Contraction of guinea pig uterus by synthetic leukotrienes

Synthetic leukotrienes (LT) C₄ and D₄ elicited concentration-dependent contractions of the guinea pig uterus between 10^?8-10^?6M, whereas LTE₄ appeared 1000-fold weaker. The potencies of LTC₄ and LTD₄ were similar to that of acetylcholine and PGF_2α but weaker than that of PGE₂. The maximal contractions elicited by LTC₄ and LTD₄ were 66.0 ± 2.1% and 63.8 ± 4.6% that elicited by acetylcholine. FPL 55712 (10^?5M) antagonized the uterine contractile activity of LTD₄, while meclofenamic acid at 10^?5M but not at 10^?6M also antagonized the LTD₄-induced contration. Radioimmunoassay of the uterine tissue bathing fluid following LTD₄ indicated the variable presence of low concentrations of PGE₂, PGF_2α and TXB₂. These results demonstrate the LTC₄ and LTD₄ possess significant uterine contractile activity, which may only partially be mediated indirectly via prostaglandin products.     

Possible function of carbohydrate on glycoproteins secreted by the pig uterus during pregnancy

Uteroferrin is a purple iron-containing acid phosphatase secreted by the porcine uterus under the influence of the hormone, progesterone. It is synthesized by the glandular epithelial cells of the uterine endometrium and during pregnancy is taken up by specialized structures (areolae) opposite each uterine gland. Uteroferrin is then released into the fetal circulation and cleared by the liver or fetal kidney. A major role in iron transport to the fetus has been proposed. Uteroferrin, as purified from uterine secretions of pigs, possesses mainly high mannose (predominately Man₅ and Man₆ chains. These oligosaccharide chains of uteroferrin appear to be responsible for its binding and uptake by reticuloendothelial cells of the fetal liver which is the major site of erythropoiesis of the fetus. Uteroferrin, although implicated in transplantal iron transport, also possesses many of the properties of a lysosomal enzyme and, when newly synthesized, carries the so-called lysosomal recognition marker, mannose 6-phosphate. The phosphate group is masked by a covering N-acetylglucosamine residue, a feature which may account for its secretion rather than retention within lysosomes. Evidence is also presented that the oligosaccharide chains of newly synthesized uteroferrin are larger than those of the mature form and are trimmed after secretion. The phosphate group is also removed. It is not clear whether uteroferrin carbohydrate is implicated in the movement of the glycoprotein across the placenta as well as its uptake by the fetal liver.     

Binding of immunoglobulins to the major progesterone-induced proteins secreted by the sheep uterus

We examined the binding of immunoglobulins to the uterine milk proteins, the major progesterone-induced proteins secreted by uterine endometrium of pregnant ewes. Binding was ascertained by measuring binding of 125 I-immunoglobulin to uterine milk proteins that were Western or dot-blotted to nitrocellulose or were coupled to Sepharose. The magnitude of binding was greatest for sheep IgM, intermediate for sheep secretory IgA, low for human secretory and serum IgA, and barely detectable for sheep IgG. Binding of IgA and IgM to uterine milk proteins was time and concentration dependent, saturable, inhibited by high ionic strength buffers, and lost due to enzymatic destruction of the Fc portion of the immunoglobulin molecule. In conclusion, the uterine milk proteins preferentially bind IgA and IgM in a species-dependent manner. Such binding may be related to the role of these proteins in the uterus and may make the uterine milk proteins a useful tool for studying or purifying sheep immunoglobulins.     

Binding of progesterone receptor by nuclear preparations of rabbit and guinea pig uterus.

Guinea pig and rabbit uterine nuclei bound [3H] progesterone in vitro only in the presence of cytosol from estrogen-stimulated uteri. Nuclei from unstimulated and estrogen-stimulated uteri bound progesterone equally well. Nuclei of nontarget tissues also bound progesterone, but to a lesser extent. The rate of nuclear bindins increased with temperature from 0-30 degrees. At 25 degrees nuclear binding remained stable for at least 3 h, but at temperatures of 30 degrees and greater, nuclear binding decreased rapidly after 15 min. Activation of the progesterone-cytoplasmic receptor complex (the change in the complex that enables it to bind quickly to nuclei at 0 degrees) took place slowly at temperatures from 0-5 degrees and rapidly at 10-25 degrees. Activation was facilitated by dilution of the cytosol. Some activation occurred in diluted cytosol in the absence of added progesterone. The cytoplasmic progesterone receptor had a sedimentation coefficient of 7 S when concentrated cytosol (20 mg of protein/ml) was incubated with progesterone at 0 degrees in 5 mM phosphate buffer. Diluting the cytosol and increasing the temperature to 20 degrees caused the sedimentation coefficient to decrease to 5.5 S. Gel filtration of guinea pig uterine cytosol on Sephadex G-100, in the absence of progesterone, yielded a progesterone-binding fraction in the void volume, with a sedimentation coefficient of 5.5 S. The complex of progesterone with the material in the void volume was taken up by nuclei at 0 degrees more rapidly than the complex of progesterone and crude cytosol. The nuclear uptake of progesterone was decreased in phosphate buffer of concentrations greater than 80 mM. Under conditions that favor the nuclear binding of progesterone, the sedimentation coefficient of the cytoplasmic progesterone receptor was 5.5 S. This may be the form of the preceptor which is taken up by nuclei. In decreasing order of effectiveness, unlabeled progesterone, 5 alpha-pregnane-3,20-dione, corticosterone 20 alpha-hydroxy-4-pregnen-3-one, testosterone, estradiol-17 beta, and cortisol competed with [3H] progesterone for binding to nuclei.     

Intrinsic innervation of the uterus in guinea pig and rat

The pattern of distribution of cholinergic and adrenergic nerves in the uterus of albino rats and guinea pigs was examined histochemically. In the albino rat, the uterus was found well-innervated by both adrenergic and cholinergic nerves with a clear regional variation. Dense innervation was demonstrated at the tubal and cervical ends of the uterus and in the cervix. Cholinergic nerves supplying the glands were more numerous than the adrenergic nerves which were relatively few. In the guinea-pigs, the uterus was richly innervated by adrenergic nerves with a clear regional variation. No cholinesterase-positive nerves or nerve cells were demonstrated.     

Patterns of electrical propagation in the intact pregnant guinea pig uterus

Previous studies have reported on propagation of individual spikes in isolated segments of the pregnant uterus, but there is no information on patterns of spike propagation in the intact organ. There is also no information on propagation of myometrial burst. The aim of this study was to record, at high resolution, patterns of propagation of electrical activities in the pregnant uterus. Sixteen timed-pregnant guinea pigs were euthanized at term, and their uteruses isolated. Fetuses were removed and replaced by an equal amount of Tyrode. A 240-electrode array was positioned at various locations along the organ, all signals were recorded simultaneously, and the electrical propagations were reconstructed. In the intact pregnant uterus at term, spikes propagated with high velocity in longitudinal (6.8 +/- 2.4 cm/s) and slower velocity in circular direction (2.8 +/- 1.0 cm/s; P < 0.01). Direction of propagation and frequency of activity were highly variable but showed similar patterns at the ovary or cervical end and along the anterior, posterior, and antimesometrial borders. Along mesometrium, spike propagation was sparse and fractionated. Migration of burst (0.6 +/- 0.4 cm/s) was significantly much slower than that of individual spikes (P < 0.001). Initial burst activity was located at variable locations along the ovarial end of the antimesometrial border, while the latest excitation occurred at the cervical end (1.2 +/- 0.9 min). In conclusion, high resolution electrical mapping of the intact pregnant uterus reveals fundamental properties in spatial and temporal patterns of spike and burst propagation that determine the contraction of the organ.     

Estrogen sulfotransferase activity in guinea pig uterus and chorion

An estrogen sulfotransferase (ST) is detectable in high speed supernatants of pregnant guinea-pig uterus and shows maximum activity between about 47 and 55 days of gestation, with a decrease toward term. No appreciable activity was apparent in the non-pregnant state or before at least 43 days of pregnancy. A considerably higher ST activity is present in chorion as early as 30 days of gestation, and this also decreases toward term. The two ST's exhibit similar KM (0.1-0.13 microM with estrone as substrate) and pI (5.8) values, as well as similar specificities. Estradiol-17 beta and estriol are sulfurylated 82 and 6% that of estrone at equimolar concn. Neither p-nitrophenol nor several neutral steroids are substrates for the enzymes. Enzyme activity is poorly expressed in the absence of thiol groups, the presence of monothioglycerol stimulating uterine and chorion enzymes by 5- and 15-fold, respectively. Stimulation is also observed in the presence of Mg2+, Ca2+ or Mn2+. Chromatofocusing on a poly buffer ion-exchanger from pH 7.4 to 4.0 resulted in elution of a sharp peak of enzyme activity, at pH = 5.8, from both tissues provided that the eluting buffer contained thiol groups and 0.25 M sucrose. This single step resulted in at least a 35- to 100-fold increase in specific activity. The partially purified enzyme from chorion exhibited a KM for estrone of 0.13 microM.     

Changes in phospholipase A2 in myometrium of the guinea pig uterus during pregnancy.

Phospholipase A2 activity has been measured in membrane and cytosolic fractions from non-pregnant and pregnant guinea pig myometrium has been studied. Enzyme activity was measured with 1-stearoyl-2- [3H]arachidonoyl-phosphatidylcholine exhibiting Michaelis-Menton kinetics with Km of 83.8 +/- 21.6 and 53.2 +/- 14.1 for membrane and cytosolic enzymes respectively. Fractionation of the myometrium from non-pregnant guinea pigs suggested that 35% of the activity was membrane associated compared with 20% (P < 0.01) in tissue from pregnant animals. In the presence of 1 mM calcium total activity rose from 3.03 +/- 0.41 to 1737 +/- 368 nmol/h per uterus between non-pregnant and late pregnancy. Calcium activated the membrane enzyme, but the effect was greater late in pregnancy with almost a 6-fold increase in activity at 1 mM calcium compared with a doubling in membrane from non-pregnant guinea pigs. The K0.5 for calcium activation was about 150 microM. Immunoblotting with anti-human-110 KDa phospholipase A2 showed in guinea pig uterus a 34 KDa form of the enzyme that, consistent with changes in activity, showed a fifteen-fold increase in quantity between non-pregnant and late pregnancy. The data are consistent with dramatic increases in the capacity for arachidonic acid release and prostaglandin production in the guinea pig myometrium late in pregnancy.     

Immunochemical and cytochemical studies of relaxin-containing cells in the guinea pig uterus

Uteri and ovaries from cycling, pregnant, and lactating guinea pigs were studied for immunolocalization of relaxin with the light microscope. Endometrial gland cells (EGC) from the same group of animals were examined in the electron microscope for the presence of secretory granules. Those EGC that exhibited high numbers of granules were stained either for relaxin with the protein A colloidal gold method or for carbohydrate with the thiocarbohydrazide technique. Relaxin was found in EGC from middle and late pregnant animals but was not detected in ovaries or uteri from cycling animals. While cytoplasmic granules were noted in most EGC from cycling animals examined, the number of granules was greatest in uteri from estrus and proestrus animals. Granules in EGC from estrus animals contained a carbohydrate-rich material but did not contain relaxin. Endometrial gland cells from animals in early to middle stages of pregnancy (days 15 and 30) contained limited numbers of granules, almost all of which contained carbohydrate. At day 45 of pregnancy, EGC containing many granules were noted. The majority of granules contained relaxin; however, a significant number of EGC contained carbohydrate-rich granules. Infrequently, EGC were noted that contained two populations of granules, and these two populations were assumed to be made up of relaxin-containing and carbohydrate-rich granules. EGC from animals on day 60 of pregnancy typically contained granules, and the majority of these contained relaxin. Carbohydrate-rich granules were observed in EGC of the day 60 animals but were smaller in diameter and were noted in much lower numbers than the relaxin-containing granules. Endometrial gland cells from lactating animals infrequently contained granules. These studies are consistent with the hypothesis that the uterus is the primary source of relaxin in the guinea pig and that relaxin plays an important role in pregnancy and parturition of this species. The observations implicate endometrial glands and their products in the physiology of the cycling animal as well as the pregnant and parturient animal.     

Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig exocrine pancreatic proteins.

A two-dimensional gel technique using slab gel isoelectric focusing in the first dimension and sodium dodecyl sulfate gradient gel electrophoresis in the second dimension has been developed for the separation of soluble proteins larger than 10,000 daltons. The technique is sensitive to 0.6 mug of protein and recovery of radiolabeled proteins averages 90%. Analysis of secretory protein from the guinea pig exocrine pancreas shows the presence of 19 distinct high molecular weight proteins. Each of these proteins has been characterized by isoelectric point, molecular weight, and proportionate mass. Thirteen of the 19 proteins have been identified by actual or potential enzymatic activity,accounting for 96% of the protein mass resolved by the two-dimensional gels.     

Steroid-induced early protein synthesis in rat uterus and prostate.

An early 'induced protein', after exposure of the rat uterus to estradiol, is detected among the soluble proteins with a double-labelling technique and electrophoretic fractionation. Efforts have been directed to establish the subcellular distribution of the induced protein, since such a protein, observable 1 h after hormone administration, may play an important role in the subsequent amplified responses, especially in terms of RNA synthesis. Moreover such an early discrete induced protein was sought in a comparable system responding to another hormone, namely prostate and seminal vesicles under androgens. The induced protein was not found in uterine nuclei of 21-day-old rats after 1 h of estradiol action in vivo and 1 h of tissue incubation with labelled leucine. This negative result summarizes a search among different nuclear protein fractions using various procedures; nor was induced protein observed in mitochondrial and microsomal pellets. Contrary to these negative findings, slight changes of histone labelling were observed under the experimental conditions used to demonstrate induced protein. In addition histone acetylation was increased after 1 h of estradiol action in vivo and 15 min tissue labelling in vitro with radioactive acetate. Furthermore, an increase in total protein synthesis between 0 and 2 h after estradiol action was observed, the relative increase of incorporation of radioactive leucine into protein of estradiol-treated vs non-stimulated uteri being corrected for variations of the acid-soluble radioactive leucine pool. Attempts to obtain an early and discrete induced protein with androgens in prostate and seminal vesicles of immature or castrated rats after different times of exposure to testosterone, androstanolone and estradiol have been unsuccessful. The contribution of both negative and positive findings in steroid-induced early protein synthesis is discussed in the context of the current knowledge of hormone action.     

Measurement of arachidonic acid release from permeabilised myometrial cells of guinea pig uterus.

A technique has been developed for prelabelling and permeabilisation of guinea pig uterine myocytes to enable measurement of arachidonic acid release/phospholipase A2 activity in cells with intact membranes. Intact cells were prelabelled with [3H]inositol or [3H]arachidonic acid for measurement of phospholipase C and A2 respectively. In intact cells 10 nM endothelin-1 or 1 microM bradykinin stimulated both inositol polyphosphate and arachidonic acid release, whilst 1 microM oxytocin, arginine vasopressin or histamine were without effect. In Streptolysin-O permeabilised myometrial cells calcium-stimulation of inositol polyphosphate and arachidonic acid release was detected between 10 microM and 1 mM free calcium. The patterns of inositol polyphosphate and arachidonic acid release were broadly similar. Responses to 1 mM calcium were not detected in intact cells not treated with Streptolysin-O. For arachidonic acid release the K0.5 for calcium activation was about 7 microM, a level above that normally likely to be found in the uterine myocyte. Hence it is concluded that unless there are high local concentrations of calcium close to the plasma membrane, calcium is unlikely alone to be the primary regulator of arachidonic acid release and phospholipase A2.