Human follicular fluid protease and antiprotease activities: a suggested correlation with ability of oocytes to undergo in vitro fertilization.

Plasminogen activator activity was determined in human follicular fluids (FFs) obtained during in vitro fertilization procedures. The fibrinolytic activity of plasminogen activator was significantly higher in fluids from follicles that contained oocytes that were later found to fertilize in vitro (group F) as compared with fluids from follicles that contained oocytes that failed to fertilize (NF). To assess whether this difference in overt plasminogen activator activity reflects differences in conversion of an inactive, latent plasminogen activator to the active enzyme, the ability of exogenous trypsin to enhance plasminogen activation was measured. The plasminogen-dependent hydrolysis of the chromogenic substrate S-2444 in presence of trasylol (Bayer, Leverkusen, Germany) was taken as a measure of plasminogen activator activity in these experiments. No activity was found in untreated FFs, while exposure to trypsin resulted in emergence of marked plasminogen activator activity. In addition, FFs exhibited trasylol-sensitive chromogenic activity indicative of serine-protease activity. Both the plasminogen activator and serine-protease levels after tryptic activation were significantly higher in NF than in F samples. Thus, while F samples have most of their plasminogen activator in an active form, NF samples have most of their plasminogen activator in a latent, trypsin-activatable form. Follicular fluids also contain inhibitory activities toward plasmin and trypsin. The inhibition of these enzymes correlates positively with the latency of plasminogen activator. These results suggest a direct relationship between the ability of oocytes to fertilize and the overt to latent plasminogen activator activity ratios in the FFs.

Plasminogen activator activity and urokinase inhibitor activity in human amniotic fluid and fetal membranes.

This study reports on the presence of latent plasminogen activator (PA) activity in human amniotic fluid (HAF). To measure PA, HAF was incubated with plasminogen, and the formation of plasmin was followed by its ability to cleave globin. The latent proenzyme in HAF was converted to active PA by treatment with sodium dodecyl sulphate (SDS) but not by tryptic digestion. The level of SDS-activatable PA activity in HAF increased with increasing gestational age. In an alternative, direct assay of PA based on its amidolytic activity upon L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide (S-2444), HAF PA activity could be demonstrated even without prior exposure to SDS. Medium conditioned with either chorion or amnion produced PA activity suggesting that HAF PA is derived from the fetal membranes. Treatment of the conditioned medium with SDS or trypsin further increased the enzyme activity. The fetal membranes also produce inhibitory activities towards exogenous trypsin, plasmin, and urokinase. The inhibition of plasmin could be separated from the inhibitory activities towards trypsin and urokinase by DEAE-sephadex ion-exchange chromatography. The function of PA in the normal physiology and in pathological processes involving HAF and the fetal membranes remains to be elucidated.

Amniotic fluid protease activity, protease inhibitory activity, and fetal lung maturity.

Amniotic fluid acid protease and acid protease inhibitory activities were examined in normal pregnancies as a function of gestational age. The acid proteolytic activity of the amniotic fluid is almost constant during gestational weeks 16-29 (26 +/- 13 micrograms globin/ml/2 hrs, mean +/- SD, n = 64). The activity sharply increases after 29 weeks in a time-dependent fashion and reaches a value of 302 +/- 89 (mean +/- SD, n = 13) at 39-40 weeks gestation. Under standard conditions, the ability of amniotic fluid to inhibit bovine pepsin declined during gestation in a linear fashion from 44 +/- 13% (mean +/- SD, n = 36) at 16-18 weeks to 9 +/- 10% (mean +/- SD, n = 41) at 36-40 weeks. A correlation coefficient of r = 0.72 was found between pepsin inhibitory activity and gestational age. No consistent change was noted in the extent of inhibition of the endogenous acid protease throughout pregnancy. In 61 amniotic fluid samples, a correlation coefficient of r = 0.70 was found between acid protease activity and the lecithin/sphingomyelin (L/S) ratio. During the course of this study, five cases of respiratory distress syndrome (RDS) were diagnosed clinically. All five infants had a low protease activity (55 +/- 22 micrograms globin/ml/2 hr, mean +/- SD) as well as a low L/S ratio (0.68 +/- 0.20, mean +/- SD). In contrast, no case of RDS of the newborn was observed among 29 pregnancies with high protease activity and a high L/S ratio. The present observations may suggest a predictive value of amniotic fluid acid protease activity in assessment of fetal lung maturity.

Suppression of plasminogen activator activity by dexamethasone in cultured cardiac myocytes.

Cardiac rat myocytes in primary culture exhibit a membrane-bound and a secreted form of plasminogen activator (PA). Growth of the cells in presence of 2 X 10(-8) M or 10(-7) M dexamethasone markedly reduced both the membrane-bound and the secreted activities of PA. The extent of reduction depended on the time of addition as well as on the length of exposure to the hormone. A similar concentration of estradiol had no effect on PA activity of the myocytes. Cardiac rat fibroblasts in primary culture showed only the particulate form of the enzyme. Exposure of the fibroblasts to 10(-7) M dexamethasone produced a marked inhibition of this activity. The inhibition of PA activity in medium conditioned by dexamethasone-treated myocytes could be relieved by treatment of the medium with 1% (v/v) sodium dodecyl sulphate (SDS). Digestion with 3.3 micrograms/ml bovine trypsin caused an increase in PA activity of media conditioned with control or dexamethasone-treated cells. The present results indicate that cardiac myocytes and fibroblasts produce PA, and that the modulation of PA by glucocorticoid either involves formation of an inactive PA-protein complex or production of an inactive proenzyme. Since glucocorticoids are often administered in conjunction with fibrinolytic enzymes to re-establish cardial perfusion after thrombosis, the present findings indicate further research to assess potential clinical effects of glucocorticoids through suppression of endogenous PA activity in the heart.

Functional assay of plasminogen activator by hydrolysis of 14C-globin.

A functional method for estimation of plasminogen activator (PA) activity is described. The end-point method follows the PA-dependent conversion of the inactive plasminogen to the potent protease plasmin. The formation of plasmin is monitored by its activity on 14C-globin to form acid-soluble radioactive products. The globinolytic assay of PA shows sensitivity similar to that of the 125I-fibrinolytic assay, and is superior in sensitivity to both the chromogenic (Pyro-Glu-Gly-Arg-pNA, S-2444) and the fluorimetric (Glu-Gly-Arg-methylcoumarin amide) methods. The assay is applicable for estimation of tissue type and urokinaselike PA activity in biologic samples, including viable cells in suspension and conditioned medium.

Plasminogen activator activity in differentiating rat myoblasts.

Primary cultures of skeletal muscle cells secrete plasminogen activator (PA) activity to the conditioning medium and display membrane-bound PA. Growth of these cells in culture in presence of 10(-7) M dexamethasone resulted in a marked reduction of the membranal and secreted PA activity. The hormone also reduced cytosolic creatine phosphokinase (CPK) activity and cytosolic protein content. However, cell viability and their ability to undergo fusion were uneffected. The extent of hormone-induced reduction in PA activity depended on the time and extend of exposure. Maximal suppression was obtained by exposing the cells to dexamethasone during the first 4 days of culture. The medium conditioned with dexamethasone-treated cells did not inhibit plasmin, endogenous PA or exogenous PA. Exposure of the conditioned medium from hormone-treated cells to sodium dodecyl sulphate (SDS) or trypsin restored the activity to values observed in media from cells not exposed to the hormone. Acidification of the medium failed to reactivate the enzyme. The myogenic cell line L-8 also displayed membrane-bound PA activity, which was of a comparable magnitude in both fusing and non-fusing L-8 cells. However, in contrast to the primary cultures, exposure of L-8 cells to dexamethasone had no effect on their PA activity whether studied under conditions which allowed or prohibited fusion. The present findings imply that PA has no conducive role in the process of fusion associated with maturation of skeletal muscle cells.

Plasminogen activator and protease inhibitor activities in isolated rat thymocytes.

Plasminogen Activator (PA) and its response to glucocorticoids and androgens was studied in viable rat thymocytes in suspension. PA was measured by its ability to convert plasminogen to plasmin, and the formed plasmin determined by cleavage of 14C-labeled globin. Using this functional assay, PA was found to be associated with the outer surface of thymic cells, and only negligible activity recovered from the incubation medium. Rat thymocytes also contain cytoplasmic and nuclear inhibitor(s) of the serine proteases plasmin, trypsin, chymotrypsin and thymic PA. Release of these inhibitors prevented determination of thymic PA activity in presence of lysed cells. The specific activity of PA in thymocytes isolated from adrenalectomized-castrated rats did not differ significantly from the specific activity associated with cells from intact animals. Furthermore, treatment of adrenalectomized-castrated rats with 0.1 mg of dexamethasone/kg for 2 days induced thymic involution without affecting thymic PA activity. These observations suggest that PA activity of thymocytes is not involved in glucocorticoid-mediated thymic involution.

Inhibition of serine proteases by steroids.

Proteolysis of 14C-labeled globin, as well as the hydrolysis of the specific substrate benzoyl tyrosine ethyl ester, by purified bovine chymotrypsin was found to be inhibited by several steroid hormones. The inhibition of chymotrypsin by the steroids was of a competitive nature, with Ki values of 9.9 x 10(-5) M for triamcinolone (9-fluoro-11 beta, 16 alpha, 17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione), 1.6 x 10(-4) M for cortisol (11 beta, 17 alpha, 21-trihydroxypregn-4-ene-3,20-dione), 3.7 x 10(-4) M for testosterone (17 beta-hydroxy-4-androsten-3-one), 5.0 x 10(-4) M for dexamethasone (9-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methyl-1,4-pregnadiene-3,20-dione) and 1.0 x 10(-4) M for epicortisol (11 alpha, 17,21-trihydroxy-4-pregnene-3,20-dione). The activity of purified bovine trypsin on its specific substrate, TAME (tosyl arginine methyl ester), also showed a similar pattern of inhibition by steroids. Both chymotrypsin and trypsin were found to bind 3H-labeled dexamethasone and cortisol. This binding was markedly inhibited by the general protease inhibitor, PMSF (phenylmethanesulfonyl fluoride), whereas the chymotrypsin-specific inhibitor, TPCK (L-[1-tosyl-amido-2-phenyl]ethylchloromethyl ketone), inhibited only the steroid binding to chymotrypsin but not to trypsin. These observations indicate that serine proteases recognize steroid hormones in a fashion similar to the recognition of their specific substrates and that the steroids inhibit activity of these enzymes at their binding sites.