Interleukin 1 stimulation of synovial cell plasminogen activator production

Addition of human monocyte interleukin 1 (IL-1) to cultured human synovial cells can cause an increase in both cell associated (30-fold) and extracellular (40-fold) plasminogen activator (PA) activity. This increase was inhibited by antibody directed against IL-1 and phenylglyoxal. PA activity could be detected 3 h after the addition of IL-1, continued to increase for 24 h and was dependent on RNA and protein synthesis. The molecular weight of the PA produced from the IL-1 stimulated synovial cells was 55,000 +/- 1,000. Mononuclear cell conditioned media (MCCM) also stimulated synovial cells to produce PA. This stimulation was partly inhibited by anti-IL-1 thus suggesting the presence of appreciable IL-1 activity in MCCM. These results could provide clues as to how immune events are linked to cartilage destruction associated with rheumatoid arthritis.     

Production of a cell-associated and secreted plasminogen activator by cultured rat granulosa cells

The localization and time-related production of plasminogen activator (PA) by ovarian granulosa cells was studied by measuring the plasmin-mediated lysis of the chromogenic substrate H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate. Granulosa cells from diethylstilbestrol-implanted immature rats produced both a cell-associated and a secreted PA, as indicated by increased hydrolysis of the substrate by the cells or extracellular medium. The formation of cellular PA was induced by FSH and was detectable as early as 2 h during a 72-h culture, with 80% of the maximal activity present by 6 h. In contrast, negligible PA activity was detected in the extracellular medium until 6-20 h of culture, after which time the secreted PA activity continued to rise throughout the 72-h culture period. Control cells also produced both cellular and secreted PA, but in lower amounts than cells stimulated by FSH. The presence of cellular PA was further indicated by a 2-fold rise in PA activity after solubilization of granulosa cells with increasing concentrations of the detergent Triton X-100. However, freshly prepared granulosa cells had no detectable PA activity in the absence or presence of detergent, suggesting that the PA was synthesized during culture. Actinomycin D and cycloheximide suppressed cellular PA production when added during the first hours of granulosa cell culture, but had little effect when added from 44-48 h of culture. In contrast, both actinomycin D and cycloheximide reduced secreted PA activity from 44-48 h. The expression of cellular PA activity was only partially dependent on the presence of fibrin, while the secreted PA fully required fibrin. These results demonstrate gonadotropin-regulated production of both cellular and secreted types of PA by granulosa cells. The cellular form is produced in the first hours of culture when it is sensitive to macromolecule synthesis inhibitors and is partially dependent on fibrin. The extracellular PA is predominantly secreted after the first 24 h of culture and requires fibrin for its activity. The differential activities of the two types of PA may be involved in the control of hormone-induced processes during granulosa cell differentiation.     

Role of alveolar macrophage plasminogen activator in the acute pulmonary responses to endotoxin

To clarify the role of alveolar macrophages (AM) in the development of endotoxin induced lung injury in rats, we examined the release of plasminogen activator (PA) by AM and fibrinolytic activity in the bronchoalveolar lavage (BAL) fluid. An intraperitoneal injection of 5 mg/kg endotoxin was followed by a rapid increase in PA release by AM and corresponding increase in fibrinolytic activity in BAL fluid. These effects reached a maximum 3 h after injection. Within 6 h after injection widening of alveolar-arterial oxygen difference (AaDO₂) and increase in alveolar capillary permeability were found. There was significant positive correlation between PA release by AM and fibrinolytic activity in BAL fluid, and also between AaDO₂ and fibrinolytic activity in BAL fluid. These results suggest that AM may contribute to the pathogenesis of endotoxin induced lung injury in rats through a PA dependent mechanism.     

Increased alveolar plasminogen activator in early asbestosis

Alveolar macrophage-derived plasminogen activator (PA) activity is decreased in some chronic interstitial lung diseases such as idiopathic pulmonary fibrosis and sarcoidosis but increased in experimental models of acute alveolitis. Although asbestos fibers can stimulate alveolar macrophages (AM) to release PA in vitro, the effect of chronic asbestos exposure of the lower respiratory tract on lung PA activity remains unknown. The present study was designed to evaluate PA activity of alveolar macrophages and bronchoalveolar lavage (BAL) fluid in asbestos-exposed sheep and asbestos workers. Forty-three sheep were exposed to either 100 mg UICC chrysotile B asbestos in 100 ml phosphate-buffered saline (PBS) or to 100 ml PBS by tracheal infusion every 2 wk for 18 months. At Month 18, chest roentgenograms were analyzed and alveolar macrophage and extracellular fluid PA activity were measured in samples obtained by BAL. Alveolar macrophage PA activity was increased in the asbestos-exposed sheep compared to control sheep (87.2 +/- 17.3 versus 41.1 +/- 7.2 U/10(5) AM-24 h, p less than 0.05) as was the BAL fluid PA activity (674.9 +/- 168.4 versus 81.3 +/- 19.7 U/mg alb-24 h, p less than 0.01). Among the asbestos-exposed sheep, 10 had normal chest roentgenograms (Group SA) and 15 had irregular interstitial opacities (Group SB). Strikingly, whereas Group SA did not differ from the control group in BAL cellularity or PA activity, Group SB had marked increases in alveolar macrophages (p less than 0.005), AM PA activity (p less than 0.02), and BAL PA activity (p less than 0.001) compared to the control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasminogen activation in synovial tissues: differences between normal, osteoarthritis, and rheumatoid arthritis joints

OBJECTIVE—To analyse the functional activity of the plasminogen activators urokinase (uPA) and tissue type plasminogen activator (tPA) in human synovial membrane, and to compare the pattern of expression between normal, osteoarthritic, and rheumatoid synovium. The molecular mechanisms underlying differences in PA activities between normal and pathological synovial tissues have been further examined.
METHODS—Synovial membranes from seven normal (N) subjects, 14 osteoarthritis (OA), and 10 rheumatoid arthritis (RA) patients were analysed for plasminogen activator activity by conventional zymography and in situ zymography on tissue sections. The tissue distribution of uPA, tPA, uPA receptor (uPAR), and plasminogen activator inhibitor type-1 (PAI-1) was studied by immunohistochemistry. uPA, tPA, uPAR, and PAI-1 mRNA values and mRNA distribution were assessed by northern blot and in situ hybridisations respectively.
RESULTS—All normal and most OA synovial tissues expressed predominantly tPA catalysed proteolytic activity mainly associated to the synovial vasculature. In some OA, tPA activity was expressed together with variable amounts of uPA mediated activity. By contrast, most RA synovial tissues exhibited considerably increased uPA activity over the proliferative lining areas, while tPA activity was reduced when compared with N and OA synovial tissues. This increase in uPA activity was associated with increased levels of uPA antigen and its corresponding mRNA, which were localised over the synovial proliferative lining areas. In addition, in RA tissues, expression of the specific uPA receptor (uPAR) and of the plasminogen activator inhibitor-type 1 (PAI-1) were also increased.
CONCLUSION—Taken together, these results show an alteration of the PA/plasmin system in RA synovial tissues, resulting in increased uPA catalytic activity that may play a part in tissue destruction in RA.

     

Elevations in synovial fluid plasminogen activator in patients with rheumatoid arthritis

Plasminogen activator (PA) activity in synovial fluid (SF) obtained from patients with rheumatoid arthritis (RA) is elevated when compared to SF obtained from patients with osteoarthritis (OA). Immunological studies and lack of evidence for a decrease in PA inhibitors, or an increase in PA stimulators, suggest that elevations in RA SF PA activity reflect increases in PA level. Although the origin(s) of SF PA was not identified, the enzyme resembles urokinase and RA synovium may be a contributing source. These observations are consistent with a possible active role of PA in the pathogenesis of RA.     

Human synovial fibroblast plasminogen activator. modulation of enzyme activity by antiinflammatory steroids

Human synovial fibroblasts in culture have been shown to have low plasminogen activator (PA) activity; however, conditioned medium from concanavalin A-stimulated peripheral blood mononuclear cells (c-MCCM) stimulates the cellular levels of this protease. The present study shows that low concentrations of a series of antiinflammatory steroids inhibit the PA activities of both unstimulated and c-MCCM-stimulated fibroblasts. Dexamethasone, the corticosteroid studied in greatest detail, suppresses both the extracellular and cell-associated enzyme activities; this inhibition is rapid, reversible, and is not due to the inhibition of cellular RNA, protein, or DNA synthesis. PA has been invoked as possibly being generally important for the processes of cell migration, tissue remodeling, and inflammation. These in vitro observations suggest that physiologic and/or pharmacologic control of the PA levels in synovial fibroblasts might also be achieved in vivo by the interacting effects of mutually antagonistic agents, namely, a product from stimulated mononuclear cells and glucocorticoids.     

Bone matrix degradation by the plasminogen activation system. Possible mechanism of bone destruction in arthritis

The observed increase in urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR) in synovial tissue of patients with rheumatoid arthritis (RA) suggests pathophysiological involvement of the plasminogen activation (PA) system in inflammatory joint disease. In the present study, we investigated the capacity of the PA system to degrade non-mineralized and mineralized bone-like matrix in vitro as a model for bone destruction. Transfected mouse LB6 cell lines, that expressed either human u-PA or u-PAR, were cultured separately and simultaneously on radiolabelled bone matrix in the presence of plasminogen. Osteoblast-like murine calvarial MC3T3-E1 cells were used to produce a well-characterized, highly organized bone-like matrix, that could be mineralized in the presence of beta-glycerol phosphate. Bone matrix degradation was followed by the release of radioactivity in the culture medium. u-PA-producing cells, in contrast to u-PAR- producing cells, degraded both non-mineralized and mineralized bone matrix. This effect could be inhibited by anti-u-PA antibodies, as well as by tranexamic acid and by aprotinin, indicating that the degrading activity is u-PA mediated and plasmin dependent. Co-cultivation of a small portion of u-PA-producing cells with u-PAR-expressing cells resulted in a marked increase in degradation activity. Reduction of this potentiating effect by suramin or the amino-terminal fragment of u- PA, both competitive inhibitors of u-PA receptor binding, shows that this synergistic effect is due to binding of u-PA to u-PAR. u-PAR must be cell associated, as binding of u-PA to a soluble u-PAR prevented this enhancement. The capability of the PA system to degrade bone matrix in vitro, and the previously demonstrated increased expression of u-PA and u-PAR in synovial tissue of patients with RA, further support a role for the PA system in the development of bone erosions.      

Evidence that interleukin-1 induction of synovial cell plasminogen activator is mediated via prostaglandin E2 and cAMP

Addition of the cyclooxygenase inhibitor indomethacin to human synovial cells in culture, at concentrations which completely block prostaglandin E2 (PGE2) synthesis, reversibly inhibited the interleukin-1 (IL-1) stimulation of cell-associated and extracellular plasminogen activator (PA) production. Results of mixing experiments suggested that the inhibition by indomethacin was not due to stimulation of production and/or activation of a PA inhibitor, but reflected inhibition of PA synthesis. Simultaneous addition of PGE2 or dibutyryl cAMP prevented the inhibition by indomethacin. Addition of the phosphodiesterase inhibitor, theophylline, the adenylate cyclase stimulator, forskolin, or dibutyryl cAMP caused an enhancement of the IL-1 induction of synovial cell PA. These results suggest that the IL-1 induction of synovial cell PA occurs via generation of endogenous PGE2 and cAMP.     

Primary hyperaldosteronism in essential hypertensives: prevalence, biochemical profile, and molecular biology

There is evidence that primary aldosteronism (PA) may be common in patients with essential hypertension (EH) when determinations of serum aldosterone (SA), plasma renin activity (PRA), and the SA/PRA ratio are used as screening. An inherited form of primary hyperaldosteronism is the glucocorticoid-remediable aldosteronism (GRA) caused by an unequal crossing over between the CYP11B1 and CYP11B2 genes that results in a chimeric gene, which has aldosterone synthase activity regulated by ACTH. The aim of this study was to evaluate the prevalence of PA and the GRA in 305 EH patients and 205 normotensive controls. We measured SA (1-16 ng/dL) and PRA (1-2.5 ng/mL x h) and calculated the SA/PRA ratio in all patients. A SA/PRA ratio level greater than 25 was defined as being elevated. PA was diagnosed in the presence of high SA levels (>16 ng/dL), low PRA levels (<0.5 ng/mL x h), and very high SA/PRA ratio (>50). Probable PA was diagnosed when the SA/PRA ratio was more than 25 but the other criteria were not present. A Fludrocortisone test was done to confirm the diagnosis. GRA was differentiated from other forms of PA by: the aldosterone suppression test with dexamethasone, the high levels of 18-hydroxycortisol, and the genetic detection of the chimeric gene. In EH patients, 29 of 305 (9.5%) had PA, 13 of 29 met all the criteria for PA, and 16 of 29 were initially diagnosed as having a probable PA and confirmed by the fludrocortisone test. Plasma potassium was normal in all patients. The dexamethasone suppression test was positive for GRA in 10 of 29 and 18-hydroxycortisol levels were high in 2 of 29 patients who had also a chimeric gene. In normotensive subjects, 3 of 205 (1.46%) had PA, and 1 of 205 had a GRA. In summary, we found a high frequency of normokalemic PA in EH patients. A high proportion of PA suppressed SA with dexamethasone, but only a few had a chimeric gene or high levels of 18-hydroxycortisol. These results emphasize the need to further investigate EH patients.     

Production of plasminogen activator and inhibition of embryonic cell aggregation by cultured human normal and neoplastic cells

The plasminogen activator (PA) production and the capacity to inhibit embryonic neural retina (NR) cell aggregation by human normal and neoplastic cell lines have been studied. The PA production was detected by both iodinated fibrin and casein lysis assays, and by changes in cell morphology at the presence of activated PA, using dog serum. Since the casein lysis assay and morphological changes proved to be less sensitive than 125I-fibrin lysis assay, a good correlation between these three assays could be observed provided that PA production measured by fibrinolysis exceeded 10--20%. The neoplastic cell lines exhibited the PA production to quite a large extent. The highest fibrinolytic activity (78%) was found in the case of bladder carcinoma cells T24, while the B-5GT cells from giant cell tumor of bone failed to produce any detectable amount of the PA. The cells from synovial sarcoma and both glioma lines exhibited fibrinolytic activity of about 10% and four sarcoma cell lines over the range 20--50%. Out of 13 normal cell lines tested, 7 were negative or exhibited very low fibrinolysis not exceeding 3% of total radioactivity. Four cell lines derived from kidneys, lungs, intestines, and from mixed embryonic tissues showed a marked fibrinolytic activity of about 10--37%, a slightly elevated fibrinolysis being found in embryonic lung cells LEP and cells from fetal skin tissue only at the presence of dog serum. The fibrinolysis detected in the neoplastic cloned cell populations showed considerable differences in the PA production between individual cell clones isolated from the same parental cell line. Unlike the normal fibroblastic cells B-41FB derived from bone, all neoplastic cell lines tested possess the capability to inhibit embryonic NR cell aggregation significantly. The results suggest the effect not to be dependent upon the PA production.     

Hormonal control of plasminogen activator secretion in ZR-75-1 human breast cancer cells in culture

Activity of secreted plasminogen activator (PA) by ZR-75-1 human breast cancer cells in culture is shown to be altered by the addition of physiologically relevant concentrations of the hormones 17 beta-estradiol (E2), insulin, and dexamethasone. After 48 h, E2 stimulated PA activity 6-fold at concentrations as low as 10(-12) M. This stimulation was prevented by the addition of actinomycin D and cycloheximide. The antiestrogen tamoxifen reduced estrogen stimulation of PA, but had slight stimulatory effects on PA secretion by itself. Insulin (5 X 10(-10) M) induced a 2-fold increase in PA activity. Effects of insulin and E2 were additive, suggesting independent sites of control of PA production. Dexamethasone (10(-8) M) decreased PA activity by 20%, but did not inhibit cell growth at the concentration tested. These data suggest that secreted PA activity is differentially regulated by hormones and that effects of PA and growth do not occur in parallel.     

Blocking ERK‐1/2 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein A

Objective

To examine the mechanism of regulation of interleukin‐18 (IL‐18) bioactivity by IL‐18 binding protein (IL‐18BP) induction.

Methods

Levels of IL‐18 and IL‐18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme‐linked immunosorbent assays (ELISAs), followed by calculation of free IL‐18. IL‐18 and IL‐18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real‐time polymerase chain reaction and ELISA, respectively, followed by IL‐18 bioactivity determination using KG‐1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL‐18BPa/IL‐18 regulation. Tumor necrosis factor α (TNFα)–induced caspase 1 activity was determined by a colorimetric assay.

Results

IL‐18BPa was lower in RA synovial fluid than in OA synovial fluid (P < 0.05; n = 8), and free IL‐18 was higher in RA synovial fluid than in OA synovial fluid. TNFα induced RA synovial fibroblast IL‐18BPa and IL‐18 in a time‐dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNFα induced IL‐18 production through the ERK‐1/2, protein kinase Cδ (PKCδ), and Src pathways, whereas IL‐18BPa synthesis was mediated through the NFκB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL‐18BPa‐Fc reduced the RA synovial fibroblast phosphorylation of ERK‐1/2 induced by TNFα.

Conclusion

These results suggest that IL‐18BPa reduces IL‐18 bioactivity induced by TNFα, by regulating the ERK‐1/2 pathway in RA synovial fibroblasts. Targeting IL‐18 bioactivity by induction or addition of IL‐18BPa may provide another therapeutic option in the management of RA.

     

The control of the synthesis and secretion of plasminogen activator by rat sertoli cells in culture

Sertoli cells in primary cultures produce plasminogen activator activity, and release it into the medium at rates greatly influenced by a variety of factors, including cell density, the presence of hormones, incubation temperature and duration of culture. In Sertoli cells maintained in culture in the presence of dibutyryl cAMP, the amounts of plasminogen activator activity secreted per cell were maximal at cell densities up to 2.5 μg DNA/cm² (350 units/μg cell DNA), and declined to 40 units/μg cell DNA at a density of 22 μg DNA/cm². Concentrations of follicle-stimulating hormone (FSH) required to elicit half-maximal stimulation of the production of plasminogen activator activity were 0.37 μg/ml for oFSH-NIH S12 and 8 ng/ml for the more purified oFSH-S1528C2. The ED₅₀ for dibutyryl cAMP was found to be 0.08 mM. Addition of an inhibitor of phosphodiesterase (3-isobutyl-l-methylxanthine) enhanced the formation of plasminogen activator by cells cultured in the presence of FSH.Addition to the culture medium of testosterone, epidermal growth factor, insulin, human chorionic gonadotropin or prostaglandins (E₁, E₂ or F_1α) did not result in increased production of PA activity by Sertoli cells. Cells in culture for as long as 14 days remained responsive to FSH or dibutyryl cAMP. Increases of cellular levels of plasminogen activator became evident within 2–4 h after addition of either FSH or dibutyryl cAMP to the medium. The stimulation by FSH or dibutyryl cAMP of the production of plasminogen activator activity was shown to be dependent upon de novo synthesis of RNA and protein. Levels of enzyme activity released by Sertoli cells maintained in culture for 48 h at 37°C were approx. 50% higher than plasminogen activator released by cells cultured at 32°C. The control of the production of plasminogen activator activity by Sertoli cells was discussed in relation to the control of plasminogen activator production by granulose cells, and the possible role of plasminogen activator in gonadal functions.     

Glucocorticoid regulation of plasminogen activator inhibitor-1 messenger ribonucleic acid and protein in normal and malignant rat osteoblasts.

Glucocorticoids exert potent inhibitory effects on bone formation. We have previously shown that glucocorticoids suppress plasminogen activator (PA) activity in normal and malignant rat osteoblasts. To clarify the mechanism of this suppression, we investigated the effects of dexamethasone on PA inhibitor-1 (PAI-1), tissue-type PA (tPA), and urokinase-type PA (uPA) expression and also on PAI-1 protein and PA activity in both normal rat calvarial osteoblasts and a clonal osteogenic sarcoma cell line, UMR 106-01. Dexamethasone increased PAI-1 mRNA and protein in both cell types. The increase in PAI-1 protein and the decrease in PA activity were obtained over the same concentration range, with a half-maximally effective concentration of dexamethasone of about 10(-9) M. The increase in PAI-1 mRNA caused by dexamethasone was retained with cycloheximide treatment, but abolished with actinomycin-D. Dexamethasone had no effect on tPA or uPA mRNA in either cell type. The glucocorticoid antagonist RU 486 prevented the effects of dexamethasone on PA activity and PAI-1 protein. Dihydrotestosterone, progesterone, and 17 beta-estradiol did not influence PA activity or PAI-1 formation. Although tPA and uPA protein could not be measured, these results suggest that glucocorticoids suppress PA activity predominantly by increasing PAI-1 synthesis in rat osteoblasts. Suppression of PA activity through actions on PAI-1 formation by glucocorticoids could contribute to the mechanisms by which glucocorticoids inhibit bone formation.     

Control of plasminogen activator activity in the thecal layer of the largest preovulatory follicle in the hen ovary.

Although the existence of plasminogen activator (PA) activity and the factors that regulate it in ovarian granulosa cells of both mammalian and avian species have been extensively documented, very little information has been generated concerning the control of PA activity in the adjacent thecal layer. This study was conducted to evaluate the effects of several physiological and pharmacological agents on PA activity in dispersed cells from the thecal layer of the largest preovulatory follicle in the hen ovary 17-16 h before ovulation. LH (50 and 100 ng) in the presence of 3-isobutyl-1-methylxanthine (0.01 mM) stimulated an approximate 25% increase in cell-associated PA activity, possibly via elevated levels of cAMP. Prostaglandin E1 and E2 (PGE1 and PGE2; 0.1 and 1 microM), but not PGI2 or PGF2 alpha (1 microM), enhanced PA activity and cAMP formation, effects that were potentiated by 0.01 mM 3-isobutyl-1-methylxanthine. Activation of Gs with cholera toxin (0.01-10 ng/tube) or adenylyl cyclase with forskolin (0.01-10 microM) stimulated cAMP formation and PA activity in a dose-dependent manner. Exposure of cells to the cAMP analog 8-bromo-cAMP (0.1-5 mM) caused similar increases in thecal cell PA activity. Incubation of cells with phorbol 12-myristate 13-acetate (PMA; 3.2-162 nM), an agonist known to activate protein kinase-C, resulted in a dose-dependent increase in PA activity. However, an equimolar concentration of phorbol 13-monoacetate (162 nM), an inactive analog of PMA that does not activate protein kinase-C, was without effect. Coincubation of cells with forskolin (1 microM) and PMA (32 nM) resulted in a synergistic stimulation of secreted PA activity, apparently via an enhancement of adenylyl cyclase activity. Treatment of cells with the calcium ionophore A23187 (0.01-1 microM) suppressed basal PA activity. However, PA activity stimulated by PMA (32 nM) was synergistically increased after coincubation with a 0.05-microM concentration of A23187, but was inhibited at doses of 0.5 and 1 microM. Taken collectively, the data indicate that PA activity is present in the thecal layer of the largest preovulatory follicle in the ovary of the domestic hen. Furthermore, several endocrine factors (i.e. LH and PGs) were found to stimulate PA activity, possibly via both the adenylyl cyclase-cAMP-protein kinase-A and phosphoinositide-protein kinase-C pathways. In light of these findings, we propose that the preovulatory increase in PGs and LH activates PA in the thecal layer of the largest preovulatory follicle, resulting in proteolytic degradation of the follicular connective tissue and, ultimately, ovulation.     

Down-regulation of epidermal growth factor receptor correlates with plasminogen activator activity in human A431 epidermoid carcinoma cells.

Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced "down-regulation" of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the t 1/2 for both processes being 3-3.5 hr. Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor down-regulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during down-regulation.     

Prolactin inhibits plasminogen activator activity in the preovulatory follicles

The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 10(2), or 10(3) ng/ml. PRL at 10(3) ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 +/- 0.08 to 28.4 +/- 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity.