-like material.     

Characterization and expression of the mouse pregnant specific uterus protein gene and its rat homologue in the intestine and uterus

The pregnant specific uterus protein gene (Psup) is a novel mouse gene expressed in pregnant uterus. This paper describes the identification and expression of the rat homologue of Psup. The gene is highly expressed in the duodenum. Expression decreases in a proximal-distal gradient in the small intestine and was not detected in the cecum and colon. The pattern of expression in the mouse was similar. Expression of Psup in the mouse was localized to the epithelial cells in the intestine and pregnant uterus by in situ hybridization. The data show tissue-specific expression of Psup.     

Cytochemical localization of hydrogen peroxide production in the rat uterus

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.     

Thromboxane and prostacyclin production in the perfused rabbit lung

We investigated whether prostacyclin formation by the isolated rabbit lung can serve as a measure of pulmonary distress. The basal TXA2 and PGI2 formation was very low, and depended on the preperfusion history of the lung (low or high flow, use of dextran or artificial perfusate). The basal prostanoid production remained unchanged over a time period of 2 h. Neither was it influenced by the serotonin uptake inhibitor chlorimipramine and by small changes in temperature (33 degrees C vs 39 degrees C). The PGI2 formation was almost independent of hemodynamic alterations such as embolism or vasoconstriction. An enhanced production was only seen after a dramatic increase in flow (from 1.7-5 ml/sec), and a transient 3-fold increase was observed after administration of 1 mM H2O2. A substantial (up to 40-fold) but transient increase in TXA2 production was measured after 1 mM of H2O2, and the TXA2 production was positively correlated to the increase in pulmonary arterial pressure. However, thromboxane production was also dramatically augmented by hemodynamic alterations such as embolism, increased flow and--to a lesser extent--vasoconstriction. We conclude that the determination of the prostanoid production (and particularly the TXA2 formation) by the rabbit lung cannot be used as a direct measure of endothelial distress. To this end it is excessively biased by hemodynamic alterations such as recruitment and shear stress.     

Further studies on prostaglandin and thromboxane production by the rat uterus during the oestrous cycle

Prostaglandin (PG) and thromboxane (TX) synthesis by uterine homogenates was measured at 4-h intervals during the 4-day oestrous cycle of rats. Production was in the order of 6-oxo-PGF-1 alpha (which reflects PGI-2 synthesis) greater than PGF-2 alpha greater than TXB-2 (which reflects TXA-2 synthesis) greater than or equal to PGE-2. Peak production occurred at 02:00 h on the day of oestrus, after which production gradually decreased, with some fluctuation on the day of metoestrus, to reach a minimum between 22:00 and 06:00 h on the days of dioestrus and oestrus, respectively. Separation of the uterine tissues showed that, on a unit weight basis, the endometrium had a much higher PG and TX synthesizing ability than did the myometrium, although this was compensated for on a total weight basis by the much greater mass of myometrium. Endometrial PG and TX production was in the order of PGF-2 alpha greater than TXB-2 greater than or equal to 6-oxo-PGD-1 alpha identical to PGE-2, with PGF-2 alpha and TXB-2 productions showing the greatest increases between 10:00 and 02:00 h on the days of pro-oestrus and oestrus, respectively. Myometrial PG and TX production was in the order of 6-oxo-PGF-1 alpha greater than PGF-2 alpha greater than PGE-2 identical to TXB-2, with 6-oxo-PGF-1 alpha and PGF-2 alpha productions showing small increases between 10:00 and 02:00 h on the days of pro-oestrus and oestrus, respectively. Myometrial PGE-2 production decreased between these two times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.     

Prostaglandin production by the early pregnant guinea-pig uterus in relation to implantation and luteal maintenance, and the effect of oestradiol

The outputs of prostaglandin (PG) F-2 alpha and PGE-2, but not of 6-oxo-PGF-1 alpha, 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-2 alpha 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-1 alpha outputs increased 2- to 3-fold between Days 7 and 15 of the cycle and of pregnancy. Endometrial PGF-2 alpha 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-1 alpha 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 micrograms s.c. daily from Days 10 to 14 of pregnancy) did not affect plasma progesterone concentrations, uterine 6-oxo-PGF-1 alpha output, and endometrial PGF-2 alpha, PGE-2 and 6-oxo-PGF-1 alpha synthesizing capacities in 9/12 guinea-pigs when examined on Day 15. Uterine PGF-2 alpha 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-2 alpha 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-1 alpha outputs also tended to be higher in these treated guinea-pigs. In these 3 guinea-pigs, endometrial PGF-2 alpha, PGE-2 and 6-oxo-PGF-1 alpha 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thromboxane production by perturbed bovine aortic endothelial cells in culture

Bovine aortic endothelial cells in culture were incubated with endotoxin. The amount of thromboxane A2 synthesized was then determined by a specific radioimmunoassay for thromboxane B2. After a lag of several hours the cells changed their shape and parallel to the change in cell shape release of thromboxane B2 occurred. At 24 h the amount of thromboxane B2 generated in response to endotoxin was 200-fold above baseline. Thromboxane B2 generation could be blocked by aspirin and the specific thromboxane synthetase inhibitor UK 37248. The endotoxin effect was dependent on protein and RNA synthesis as evidenced by the inhibitory action of cycloheximide (1.5 microM) and actinomycin D (2 micron).     

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).     

Hypothyroid state reduces calcium channel function in 18-day pregnant rat uterus

Hypothyroidism significantly reduced the mean amplitude and increased the mean frequency of spontaneous rhythmic contractions in 18 day pregnant rat uterus. Nifedipine (10(-12)-10(-9) M) and diltiazem (10(-10)-10(-6) M) caused concentration related inhibition of the myogenic responses of the uterine strips obtained from both pregnant and hypothyroid state. However, nifedipine was less potent (IC50:2.11 x 10(-11) M) in pregnant hypothyroid state as compared to pregnant control (IC50: 3.1 x 10(-12) M). Similarly, diltiazem was less potent (IC50: 3.72 x 10(-9) M) in inhibiting the uterine spontaneous contractions in hypothyroid than in pregnant rat uterus (IC50:5.37 x 10(-10) M). A similar decrease in the sensitivity to nifedipine and diltiazem for reversal of K+ (100 mM)-induced tonic contraction and K(+)-stimulated 45Ca2+ influx was observed with these calcium channel antagonists in uterus obtained from hypothyroid pregnant rats compared to the controls. Nifedipine-sensitive influx of 45Ca(2+)-stimulated either by K+ (100 mM) or by Bay K8644 (1,4-dihydro-2,6-methyl-5-nitro-4-[2'-(trifluromethyl)phenyl]-3-pyridine carboxylic acid methyl ester) (10(-9) M) was significantly less in uterine strips from hypothyroid rats compared to controls. The results suggest that the inhibition of uterine rhythmic contractions may be attributable to a reduction in rat myometrial Ca2+ channel function in the hypothyroid state.