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A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Brännström et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH.

A method is described by which successive expiratory tidal and minute volumes are measured by a flow-resistance device (pneumotachometer) which is automatically calibrated up to three times per minute by intermittent collection of gas in a spirometer. Records of any duration, at rest or during exercise, can be made without interruption. The method automatically corrects for the composition of gas flowing through the pneumotachometer, and is suitable for minute-to-minute or breath-by-breath studies of gas exchange or respiratory timing.

Non-uniform composition of gas in the lungs may arise because of uneven dilution of alveolar with inspired gas by convection (bulk flow) or because of the finite rates of molecular diffusion in the small air-spaces. Such non-uniformity represents 'mixing inefficiency' which may be expressed by the difference between the effective dead-space ('ventilatory dead-space', VDV) and the actual airway dead-space ('Fowler dead-space', VDF). This expression of mixing inefficiency does not distinguish between convective and diffusive mechanisms. We show, from simulated gas wash-out of an asymmetrical, two-compartment model of the lung, that a third dead-space ('wash-out dead-space', VDW) enables this distinction to be made: VDV-VDW reveals convective, and VDW-VDF diffusive, mixing inefficiency. The position of the branch point subtending the two compartments greatly affects the outcome, however. When the branch point is relatively peripheral, inefficiency is mainly diffusive; when it is relatively central, inefficiency is mainly convective. These findings indicate how mixing efficiency may be measured more selectively than at present in real lungs.

This study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the collagen gels. HFF cultured in Dulbecco's modified Eagle's (DME) medium supplemented with bovine serum albumin (BSA) and either endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices. HFF-mediated collagen contraction was inhibited by prostaglandins E1 or E2, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils.

Cultured cells were found to be highly useful for investigating intracellular storage of amphiphilic compounds using disobutamide as a model agent. To select types of cultured cells most suitable for investigations, cells of dog coronary artery muscle, rabbit aorta muscle, rat urinary bladder carcinoma, rat basophilic leukaemia, human skin fibroblasts, bovine aorta endothelium, Chinese hamster ovary tumour and mouse fibroblasts were incubated with 0, 1, 2, 4, 6, 8 and 10 x 10(-4)m-disobutamide for 24 hr. Cultures were examined in situ by phase light microscopy for the presence of clear cytoplasmic vacuoles, cell death (cell detachment), and for drug effect on confluency/cell count. Disobutamide induced vacuoles in all cell types except rat leukaemia. The drug induced cell death and reduction in confluency or cell count in cultures of all cell types except rat carcinoma and rabbit aorta muscle. Release of lactic dehydrogenase from cells confirmed the relative resistance of the rat carcinoma and rabbit cells, and susceptibility of rat leukaemia, to drug-induced cell death. By means of electron microscopy of rat carcinoma and rabbit cells, it was established that vacuoles were membrane-bound and their content was predominantly electron-lucent.

The dose-dependent effects of heavy metals on cell proliferation, collagen synthesis, and non-collagen protein synthesis were studied in early passage cultures of human synovial cells exposed to 1-100 microM concentration of gold, silver, mercury, cadmium or lead for 5 days. The incorporation of [3H]thymidine into trichloroacetic acid insoluble material was inhibited 50% by each of the heavy metals at concentrations between 1 and 10 microM. Gold, lead and mercury (10 microM) decreased the DNA content of the cultures by less than 15%; silver (10 microM) and cadmium (10 microM) resulted in decreased DNA content, which was attributed to cytotoxicity. A dose-dependent inhibition of [3H]proline incorporation into bacterial collagenase resistant (non-collagen) protein was observed after incubation with 10 microM mercury, lead and silver. During incubations with 10 microM gold and cadmium, collagenase resistant protein accumulation increased. All the heavy metals except for gold inhibited collagen accumulation to a greater extent than non-collagen protein accumulation. Gold (10 microM) stimulated the amount of collagen produced per cell, and the percentage of collagen to total protein was increased 50%. The rate of collagen accumulation in medium decreased during incubation with 10 microM silver, mercury, cadmium and lead. The stimulation of collagen synthesis may be a unique property of gold related to the therapeutic indices of gold, compared to other heavy metals, in rheumatoid arthritis.

Prolyl hydroxylase and an immunologically related protein (CRP) were purified from neonatal rabbit skin at various time periods following administration of 3H-leucine. The peak incorporation of label into prolyl hydroxylase was found to be 12 hours, while peak incorporation into CRP occurred within 2 hours. Semi-log plots of the loss of radioactivity from these protein pools against time indicated an apparent T 1/2 for prolyl hydroxylase of 78 hours, and a T 1/2 of CRP of 44 hours. Calculated Kd values indicate that that breakdown of active enzyme does not account for the amount of CRP found in tissues.

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.

Serum galactosylhydroxylysyl glucosyltransferase (S-Glu-Gal-Hyl-Tase), liver galactosylhydroxylysyl glucosyltransferase (L-Glu-Gal-Hyl-Tase), liver hydroxylysyl galactosyltransferase (L-Gal-Hyl-Tase), and liver prolyl hydroxylase (L-PH) activities were measured in rats during the development of CCl4-induced cirrhosis (0.2 ml of 33% CCl4 in light mineral oil two times weekly for 10 weeks followed by 6 weeks of no treatment). Serum and liver markers of collagen synthesis increased in a time-dependent manner reaching maximum activity at 6 weeks (S-Glu-Gal-Hyl-Tase, two times; L-PH, two times). These enzyme levels returned to normal during the 4-week recovery period. In a separate 4-week experiment, colchicine (10 micrograms/rat/day) was administered with CCl4. Colchicine prevented the increase in S-Glu-Gal-Hyl-Tase, L-Glu-Gal-Hyl-Tase, and L-Gal-Hyl-Tase induced by CCl4 and resulted in a smaller increase in L-PH. These results demonstrate that S-Glu-Gal-Hyl-Tase elevation occurs following CCl4 because of increased liver collagen synthetic activity and the hepatocellular injury produced by CCl4.

Prolyl hydroxylase [EC 1.14.11.2] was shown to be inhibited by an ultrafiltrate (less than 30,000 molecular weight) fraction isolated from skin and blood of neonatal and adult rabbits. This fraction also inhibited two other alpha-ketoglutarate requiring mixed function oxidases, lysyl hydroxylase [EC 1.14.11.4] and alpha-butyrobetaine hydroxylase [EC 1.14.11.1] but not the amine oxidase, lysyl oxidase. Purification of the skin ultrafiltrate on Sephadex G-25 demonstrated a peak of prolyl hydroxylase inhibitory activity which chromatographed at a molecular weight corresponding to approximately 3,000. Chromatography of a blood ultrafiltrate separated a similar peak of material which was inhibitory for prolyl hydroxylase.

The effects of the stable endoperoxide analog U46619 (U) on the regulation of prostacyclin (PGI2) formation and cyclic adenosine monophosphate (cAMP) were investigated in cultured bovine aortic endothelial (BAE) cells. Incubation of U (0.3, 3.0 and 30 microM) with BAE cells for 5 min results in a dose-dependent increase in PGI2. Cyclic AMP levels were not changed at 0.3 and 3.0 microM but were stimulated at 30 microM U. When cells were exposed to U for a second and third 5 min period, PGI2 formation at 0.3 and 3.0 microM U remained stimulated while at 30 microM, PGI2 was not increased. Five min incubation of BAE cells with the cyclooxygenase inhibitor indomethacin blocked the stimulation of PGI2 at all concentrations of U and also prevented the increase of cAMP levels at 30 microM. In cells prelabeled with 3H-arachidonate, U stimulated release of labeled products at 0.3 and 3.0 microM but not at 30 microM U. In cells treated with bradykinin in the presence of U, PGI2 production was stimulated at 0.3 and 3.0 microM but not 30 microM U. When cells were exposed to U and stimulated with PGI2 (with and without phosphodiesterase inhibition), U caused significant increases in cAMP. We conclude that incubation of BAE cells with U results in an initial dose-dependent increase in PGI2 formation. Cyclic AMP levels are increased at high concentrations of U. This increase in cAMP is mediated by the initial stimulated PGI2 and results in decreased PGI2 on further exposure to U. Data suggest that U stimulates phospholipase activity and, at high concentrations, inhibits phosphodiesterase.

Serum galactosylhydroxylysyl glucosyltransferase (S-GGT), liver galactosylhydroxylysyl glucosyltransferase (L-GGT), liver hydroxylysyl galactosyltransferase (L-GGal-T) and liver prolyl hydroxylase (L-PH) activities were measured in CF1 female mice each week for 10 weeks after infection with Schistosoma mansoni (50 cercariae per mouse). None of the enzyme activities measured were significantly increased in the first six weeks following the infection. However, the activities increased thereafter reaching a maximum of seven-fold for S-GGT, five-fold for L-GGT, three-fold for L-GGal-T and ten-fold for L-PH compared with control animals. The increase in S-GGT activity was positively correlated with L-GGT (r = 0.788), L-GGal-T (r = 0.774) and L-PH (r = 0.800). The present data suggest that during the first six weeks of the development of murine hepatic schistosomiasis mansoni infection, few eggs bombard the liver. The biochemical changes during this period are mild. During weeks 7 to 10, which is the active fibrotic stage, the liver biochemical markers of collagen synthesis increase rapidly and lead to fibrotic morphological features that are the chronic irreversible hepatic changes characteristic of this infection. The data reported here indicate that the aggressive fibrotic changes in liver during weeks 7 to 10 of murine schistosomiasis infection are predicted by S-GGT activity.

Prolyl hydroxylase activity was determined in liver biopsy samples obtained from 10 patients. The liver prolyl hydroxylase values in patients with active hepatitis distribute into two numerical populations based on the extent of elevation over control. The first of these groups includes those with enzyme levels elevated approximately 2.5-fold over normal. Included in this group are patients with active (but nonagrressive) hepatitis and patients where advanced portal fibrosis is already established. The second group where prolyl hydroxylase is elevated approximately nine-fold is comprised of two patients with advanced clinical symptoms of active alcoholic hepatitis with evidence of aggressive cirrhosis but with only early minimal evidence of existing fibrosis.

The in vitro removal of six barbiturates from pH 2 donor solutions by liquid membranes with pH control was evaluated. More than 90% of amorbarbital, phenobarbital, and secobarbital were removed within 10 min by the liquid membranes. Drug transport obeyed first-order kinetics initially, and Fick's law was obeyed. The transport rate of phenobarbital by a liquid membrane was temperature dependent. An Arrhenius plot revealed that the activation energy was 10 kcal/mole. The liquid membranes showed some instability in the presence of bile salts.

Hepatic prolyl hydroxylase activity and collagen synthesis were measured in patients with alcoholic liver disease to determine the feasibility of using the enzyme prolyl hydroxylase as a marker of hepatic fibrogenesis. Alcoholic patients with liver histopathology consistent with normal, steatosis, alcoholic hepatitis, early cirrhosis, or advanced cirrhosis were analysed for liver prolyl hydroxylase activity and in vitro collagen synthesis. Prolyl hydroxylase activity and the rate of in vitro collagen synthesis were correlated when these parameters were measured in samples of the same liver biopsy. Mean prolyl hydroxylase activity was significantly raised in all groups of alcoholic patients with alcoholic liver disease, except those with steatosis, when compared with alcoholic patients with normal morphology. Alcoholic patients with early cirrhosis had enzyme activity (mean +/- SE: 1.367 +/-0.162 mU/mg protein) significantly raised over all other groups. Mean enzyme activity was less raised (0.985 +/- 0.097 mU/mg protein) in patients with advanced cirrhosis. The percentage of collagen synthesis in patients with early or advanced cirrhosis was also raised compared with alcoholic patients with normal morphology. Prolyl hydroxylase activity and the rate of collagen synthesis are significantly correlated (r=0.62). These findings suggest that hepatic prolyl hydroxylase activity is a useful indicator of hepatic fibrogenesis and its measurement on available liver biopsy tissue should be a potent diagnostic tool reflecting active fibrogenesis and predicting progression of alcoholic liverdisease.

These studies, conducted on early passage synovial cell mono-layers (derived from explant cultures of tissue obtained from patients with rheumatoid arthritis), have established that gold sodium thiomalate (GST) exposure results in dose-dependent changes in cell proliferation and protein synthesis as a consequence of the cellular accumulation of gold. The amount of gold found in the cell layer is correlated with the degree of inhibition of [3H]thymidine incorporation. Gold remains in the cell layer of treated cells after they have been subcultured twice in the absence of GST. Exposure of cells to a concentration of GST of 100 muM for 4 days results in 50% inhibition of [3H]thymidine incorporation. This antiproliferative effect is reversible at concentrations of 10 muM GST or less. Only partial recovery is observed after exposure to higher concentrations of GST which may be related to retained gold. The amount of collagen and noncollagen protein synthesized per cell increases at concentrations of GST of 10 muM and below but decreases with concentrations above 10 muM. A dose-dependent decrease in protein synthesized per flask and a decrease in the commitment to synthesize collagen relative to total protein synthesis follows exposure to GST in excess of 10 muM for 20 days which recovers partially after synovial cells are grown in GST-free medium for 10 days. An observed decrease in the percentage of type III collagen synthesized by synovial cells after GST exposure was not observed in cells grown in GST-free medium for 5 days after exposure, indicating that this effect of collagen synthesis is reversible. The reversible biochemical changes resulting from the exposure of cultured human synovial cells to GST are discussed as a mechanism of action of this drug on the proliferative synovitis that characterizes diseases such as rheumatoid arthritis.

Synthesis of collagen in the vasculature and other tissues of normotensive rats is markedly reduced by reserpine. Hypophysectomy produces similar effects. The effects of reserpine on collagen biosynthesis may be mediated through the hypothalamus.

Human mononuclear cell supernatants were obtained by incubating 3 X 10(6) cells per ml of Dulbecco's modified Eagles medium at 37 degrees C for 24 h or 48 h, either in presence or absence of phytohaemagglutinin. After removal of intact cells, the supernatants were dialysed and diluted (1:1, 1:2, 1:5, 1:10) again using the above medium. The diluted supernatants, containing a final concentration of 10% (v/v) human platelet-factor poor serum, were found to stimulate the proliferation of human synovial cells in culture and to increase both the total amount of collagen and the percentage of Type III collagen synthesized by these cells. Incubation of the mononuclear cells in presence of phytohaemagglutinin appeared to further enhance the stimulatory effects of the supernatants upon the synovial cells. Since activated mononuclear leucocytes such as lymphocytes and macrophages are present in rheumatoid synovia, this study suggests that factors released from activated mononuclear leucocytes may play an important role in the proliferation of rheumatoid synovial tissue and development of the pannus.

The contribution of collagen degradation, as measured by collagenase activity, to the accumulation of collagen during hepatotoxic fibrogenesis was examined using a carbon tetrachloride rat model. Both active and inactive enzyme forms were determined with the inactive form quantitated following activation by limited trypsin digestion of the liver homogenate. The rate of collagen biosynthesis was monitored by quantitating hepatic prolyl hydroxylase activity and accumulation of collagen. In one study CCI4 was administered twice weekly, i.p. (0.2 mL, 33% v/v in light mineral oil) for sixteen weeks, with animals sacrificed every two weeks. Histologic examination of liver sections and serum alanine transaminase levels indicated a progressive necrosis and fibrosis which was confirmed by the increase in hepatic hydroxyproline content from 0.303 to 4.84 micrograms/mg wet wt. tissue. Prolyl hydroxylase activity increased in a time dependent manner to a maximum of 3.7 X control. This increase was accompanied by a large increase in both active collagenase (15.0-40.8 mU/g protein) and latent collagenase activity (69.8-191.7 mU/mg protein). These increases were maintained through the transition to irreversible fibrosis. The increase in active collagenase activity was positively correlated with collagen content. A second short term CCI4 treatment study confirmed a transient alteration in the active to latent collagenase ratio early in the fibrotic process. These results demonstrate the dynamic changes in hepatic collagenase levels and indicate that the fibrotic lesion is not dependent on decreased collagenase levels for collagen accumulation.

Cloned bovine aortic endothelial cells (BAEC) were grown to confluence then treated for 24 hours with dibutyryl cyclic AMP (dbcAMP) or dibutyryl cyclic GMP (dbcGMP) in serum free medium. Submillimolar concentrations of dbcGMP caused a significant enhancement of thromboxane (TXB2) synthesis in washed cells exposed to arachidonate. DbcAMP had no effect on the production of either metabolite. TXB2 synthesis was inhibited by 3 micrograms/ml cycloheximide, whether or not the cells were pretreated with dbcGMP. Prostacyclin production was inhibited to a much lesser extent by cycloheximide. We conclude that dbcGMP elevates TXB2 production by increasing the amount of thromboxane synthetase available, and that this effect is inhibited by cycloheximide. Data are described which suggest that dbcGMP increases the degradation rate of TXB2 by BAEC, so that the observed increase in TXB2 may be an underestimate of the true effects of dbcGMP on TXB2 production.

Collagen synthesis is increased in the aortas, mesenteric arteries, and to a lesser extent, in the hearts of rats either made hypertensive with desoxycorticosterone acetate-salt or that are spontaneously hypertensive. Several markers of collagen biosynthesis were shown to be increased, including prolyl hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase), prolyl hydroxylase-related antigen, total collagen content, and the incorporation of [(3)H]proline into total protein and into collagen. The antihypertensive agents chlorothiazide and reserpine, when administered before the onset of hypertension in the rats treated with desoxycorticosterone acetate-salt, prevented or diminished the increase in collagen biosynthesis. When reserpine was given after the onset of hypertension, prolyl hydroxylase activity was decreased concomitant with the decrease in blood pressure. Treatment with reserpine is particularly effective in diminishing arterial prolyl hydroxylase activity.

Increased aortic and liver prolyl hydroxylase activity has been suggested as an early biochemical indicator of the fibrotic changes which occur in rabbits with injury induced arteriosclerosis. Daily administration of epinephrine (0.025-0.050 mg/kg, i.v.) and thyroxine (0.050 mg/kg, i.p.) to rabbits for 3 weeks produced aortic fibrous plaques with a 4-fold increase in aortic prolyl hydroxylase and also a 5-fold increase in liver prolyl hydroxylase. Histopathologically, the livers of these rabbits show subcapsular areas of necrosis. When total prolyl hydroxylase related antigen was measured. the increase in liver prolyl hydroxylase activity accounted for only a small portion of the total prolyl hydroxylase antigen. However, in the aorta a majority of the increase in antigen is due to the increased amount of enzyme. DNA content per aorta was unchanged and RNA content increased in the aortic tissue of the arteriosclerotic rabbits. However DNA and RNA levels increased 60% in the livers of arteriosclerotic rabbits. In vitro incorporation of radioactively labeled proline into collagenase digestable protein was at least 2-fold greater in aorta and liver minces from arteriosclerotic rabbits. Michaelis--Menten kinetic parameters were obtained for the liver prolyl hydroxylase purified by affinity chromatography from arteriosclerotic rabbits. The Km for the enzyme from treated animals was not significantly different from control. However, the Vmax of the enzyme purified from diseased liver was 4-fold greater when compared to controls.

Smooth muscle cells released by protease from the coronary artery of dogs were cultured in M199 supplemented with 10% fetal calf serum. Cells were grown on glass coverslips to semiconfluence and exposed to disobutamide at 0, 1, 2, 3, 6, 8, and 10 X 10(-4) M for 24, 48, and 72 h, examined in situ by light microscopy, then fixed in 100% methanol, stained with May-Grünwald Giemsa and examined by light microscopy. Cells were also exposed to drug for 24 h, pelleted, fixed and prepared routinely for electron microscopy. Control cells and cells exposed to 6 X 10(-4) M disobutamide were examined. Vacuole formation was dose and time dependent between 2 X 10(-4) M and 10(-3) M. By light microscopy, morphologic alterations induced by the drug were clear cytoplasmic vacuoles in live cells, vacuoles in dead cells and dead cells without vacuoles. Small round vacuoles were an early change. Dark granules were dispersed among the vacuoles. The vacuoles increased in size at higher doses or longer times. Eventually all the cytoplasm was occupied by vacuoles and the cells were enlarged. By electron microscopy, the vacuoles were round, primarily membrane-bound, contained mostly electron-lucent material and occasionally small flocculent bodies. There was vacuolar coalescence. The dose response of vacuole induction in confluent and semiconfluent cells was similar. Cytoplasmic vacuoles without cell death can be induced by disobutamide at 2 or 4 X 10(-4) M during a 3-day exposure. Cell culture is a suitable biological system for studying cytoplasmic vacuoles of the type induced by disobutamide.

The invasive activities of some malignant cells appear to be activated by contact with laminin. This protein occurs solely in basement membranes and the binding of malignant cells to the surface of this extracellular matrix initiates the invasion process. Passage of the cells across basement membrane requires degradative activity and laminin induces the production of collagenase IV which lyses the collagen IV network. The motility of the cells is enhanced by chemo-attractants and by matrix molecules. Peptides that inhibit the binding of tumour cells to laminin, inhibitors of collagenase IV, and inhibitors of specific pathways of arachidonic acid metabolism prevent invasion as well as the metastasis of malignant cells and could be employed to stop the spread of cancer.

Synovial cells derived from patients with either rheumatoid arthritis, or simple joint-trauma were grown in tissue culture. The rheumatoid osteoarthritic and non-arthritic synovial cells in cultured all had similar levels of prolyl hydroxylase activity. Following a 3 hour incubation with ascorbate (10(-4)M), prolyl hydroxylase activity was elevated to a similar extent in all synovial cell cultures examined. The activation of prolyl hydroxylase by ascorbate (10(-4)M) was accompanied by increased radioactive hydroxyproline formation and secretion into the media. Increased amounts of collagenase degradable radioactive protein were also secreted into the media, but no changes in total collagen synthesis (media plus cell layer) were observed as a result of ascorbate supplementation using this assay system.

This rapid, sensitive method of determining collagenase (EC 3.4.24.7) activity incorporates several advantages of previous methods. Soluble [14C]acetylated collagen is prepared as the enzyme substrate and collagen-cleavage products are separated from noncleaved collagen by precipitation with dioxane/methanol. The assay is more reproducible than previous methods and has a lower detection limit, 15 mU of enzyme activity. We used the method in a competitive substrate assay with isolated extracellular hepatic matrix from cirrhotic and normal rat liver. Purified collagenase was consistently bound to normal rat matrix to a greater extent than to cirrhotic matrix, suggesting that in hepatic fibrosis the extracellular matrix is not as susceptible to collagenase degradation as that in normal liver.

The activities of prolyl hydroxylase (Pro-OHase), galactosylhydroxylysyl glucosyltransferase (Glu-Gal-Hyl-Tase), and hydroxylysyl galactosyltransferase (Gal-Hyl-Tase) were assayed in lung tissues of hamsters with bleomycin-induced experimental pulmonary fibrosis. Serum Glu-Gal-Hyl-Tase and aspartate transaminase (Asp-NH2-Tase) were measured in the same animals. Lung fibrosis was induced by intratracheal bleomycin instillation, and the enzyme activities were assayed 2, 3, and 4 weeks after bleomycin administration. The activities of the three lung enzymes increased significantly after bleomycin instillation. However, no difference in the values of serum Glu-Gal-Hyl-Tase or Asp-NH2-Tase were observed. Histologic examination of lung sections indicated progressive fibrotic foci. These results thus indicate that the activities of collagen processing enzymes are elevated in the fibrotic lung tissues as a reflection of the increased rate of collagen synthesis during the period of active fibrogenesis, but unlike liver fibrosis, the elevation of tissue Glu-Gal-Hyl-Tase is not predicted by a corresponding increase in serum levels of this enzyme.

Prostacyclin has been used as a substitute for heparin in renal dialysis because of its potent antiplatelet activity and its short half-life. However, it is difficult to use because of its instability in neutral buffer and its hypotensive activity at antithrombotic doses. SC-39902, a 5-F prostacyclin analogue, has an ED50 of 17.7 micrograms/kg i.v. against ADP-induced thrombocytopenia in rats. The ED25 for hypotensive activity is 7.0 micrograms/kg i.v. The therapeutic index (blood pressure ED25/antiplatelet ED50) of 0.40 is 3.6 times better than that of PGI2 (0.11). SC-39902 supports clot-free dialysis in dogs at 4.0 micrograms/kg/min (ED100 for dialysis) with a 26% change in blood pressure at the 100% effective dose as compared with 0.052 microgram/kg/min required for PGI2 with a 32% change in blood pressure. The authors conclude that SC-39902 will provide the desirable antiplatelet/antithrombotic activities of PGI2 without instability and buffer incompatability problems. However, at the ED100 for dialysis, SC-39902 does have haemodynamic activity and, thus, a therapeutic index which is greater than four times that of PGI2 in rats is required for a PGI2 analogue to have less hypotensive activity during dialysis.