OBJECTIVE: To study if spontaneous contractions augmented by proteinase-activated receptor-2 (PAR-2)-activating peptide serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL) involve coactivation of membrane chemoceptors and are associated with expression of PAR-2 mRNA in non-pregnant and pregnant rat myometrium. MATERIALS AND METHODS: Non-pregnant, mid-pregnant, and late pregnant rat uterine horn and small intestine segments were snap-frozen in liquid nitrogen to determine PAR-2 mRNA levels by real time polymerase chain reaction (PCR). Uterine rings were used for isometric tension recording. Effect of SLIGRL (0.1mM) on spontaneous contractions before and after exposure to ibuprofen (cyclooxygenase inhibitor, 1.0muM), SQ-29548 (thromboxane A(2) receptor inhibitor, 1.0muM), ketotifen (histamine 1 receptor inhibitor, 10muM), WEB-2170BS (platelet-activating factor (PAF) receptor inhibitor, 10muM), atropine (muscarinic receptor inhibitor, 0.1muM), or ketanserin (serotonin receptor inhibitor, 10muM) were compared. Paired t-test and one-way ANOVA followed by Dunnett's or Newman-Keuls post hoc tests were used for statistical analysis when appropriate. SIGNIFICANCE: P<0.05. RESULTS: The agents did not significantly affect time-associated decay in spontaneous contractile activity in any group of the tissues. Activation of spontaneous contractions induced by SLIGRL in non-pregnant rat myometrium did not involve coactivation of membrane chemoceptors, while in mid-pregnant rat myometrium coactivation of prostanoid, histamine, and serotonin receptors and in late pregnant rat myometrium coactivation of thromboxane receptors was noted. Expression of PAR-2 mRNA was similar in non-pregnant, mid-pregnant, and late pregnant rat myometrium. CONCLUSIONS: Expression of PAR-2 in rat myometrium is not dependent on gestational age. Stimulation of PAR-2 is associated with production/release of cyclooxygenase pathway product(s) activating thromboxane/prostaglandin H2 receptors, partial involvement of histamine H1 receptors and serotonin receptors in midpregnancy and thromboxane A2/prostaglandin H2 receptors in late pregnancy.

1. The mechanism of trypsin-induced contraction in the rat myometrium was investigated using front-surface fluorimetry on fura-PE3-loaded strips. The expression of protease-activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT - PCR). 2. In non-pregnant rats, 10 microM trypsin developed a force of up to 30.5 +/- 5.1% of that obtained during the 40 mM K(+)-depolarization-induced contraction. In pregnant rats, the maximal level of the cytosolic Ca(2+) concentration and tension obtained with 3 microM trypsin was 143.2 +/- 6.0% and 63.2 +/- 7.9%, respectively. The depletion of the extracellular Ca2+ abolished the trypsin-induced contraction. 3. Trypsin-induced contraction was abolished by the pre-treatment of a serine protease inhibitor. PAR1-activating peptide (PAR1-AP) caused a potent contraction of the myometrium, while neither PAR2-AP nor PAR4-AP induced any contraction. 4. RT - PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium. 5. Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the pre-stimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1-AP remained after the pre-stimulation with thrombin and trypsin. 6. In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

In the present study, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17beta-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

1. A new serine proteinase, tryase, was isolated from the membrane fraction of a post-nuclear supernatant of rat liver homogenate. The enzyme was solubilized with 1 M-MgCl2 and purified to homogeneity by DEAE-cellulose chromatography and affinity chromatography with soya-bean trypsin inhibitor linked to Sepharose 4B. 2. The enzyme was identified on sodium dodecyl sulphate/polyacrylamide gels by reaction with radiolabelled di-isopropyl phosphorofluoridate. Unreduced its molecular weight was 32 500, reduced it was 28 000. 3. The enzyme readily hydrolysed azocasein and tripeptide nitroanilide substrates with an arginine or lysine residue adjacent to the leaving group. D-Pro-Phe-Arg-NPhNO2 was used routinely (Km = 0.25 mM). Tryase showed little activity on blocked arginine esters or amides. 4. It was inhibited by di-isopropyl phosphorofluoridate, benzamidine, aprotinin, soya-bean and lima-bean trypsin inhibitors, Ile-Leu-Arg-CH2Cl and Phe-Ala-Arg-CH2Cl. It was not inhibited by Tos-Lys-CH2Cl. 5. Subcellular-fractionation studies showed that tryase was associated with particles similar in their sedimentation properties to lysosomes, but, since it was not present in tritosomes, it was not in the classical lysosome. 6. Rat liver contained other neutral proteinases; one of these was a serine proteinase with an apparent molecular weight of 90 000 on gel chromatography.

Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors.

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.

Plasminogen activator and nonspecific proteolytic activity in transplantable squamous cell rat prostate tumor 11095 were measured by a fluorometric method. Prostate tumors which regressed after treatment with D-Trp-6-LH-RH had 10-fold lower concentrations of plasminogen activator(s) per mg of protein, and considerably higher levels of nuclear androgen receptor. On the other hand, there were no significant changes in nonspecific proteolytic activity in tumor tissue between untreated and D-Trp-6-LH-RH treated rats. The prostate tumor had at least three different plasminogen activator-like bands, as determined by polyacrylamide gel electrophoresis with plasminogen as substrate. The decreased activity of plasminogen activator(s) and considerably higher levels of nuclear androgen receptors correlate with the regression of prostate tumors induced by the treatment of rats with D-Trp-6-LH-RH.

The proteinase extracted from the myofibrillar fraction of (a) primary rat myocytes and (b) the L-8 myogenic cell line, both maintained in culture, was identified by immunochemical analysis as chymase, the chymotrypsin-like serine proteinase of rat mast cells. Chymase would therefore appear to be an intrinsic protein in the rat myocyte also.

The role of plasminogen activators (PAs) as potential mediators of involution of the rat ventral prostate was investigated by using an approach involving the administration in vivo of anti-PA drugs. The prostates of castrated rats, which had been injected daily for 7 days with the anti-PA drugs 6-aminohexanoic acid, tranexamic acid, aprotinin and cortisol, were assayed for PA activity, weight and cell number. In the prostates from the castrated controls, there was a 10-fold increase in the mean PA activity and a 7-fold decrease in cell number relative to that of the non-castrated animals. Although this rise in enzyme activity could be decreased to some extent by all the drugs except aprotinin, only treatment with high doses of tranexamic acid or cortisol had a statistically significant effect. A similar pattern was observed with respect to the relative potency of the drugs in preventing the loss of prostatic weight and cell number after castration. The effects of cortisol were dose-dependent, with complete inhibition of both the rise in PA activity and cell loss occurring at a dose of about 15 mg/day. Since the concentration of the principal intranuclear androgen, dihydrotestosterone, was the same in the prostates from treated and untreated castrated rats, the effects of cortisol are not due to increased retention of this androgen. Rather, the high inverse correlation (r = 0.86) between the cellular concentration of PA activity and the cell population of the prostate implies that PAs are directly associated with prostatic involution and that cortisol, and to a lesser extent tranexamic acid, blocks the involution process through inhibition of PAs.

We had shown previously that the serine protease inhibitors, antipain and leupeptin, restrict uterine DNA snythesis and function in adult mice. The present study is an extension, focusing on the perimaturation period. The protease inhibitors, antipain and elastatinal were tested and the end points were uterine development and peroxidase activity in the uterus. Four week old mice were treated with eigher antipain (3 mg), elastatinal (5 mg) or vehicle (control) twice daily for 3 weeks. The uteri were excised, weighed and homogenized. Subcellular fractions were prepared by differential centrifugation. Each fraction was analyzed for peroxidase activity. The weights and protein content of uteri from control mice averaged greater than twice those in either antipain or elastatinal treated mice (p less than 0.001). Peroxidase activity was sharply curtailed by both inhibitors, antipain being more active than elastatinal. Mechanistically, antipain and elastatinal may act directly on the uterus either by inhibiting key enzymes or by preventing the formation of macromolecules crucial to normal function. Alternatively, the protease inhibitors may interfere with ovarian function, thereby restricting normal uterine development.

As in rats, administration of estradiol to ovariectomized mice results in a trypsin-like proteolytic activity in the uterus. After fractionation of uteri from estradiol-treated ovariectomized mice the protease activity was found in the 12,000 times g pellet and the nucleus, appearing first in the former. Further fractionation of the pellet by discontinuous sucrose gradient centrigugation resulted in sedimentation of the protease with 5'-nucleotidase, a marker enzyme for plasma membrane and separate from mitochondrial and lysosomal enzyme markers. Solubilization was best accomplished by lysis at 37 degrees. The soluble enzyme from mouse uterus had optimal activity at about 43 degrees and pH 8.3 and was inhibited by diisopropylfluorophosphate, tosylarginine methyl ester, antipain, and leupeptin, but not by soybean trypsin inhibitor. Inhibition in vitro by antipain and leupeptin, two low molecular weight peptides, prompted the study of their effect in vivo on the mouse uterus. After intact, cycling female mice received subcutaneous injections of antipain and leupeptin for 16 days, their uteri showed significant diminution in weight and total DNA when compared to untreated controls. Fertility rates were also diminished. Trypsin-like protease activity may be essential to normal uterine metabolism and function.

Bile acid conjugates are found in human breast cyst fluid in average concentrations about 50-fold greater than those in blood. Because epidemiologic studies have linked colon and breast cancer and aberrant bile acid profiles are associated with colon cancer risk, we decided to study the influence of bile acid conjugates (glycochenodeoxycholic acid, glycodeoxycholic acid, glycocholic acid, and glycolithocholic acid) on thymidine incorporation into DNA in cancer (MCF-7) and noncancer (MCF-10A) human mammary cell lines. The two lines responded differently. In MCF-7, bile acids, except for glycolithocholic acid, stimulated thymidine incorporation. Estradiol caused even greater stimulation, an effect that was not influenced further by the addition of bile acids. Bile acids suppressed incorporation in MCF-10A cells. Estradiol at 1 nM had no effect, but 10 nM estradiol was stimulatory. In most cases bile acids appeared to diminish the incorporations observed with estradiol alone, but not significantly. The relevance of these studies to the possible impact of bile acids on the course of fibrocystic disease of the breast would require further investigation.

The concentrations of hydrogen peroxide (H2O2) and peroxidase activity (PA) were measured in mouse uterus during the perimaturation period (4-8 weeks of age) and the estrous cycle. The onset of maturation was accompanied by a spurt in PA but not in H2O2. Neither H2O2 nor PA varied significantly during the estrous cycle. These results differ from those reported in the rat where positive correlations for both with the estrogen state of the uterus were observed.