Matrix-bound plasminogen activator inhibitor type 1 inhibits the invasion of human monocytes into interstitial tissue.

Invasion of tissue by monocytes in the course of cellular immune reactions is a multistep process that is thought to be based on the action of urokinase type plasminogen activator (u-PA), an ubiquitous serine protease able to convert the zymogen plasminogen into the active protease plasmin. Expression and occupation of urokinase-type plasminogen activator receptors (u-PA-R) are known to be up-regulated by IFN-gamma and TNF-alpha, and endogenously occupied u-PA-R were found to be instrumental in monocyte invasiveness. We used the amnion invasion assay to investigate whether monocyte invasiveness is affected by matrix-bound plasminogen activator inhibitors (PAI) and by fluid phase u-PA. We show in this study that preincubation of amnion membranes with 1.5 U/cm2 PAI-1 decreases invasion of IFN-gamma activated monocytes by 70% compared with controls. Anti-vitronectin antibodies, which block PAI-1 binding to the matrix, abrogate the inhibitory effect of PAI-1 on monocyte invasiveness, indicating that active PAI-1 is bound via matrix-associated vitronectin. In contrast, preincubation of the amnion membrane with PAI-2 which does not bind to the extracellular matrix has no effect on monocyte invasiveness. Finally, the inhibitory action of matrix-bound PAI-1 can be abrogated by addition of 5 IU/ml u-PA to the monocytes in the invasion chamber. These findings indicate that monocyte invasiveness might be regulated not only by expression and occupation of u-PA-R but also by matrix-bound PAI-1.     

Plasmin regulating system from embryonal carcinoma F9 cells: plasminases A, B and embrinogen

Two plasmin inactivators, plasminase A and B, and their inhibitor embrinogen were isolated from embryonal carcinoma F9 cells by preparative two-dimensional electrophoresis. Plasminases A and B have molecular weights of 160,000 and 82,000, respectively. Both are serine proteinases which digest the light chain of plasmin in a time dependent inactivation process. The heavy chain of plasmin is not affected by this action. Plasminases A and B show similar specificity towards synthetic and natural polypeptide inhibitors. The interaction of the two enzymes leads to their inhibition. Embrinogen (m.w. 84,000) inhibits both plasminases A and B as well as urokinase and plasmin. Its activation by trypsin creates embrin, a proteinase directed against plasmin heavy chain.     

Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

Inhibition of fibrinolytic enzymes by thrombin inhibitors

Thrombin inhibitors have recently advanced to the stage of preclinical testing as anticoagulants. However, little is known about the effects of these inhibitors on the enzymes of the fibrinolytic system. In the present study we evaluated the effect of two protein and two synthetic inhibitors of thrombin on tissue plasminogen activator (tPA), urokinase, and plasmin. We found that hirudin inhibited the amidolytic activity of plasmin but had no effect on tPA or urokinase. Antithrombin III inhibited plasmin and urokinase but had no effect on tPA. D-Phe-Pro-Arg-CH2Cl inhibited plasmin and tPA but had no effect on urokinase. Thromstop inhibited all three fibrinolytic enzymes: plasmin, urokinase, and tPA. Thus each thrombin inhibitor tested had different inhibitory effects on the fibrinolytic enzymes. These effects should be carefully considered when thrombin inhibitors are used as antithrombotic drugs.     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.     

Platelet-activating factor as a stimulus of exocytosis in human neutrophils

The properties of platelet-activating factor (PAF-acether) 42–48ulus of exocytosis in human neutrophils have been re-investigated with particular attention to effects on cells that were not pretreated with cytochalasin B. Release of gelatinase, the most sensitive marker of exocytosis, was determined in addition to that of vitamin B-12-binding protein and β-glucuronidase. Superoxide production was assayed as a measure of the respiratory burst. The effects of PAF-acether were compared to those of leukotriene B₄ and N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). Our results show that PAF-acether elicits marked secretion in untreated human neutrophils, and refute the prevalent view that cytochalasin B treatment is required for responsiveness. PAF-acether induced abundant release of gelatinase, increasing on average from 20% at 10 nM to 35% at 1 μM. This release was very rapid, i.e., almost complete after 2 min. fMet-Leu-Phe induced the same maximum response already at 0.1 μM, but release was considerably slower. Leukotriene B₄ was less potent with a maximum release of 20%. Exocytosis of gelatinase was always paralleled by liberation of smaller but significant amounts of vitamin B₁₂-binding protein from the specific granules. In contrast to their effect on exocytosis, PAF-acether and leukotriene B₄ were very weak stimuli of the respiratory burst when compared with fMet-Leu-Phe.     

Down-regulation of plasmin receptors on human sarcoma cells by glucocorticoids

After incubation of confluent monolayer cultures of human HT-1080 fibrosarcoma cells with purified native human plasminogen in plasminogen-depleted serum-containing medium, bound plasmin activity could be specifically eluted from the cells with tranexamic acid, an analogue of lysine. Dexamethasone reduced the amount of recoverable bound plasmin activity in a dose-dependent manner. Dexamethasone was also found to induce a time- and dose-dependent decrease in the ability of the cells to bind added plasmin. Untreated HT-1080 cells bound added plasmin with a high capacity (600,000 molecules bound per cell), and this decreased to an undetectable level after treatment with 100 nM dexamethasone. The kinetics of the loss of plasmin binding by the dexamethasone-treated sarcoma cells, a clear decrease after 4 h, correlated with those for the loss of cell-bound urokinase (u-PA) activity. Plasmin was not, however, bound to the active site of u-PA: an anti-catalytic monoclonal antibody to u-PA had no effect on plasmin binding. Other glucocorticoids, such as hydrocortisone and corticosterone, had a similar effect to dexamethasone on plasmin binding to HT-1080 cells. The effect of glucocorticoids on the plasmin receptor seemed to occur at least partly via a decrease in the affinity for plasmin, since the Kd for plasmin with untreated cells was 5.4 x 10(-9) M, and with cells treated with 5 nM dexamethasone, the Kd value for plasmin was 1.2 x 10(-7) M. These results show that glucocorticoids induce down-regulation of plasmin receptors on the surface of HT-1080 cells: a novel mechanism, in addition to the known effects of glucocorticoids on u-PA and PA inhibitors, by which human tumor cells may be disarmed of their pericellular proteolytic activity.     

A novel and simple immunocapture assay for determination of gelatinase-b (MMP-9) activities in biological fluids: Saliva from patients with Sjögren's Syndrome contain increased latent and active gelatinase-b levels

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys↓IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLueGly↓IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can measured directly using a chromogenic peptide substrate for urokinase. The assay has been specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this body MMp-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed.We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 ± 4.9 vs 12.2 ± 2.5 × 10⁴ cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 ± 1.9 × 10⁴ cmp/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 ± 2.5 U/mg, controls 1.0 ± 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 ± 9.8 U/mg, controls 16.5 ± 2.6 U/mg (p = 0.01).     

Plasminogen activator from cells transformed by an oncogenic virus: inhibitors of the activation reaction.

This paper describes an assay for direct measurement of plasminogen activation and its application for determining the kinetic constants and for screening potential inhibitors of the reaction. The assay is based on the conversion of the single chain of 125I-labelled plasminogen to the two chains of 125I-labelled plasmin (EC 3.4.21.7), the latter then being separated from each other and from the plasminogen substrate by electrophoresis under reducing conditions in SDS-polyacrylamide gels. The Km of activator from transformed murine cells for human plasminogen was 180 nM. A broad range of compounds was tested as potential inhibitors of plasminogen activation and of plasmin-catalyzed fibrinolysis respectively, and the two reactions differed qualitatively and quantitatively in their response to previous agents. The principal qualitative difference was in the susceptibility of the reactions to a spectrum of naturally-occurring macromolecular inhibitors: all of the macromolecular inhibitors that blocked the action of plasmin were without effect on murine activator or human urokinase (EC 3.4.99.26). A variety of small molecules inhibited both of the reactions tested, and showed significant quantitative differences; some of these were active at micron concentrations. The exacting specificity of plasminogen activators for macromolecules, both substrates and inhibitors, encourages the expectation that effective inhibitors of great specificity may be isolated from as yet undiscovered natural sources.     

Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation.

The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.     

Human inter-alpha-trypsin inhibitor. Limited proteolysis by trypsin, plasmin, kallikrein and granulocytic elastase and inhibitory properties of the cleavage products.

The acid-labile inter-alpha-trypsin inhibitor is cleaved enzymatically in vivo, liberating a smaller acid-stable inhibitor. The molar ratio of native inhibitor to this smaller inhibitor in plasma is significantly changed in some severe cases of inflammation and kidney injury. To clarify this observation on a molecular basis, the action of four different types of proteinases (trypsin, plasmin, kallikrein and granulocyte elastase) on the inter-alpha-trypsin inhibitor was studied. The initial rate of cleavage of the inter-alpha-trypsin inhibitor by a 1.3-fold molar excess of proteinase over inhibitor was found to be 4375 nM x min-1 with granulocyte elastase, 860 nM x min-1 with trypsin, 67 nM x min-1 with plasmin, and 0.3 nM X min-1 with kallikrein. Obviously, of the enzymes studied so far, the granulocyte elastase known to be released during severe inflammatory processes is by far the most potent proteinase in the transformation of the inter-alpha-trypsin inhibitor. The inter-alpha-trypsin inhibitor and its cleavage products inhibit bovine trypsin very strongly (Ki = 10(-9)--10(-11) M), porcine plasmin much less strongly, human plasmin very weakly and pancreatic kallikrein practically not at all.     

N-oleoyl heparin inhibits the amidolytic activity of plasmin and urokinase.

N-desulphated heparin, partially N-acylated on average with three oleoyl chains per molecule, inhibits the amidolytic activity of plasmin (IC50 16 nM) and urokinase (IC50 10mM) when assayed on N-p-tosyl-Gly-Pro-Lys-4-nitroanilide and benzoyl-Ala-Gly-Arg-4-nitroanilide substrates respectively. N-desulphated heparin is not inhibitory. This effect requires the covalent binding of oleoyl residues to heparin and it decreases with increasing concentration of Tris-HCl and non-ionic detergents.     

Stimulation of human breast carcinoma cell invasiveness and urokinase plasminogen activator activity by glucose deprivation

Glucose deprivation has been shown to increase the invasive and metastatic potential of tumour cells. In the present study, we determined whether the enhanced tumour cell invasiveness resulting from glucose deprivation is linked to increased activity of enzymes required for extracellular matrix degradation. Results of in vitro invasion assays revealed that the invasiveness of human MDA-MB-231 and MCF-7 breast carcinoma cells and MCF-10A1 normal breast cells was, respectively, 3.9-, 2.9-, and 2.1-fold higher when they were incubated under glucose-deprivation (0.2 mM glucose) than when incubated under physiological blood glucose levels (5 mM). This effect of glucose deprivation on invasion correlated with increased urokinase plasminogen activator (uPA) and plasmin activity. Glucose deprivation did not increase the levels of gelatinase and plasminogen activator inhibitor-1 secretion, or the expression of cell-associated uPA receptor. To determine whether the increased invasiveness resulting from glucose deprivation is causally linked to increased uPA activity, invasion assays were conducted using MDA-MB-231 cells incubated in 0.2 mM or 5 mM glucose in the presence of a neutralising anti-uPA antibody. Results revealed that the anti-uPA antibody significantly inhibited invasion in a dose-dependent manner and to a much greater extent in cells incubated in 0.2 mM glucose than in cells incubated in 5 mM glucose. These results suggest that low glucose levels in malignant cancers increase tumour cell invasiveness by stimulating uPA and plasmin activity.     

A transitional state of pro-urokinase that has a higher catalytic efficiency against glu-plasminogen than urokinase.

Plasminogen activation by single-chain urokinase-type plasminogen activator or pro-urokinase (pro-UK) is accompanied by the generation of two-chain urokinase (UK) by plasmin which provides a positive feedback. In the present study, the time course of the activation of Glu-plasminogen and of Lys-plasminogen (10 microM) by pro-UK (1.0 nM) was studied. In the presence of native plasminogen (Glu-plasminogen), three distinct phases with different rates of plasmin generation were observed. The initial phase was slow and corresponded to the intrinsic activity of pro-UK as reflected by the activity of a plasmin-resistant mutant (Lys158----Ala). This was followed by a second phase which had the most rapid rate. The third phase had a plasminogen activation rate which was significantly slower than the second and paralleled the rate of activation by UK (1.0 nM). The second phase coincided with the time at which there was only about 50% conversion of pro-UK to UK, whereas the final phase coincided with essentially complete conversion. In the presence of fibrin fragment E-2 (20 microM), previously shown to strongly promote plasminogen activation by pro-UK, the identical phenomenon was observed, but at one-tenth the concentration of pro-UK. The most rapid rate of plasmin generation again coincided with transitional (25-60%) pro-UK to UK conversion. To further examine this phenomenon, the rate of pro-UK to UK conversion was controlled by using kallikrein in the presence of a plasmin inhibitor. In this experiment, the activation of Glu-plasminogen bound to solid-phase fibrin was measured. A similar three-phase sequence was observed, the highest rate of plasmin generation coinciding with about 45% conversion of pro-UK to UK. A mechanism for this transitional state phenomenon was postulated based on the established significantly higher affinity of pro-UK than of UK for Glu-plasminogen. This exceptional property for a proenzyme may enable a transient activity to be generated during the transition from pro-UK to UK corresponding to the more favorable KM of pro-UK and the kcat of UK. This hypothesis was supported by the results from experiments in which Lys-plasminogen was substituted for the Glu form. No transitional state activity was observed, consistent with the relatively high KM of pro-UK against Lys-plasminogen.     

Glucose- and concentration-dependence of vasopressin-induced hormone release by mouse pancreatic islets.

The effects of arginine-vasopressin (AVP) on hormone release by the endocrine pancreas have been studied with incubated islets from normal mice. A wide range of AVP concentrations (1 pM-100 nM) were tested in the presence of various glucose concentrations. AVP did not affect somatostatin release in a glucose-free medium but increased it in the presence of all tested glucose concentrations (3-30 mM). The lowest effective concentration was 1 mM and the effect was not yet maximal at 100 nM AVP. AVP markedly increased glucagon release in the absence of glucose. Its effect was attenuated but not abolished when glucagon release was inhibited by glucose. Surprisingly, the attenuation of the effect of AVP was stronger in 3-10 mM than in 15-30 mM glucose. The lowest effective concentration was 1 nM and the effect was not yet maximal at 100 nM AVP. AVP was ineffective on basal insulin release (0, 3 and 7 mM glucose), but potentiated the effect of 10, 15 and 30 mM glucose. The lowest effective concentration was 0.1-1 nM AVP and the maximal effect was produced by 10-100 nM AVP. The results suggest a direct action of AVP on each of the three islet cell types which display a roughly similar sensitivity to the peptide. This sensitivity is too low to make islet cells a possible target for circulating AVP under physiological conditions. On the other hand, the presence of AVP in the pancreas suggests that it might be involved in the peptidergic control of islet function.     

Effects of neuromedin B on insulin and glucagon release from the isolated perfused rat pancreas

The effect of neuromedin B (NMB) on insulin and glucagon release was studied in isolated perfused rat pancreas. Infusion of NMB (10 nM, 100 nM and 1 microM) did not affect the insulin release under the perusate conditions of 5.5 mM glucose plus 10 mM arginine and 11 mM glucose plus 10 mM arginine, although 10 nM NMB tended to slightly suppress it under the perfusate condition of 5.5 mM glucose alone. The degree of stimulation of insulin release provoked by the addition of 5.5 mM glucose to the perfusate was not affected by the presence of 10 nM NMB. The glucagon release was slightly stimulated by the infusion of 100 nM and 1 microM NMB but not by 10 nM NMB under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The effect of C-terminal decapeptide of gastrin releasing peptide (GRP-10) was also examined and similar results were obtained; 10 nM and 100 nM GRP-10 did not affect insulin release and 100 nM GRP-10 stimulated glucagon release under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The present results concerning glucagon release are consistent with the previous results obtained with isolated perfused canine and porcine pancreas. However, the results regarding insulin release are not. Species differences in insulin release are also evident with other neuropeptides such as substance P and the mechanism of such differences remains for be clarified.     

Intracellular localization of human monocyte associated interleukin 1 (IL 1) activity and release of biologically active IL 1 from monocytes by trypsin and plasmin

The production of interleukin (IL 1) by normal human peripheral blood monocytes purified by Ficoll-Hypaque density sedimentation, Percoll-gradient sedimentation, and plastic adherence can be detected as early as 30 min intracellularly, and extracellularly within 1 hr after stimulation with lipopolysaccharide (LPS). Production of mRNA coding for the isoelectric point 7.0 species of IL 1 was also detected as early as 1 hr after LPS stimulation and reached a maximum level at 6 hr. Cell-associated IL 1 activity could be extracted with CHAPS detergent from every cell fraction (i.e., membranes, cytosol, and particulates), but was present mainly (greater than 95%) in the cytosol of LPS-activated monocytes and the myelomonocytic cell line, THP-1. The apparent m.w. of IL 1 activity on high pressure liquid chromatography gel filtration in every cell fraction was approximately 23,000 daltons, with a minor peak at 31,000 daltons, whereas the IL 1 activity in the culture supernatants was 17,000 daltons. Western blotting analysis of LPS-stimulated monocyte extracts showed two forms of IL 1 corresponding to 31,000 daltons and 25,000 daltons. Exposure of viable cells to trypsin and plasmin released biologically active 23,000 dalton IL 1 only from IL 1-producing cells such as activated monocytes and IL 1-producing Ebstein-Barr virus B lymphocyte cell lines. Consequently, biologically active IL 1 is presumably exposed on the outer surface of cell membranes. Furthermore, IL 1 release by human monocytes in plasminogen-depleted fetal calf serum was considerably decreased. Conversely, supplementation of plasminogen-depleted serum with purified plasminogen restored the IL 1 production, suggesting that plasmin or plasmin-like factors may be involved in the regulation of the release of IL 1 from IL 1-producing cells. In conclusion, the results suggest that IL 1 is rapidly produced, is pooled in the cytosol, and in part is processed by enzymes, is transferred to the plasma membranes, and is then released from the cells. Tissue plasminogen activator and serum enzymes such as plasmin may therefore be involved in the release of IL 1 from IL 1-producing cells.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Vitamin K1 reduction in human liver. Location of the coumarin-drug-insensitive enzyme.

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.