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Aims: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease with unclear etiology. Different receptors play a role in the pathophysiology including protease activated receptors (PARs). The present study aimed to investigate the subtypes and the effects of PARs on contractility using permeabilized detrusor smooth muscle strips in IC/BPS., Main Methods: IC/BPS was induced by cyclophosphamide injection. Histopathological analysis, PCR for detecting PAR proteins, western blotting for indicating PAR2 protein expression levels and myograph recording for measuring contractile force were used., Key Findings: The present study reveals that in rat bladder PAR1 and PAR2 but not PAR4 were found to be expressed. The first evidence was revealed where trypsin-induced contractions in rat permeabilized detrusor were potentiated in CYP-induced cystitis. Moreover, the functional inhibition of trypsin-induced contractions by selective PAR2 antagonist (ENMD-1068) and the supporting immunoblotting results emphasized that the main PAR subtype involved in IC/BPS model in rat bladder is PAR2. Our data emphasize the prominent role of IP 3 in cystitis pathology besides ryanodine channels. Trypsin-induced Ca 2+ sensitization contractions were also higher in cystitis. Both Rho kinase and protein kinase C played a role in this increased Ca 2+ sensitization situation., Significance: The present paper highlights the intracellular pathways that are involved in trypsin-induced contractions mainly via PAR2 in permeabilized bladder detrusor smooth muscle in a rat model of IC/BPS., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The snake venom may be considered as a potent source of untapped therapeutic proteins and peptides. The peptide mass fingerprinting and N-terminal sequence alignment of a 6.9kDa peptide named Rusvikunin from Daboia russelii russelii venom show the presence of putative conserved domains of the KU superfamily. Further, BLAST analysis of two of the de novo peptide sequences of Rusvikunin demonstrates significant sequence homology with serine proteases reported in the NCBI database. Rusvikunin possesses conserved cysteine residues and Arg15 at the P1 position. It inhibits amidolytic activity of trypsin (IC50=50nmol/l), plasmin (IC50=1.1μmol/l), and fibrinogen clotting as well as plasma clotting activity of thrombin (IC50=1.3μmol/l); however, it does not inhibit the amidolytic activity of chymotrypsin, thrombin, factor Xa, and tissue plasminogen activator. Rusvikunin is a glycoprotein, demonstrates dose-dependent BAEE-esterase activity. It does not show lethality in mice or in vitro cytotoxicity against mammalian cells but shows in vivo anticoagulant activity 6h after i.p. injection in the mouse model. The commercial polyvalent and monovalent antivenom failed to inhibit the functional properties of Rusvikunin. The possible biomedical applications of Rusvikunin in the treatment and/or prevention of cardiovascular disorders such as thrombosis and trypsin-induced inflammation are suggested., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Acute experimental pancreatitis induced by injection of trypsin into the pancreatic tissue exhibited characteristics of fulminant hemorrhagic pancreonecrosis (intense exudation of interlobular stroma, massive plasmo- and hemorrhages, foci of acinar cell autolysis involving by the end of day 1 an appreciable portion of the organ with formation of fields of necrosis and hemorrhagic imbibition of the glandular parenchyma, virtually completely without demarcation cellular reaction). Marked microcirculatory disorders and degenerative and necrobiotic changes in the duodenal mucosa and liver reflected the polyorgan nature of the pathological process. This model of hemorrhagic pancreonecrosis corresponded to the most severe forms of this conditions observed clinically.

This study investigated the effects of pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K)γ in the pruriceptive, inflammatory, and nociceptive responses induced by trypsin in mice. The animals were orally treated with the selective PI3Kγ inhibitor AS605240 (0.3-30 mg/kg) 30 minutes beforehand. In separate groups, AS605240 was given by intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes. The control groups received saline at the same schedules. The effects of PI3K blocking were assessed in different experimental assays. The oral treatment with AS605240 produced a marked reduction of scratching behavior elicited by trypsin. Moreover, AS605240 (1mg/kg) was able to produce a partial but significant inhibition of the scratching bouts elicited by CP 48/80. Interestingly, the i.c.v. and i.t. injection of AS605240 also reduced trypsin-induced itching. The oral administration of AS605240 was found effective in producing a significant and dose-dependent reduction of trypsin-induced paw edema and tumor necrosis factor α production, as well as the neutrophil recruitment, according to myeloperoxidase activity assessment. Likewise, oral AS605240 (1mg/kg) promoted a significant reduction of spontaneous nociception induced by trypsin in the mouse paw. In contrast, the oral administration of AS605240 did not significantly modify capsaicin-evoked nociception, although this inhibitor was effective when dosed by i.c.v. route. Noteworthy, AS605240 (1mg/kg) was able to prevent c-Fos and phospho-Akt immunopositivity at the spinal cord of trypsin-injected mice, either into the back of the neck or the paws. To conclude, PI3Kγ inhibition might well represent a valuable alternative for treating inflammatory and painful conditions, as well as pruritus., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

The present investigation was carried out to study the effect of rebamipide in experimentally induced bronchial asthma in mice. Trypsin and egg-albumin induced chronic model of asthma was used and various parameters were measured on the 35th day. The asthmatic control group showed lower level of haemoglobin saturation with oxygen, tidal volume, airflow rate and higher respiratory rate, serum bicarbonate level, eosinophil count in bronchoalveolar lavage fluid and histamine level compared to the normal control group. Dexamethasone and rebamipide treated groups showed the return of all the above parameters towards normal values. Histopathological examination of lungs showed more prominent alveolar and muscular layer destruction in the asthmatic control group than in dexamethasone and rebamipide treated groups. Rebamipide showed a beneficial effect and might be used for the treatment of bronchial asthma.

The consistently high proportion of early deaths in patients with severe acute pancreatitis (SAP) has been associated mainly with the development of multiple organ dysfunction syndromes (MODS). So far, scholars believed that the main reasons of MODS with SAP are systemic microcirculation dysfunction and inflammatory mediator induced cascading effect on the basis of pancreas digesting itself. However, there is some special pathological phenomenon in the process of SAP which could not be explained by current theories. First, it has been evident that the pancreatic tissue bleeding and necrosis is special pathological change in pancreas autodigestive effect from digestive enzymes such as trypsin in SAP. However, we found that the liver, the lung, the intestine, the brain and the kidney have the same pathological changes in experimental animal models of SAP. Secondly, unlike the general inflammatory response, a significantly amount of bloody ascites and pleural effusion was often in patients with SAP and in experimental SAP animal models. It indicates that the vascular permeability significantly increased leading to the red blood cells extravasation. Thirdly, apart from dual blood supply, liver bears a strong compensatory function. However, liver has the highest incidence of injury in SAP when compared with other organs. In addition, we found a very interesting phenomenon after reading texts and clinical records. From the pancreatic venous drainage from the point of view, the farther the organ from the pancreas, the lower injury incidence rate observed. How to explain these mysteries? We postulate that the trypsin is the culprit of multiple organs dysfunction in SAP. The activated trypsin destroy the pancreas itself, causing pancreatic tissue bleeding and necrosis, at the same time, through venous flow it flow into the blood circulation and destroy the vascular endothelial barrier, leading to highly increased vascular permeability. So, a large number of bloody exudates leakages from the vessels, resulting in patients early circulatory disorders, multiple organ bleeding, bloody pleural effusion and ascites. This pathological change is the most apparent in the liver because the liver is the first organ to receive the pancreatic venous flow having the highest concentration of trypsin. Thus, if the quantity of trypsin decreases in blood, its ability to damage other organs also shows a trend of gradually reducing. These mysteries can be explained by this hypothesis and might help us to understand more clearly about the mechanism of SAP-associated MODS.

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.

In mice and humans, a normal offspring can be obtained by injecting a single spermatozoon into an oocyte, the process called intracytoplasmic sperm injection (ICSI). When three or more mouse spermatozoa with intact acrosomes were injected into individual mouse oocytes, an increasing proportion of oocytes became deformed and lysed. Oocytes did not deform and lyse when acrosome-less spermatozoa were injected, regardless of the number of spermatozoa injected. Injection of more than four human spermatozoa into a mouse oocyte produced vacuole-like structures in each oocyte. This vacuolation did not happen when spermatozoa were freed from acrosomes before injection. Hamsters, cattle, and pigs have much larger acrosomes than the mouse or human. Injection of a single acrosome-intact hamster, bovine, and porcine spermatozoon deformed and lysed many or all mouse oocytes. This deformation did not happen when these spermatozoa were freed from acrosomes before ICSI, regardless of the number of spermatozoa injected. Because trypsin and hyaluronidase mimicked the action of acrosome-intact spermatozoa, it is likely that the acrosomal enzymes deform and lyse the oocytes. Injection of small amounts of trypsin and hyaluronidase into normally fertilized mouse eggs disturbed their pre- and postimplantation development. In view of potentially harmful effects of acrosomal enzymes on embryo development, the removal of acrosomes before ICSI is recommended for animals with large sperm acrosomes. The removal of acrosomes may increase the efficiency of ICSI in these animals. Although human and mouse spermatozoa do not need to be freed from acrosomes, the removal of acrosomes before ICSI is theoretically preferable.

Enteral administration of oxymethyluracil in non-inbred albino rats decreased the inflammatory reaction to histamine, serotonin, bradykinin, carrageenan and trypsin, (being similar to voltaren) and reduced granulomatous inflammation, but influenced the proliferation to a lower extent than did voltaren. In contrast to voltarcen, oxymethyluracil also exhibited gastroprotective antiulcer properties.

Human keratinocytes are known to express the protease-activated receptors, PAR-1 and PAR-2. Activation of PAR-1 results in increased proliferation, whereas PAR-2 activation results in decreased keratinocyte proliferation. Trypsin activates PAR-1 and in higher concentrations, PAR-2. The aim of this study was to evaluate the overall effect of trypsin on keratinocyte proliferation in a mouse in vivo and in vitro model. Daily topical application of 0.3-300 pmol trypsin/cm2 on hairless mouse skin induced dose-dependent epidermal hyperproliferation as determined by an increase in 5-bromo-2'-deoxyuridine incorporation of up to eight-fold in basal keratinocytes and an up to three-fold increase in keratinocyte layers. This was accompanied by an increased transepidermal water loss. These effects of trypsin were abolished by the addition of the trypsin inhibitor n-p-tosyl-l-lysine-chloromethyl ketone. Histological analysis revealed acanthosis, hypergranulosis, and spongiosis in the epidermis as well as vasodilatation and an inflammatory infiltrate in the upper dermis. In the murine keratinocyte cell line PAM-212 activation of PAR-1 with specific activating peptides resulted in a calcium influx and an increase of proliferation, whereas activation of PAR-2 caused a diminished proliferation. Incubation with trypsin, PAR-1-, and PAR-2-activating peptides induced cytokine-induced neutrophil chemoattractant (KC) mRNA expression as a marker for inflammation in PAM-212 in a dose-dependent manner. In conclusion, our results suggest that trypsin induces in vivo epidermal proliferation and inflammation. Proliferation seems not to be signaled by PAR activation, but PAR-2-induced KC chemokine expression may contribute in part to trypsin-induced inflammation.

Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.

The catalytic activity of type II phospholipase A2 (PLA2) in blood has been reported to increase in acute pancreatitis and to reflect the severity of pancreatitis. In this study, we evaluated the effects of a new inhibitor of type II PLA2, S5920/LY315920Na, on trypsin-taurocholate-induced pancreatitis in rats. Hemorrhagic pancreatitis was induced by an infusion of a mixture of trypsin and taurocholate into the pancreatic duct. S5920/LY315920Na was administered subcutaneously at 0 h and 3 h after the induction of pancreatitis. Survival rates for 24 h in rats treated with 0.1 and 1 mg/kg of S5920/LY315920Na were significantly higher than that in untreated rats (71 and 86% vs. 14%). Serum levels of amylase and lipase in rats treated with 1 mg/kg of S5920/LY315920Na were significantly lower than those in untreated rats (amylase, 6,903 vs. 32,516 U/L; and lipase, 514 vs. 6,710 U/L) at the time of death or 24 h after the induction of pancreatitis. Plasma levels of S5920/LY315920Na were enough to inhibit catalytic activity of PLA2 in plasma for 9 h. A new inhibitor of type II PLA2, S5920/LY315920Na, inhibited catalytic activity of PLA2 and improved the survival rate in experimental hemorrhagic pancreatitis in rats.

The effect of methylcarbonylmethyl 2(S)-14-(4-guanidino-benzoyloxy) phenyl] propionate methanesulfonate (TT-S24) on experimental pancreatitis in rats was examined in comparison with that of camostat. TT-S24 showed a preventive effect on increases in plasma amylase activity and pancreatic weight induced by cerulein injection. TT-S24 also reduced an increase in plasma amylase activity induced by taurocholate. TT-S24 effectively prevented the mortality induced by an injection of a mixture of trypsin and taurocholate. TT-S24 showed no effect on an increase in amylase activity 6 h after duodenum ligation (closed duodenal loop pancreatitis), indicating that the drug had no effect on the initiation and propagation step of closed duodenal loop pancreatitis. On the other hand, TT-S24 reduced an increase in amylase activity 6 h after release of the duodenum ligation. TT-S24 showed anti-trypsin, anti-kallikrein, anti-thrombin and anti-plasmin activities. The effect of TT-S24 on some experimental pancreatitis models was nearly equal to or somewhat more potent in most instances to that of camostat. Therefore, TT-S24 should be useful in the clinical treatment of pancreatitis.

This study investigated the possible beneficial effect of intraperitoneal proteolytic enzyme administration on the development of hypertension-induced renal injury in the rat model of 2-kidney 1-clip (2K1C) Goldblatt hypertension. Male Wistar rats (120-150 g) underwent either sham surgery (control, n = 5) or clipping of the left renal artery. From day one 2K1C rats were randomized into 2 groups, placebo treatment (n = 7), and proteolytic enzyme treatment (n = 9). To the verum group a fixed mixture of trypsin (2.42 mg), bromelain (4.54 mg), and rutin (5.04 mg) dissolved in 2 ml of sterile 0.9% NaCl was administered intraperitoneally daily, while the placebo group received only vehicle. Rats were pair-fed. The duration of the study was 7 weeks. All 2K1C rats developed hypertension and the mean values of systolic blood pressure (SBP) did not differ significantly between the groups at any time recorded (SBP at sacrifice: controls 122.0 +/- 8.5 mm Hg; placebo 191.4 +/- 7. 6 mm Hg; enzyme 180.5 +/- 6.5 mm Hg). Enzyme treatment prevented the rise in proteinuria (controls 12.4 +/- 2.6 mg/24 h; placebo 19.7 +/- 3.9 mg/24 h; enzyme 12.2 +/- 1.3 mg/24 h; p < 0.05) and ameliorated the increase in serum urea concentrations. Histomorphologically, signs of malignant nephrosclerosis were not found in control rats, while they were present in 4/7 (57%) of placebo-treated rats, but only in 1/9 (11%) of the enzyme-treated group. The volume fraction of renocortical interstitium was increased in both 2K1C groups in comparison with controls; however, enzyme treatment decreased the accumulation of interstitial tissue significantly (-22%) compared to placebo treatment. Cellular infiltration with mononuclear cells was also lower in the protease-treated group. To summarize, in the rat model of 2K1C hypertension, systemic treatment with proteases ameliorates the severity of nephrosclerosis and tubulointerstitial fibrosis in the non-clipped kidney, as well as proteinuria, without affecting high blood pressure.

This study investigated whether protease treatment ameliorates the progressive course of chronic failure in the rat model of subtotal nephrectomy. Fourteen male Wistar rats underwent 5/6 nephrectomy, and were randomized into a control group (C, n = 7) given 2 ml of 0.9% NaCl intraperitoneally (i.p.) daily, and a study group (P, n = 7) treated with 12 mg Phlogenzym (combination of trypsin, bromelain, and rutosid) in 2 ml saline i.p. daily. After 6 weeks treatment, the Phlogenzym group showed lower proteinuria (C: 19.6 +/- 9.1 vs. 10.2 +/- 6.2 mg/24 h, p < 0.05). Endogenous creatinine clearance was higher (C: 192.3 +/- 99.4, P: 300.5 +/- 47.9 microliters/min per 100 g, p < 0.05), while plasma creatinine was decreased (C: 106.7 +/- 33.9, P: 76.0 +/- 6.3 mumol/l, p < 0.01). Blood urea nitrogen levels did not change, although urea clearance tended to a higher level in the protease-treated rats. Decreased renal formation of cytokines was reflected by a lower urinary excretion ratio of transforming growth factor (TGF)-beta/ creatinine (C: 0.363 +/- 0.183, P: 0.232 +/- 0.085 ng TGF-beta/mg creatinine, p < 0.05). Renal morphology revealed less infiltration of mononuclear cells and an amelioration of interstitial fibrosis as expressed by the volume index of the cortical region (C: 17.17 +/- 1.43; P: 12.3 +/- 0.5%, p < 0.001). In addition, the activities of lysosomal proteinases (cathepsin B, L + B, and H), which are decreased in the remnant kidney model of chronic renal failure, were significantly higher in the enzyme-treated group both in isolated glomeruli and proximal tubules. The body and kidney weight tended to be lower, probably due to a catabolic action of the enzymes. In summary, we provide evidence that protease treatment may be beneficial in a nonimmune mediated renal disease. Phlogenzym ameliorated the course of chronic renal failure in the rat model of subtotal nephrectomy and retarded the development of tubulointerstitial fibrosis. Decreased cytokine formation in the remnant kidney is supposed to play a key role.

Intravenous injection of trypsin in the rat induces early lung leucostasis and emphysema of delayed onset. This report confirms that this emphysema is not rat-specific and that the leucostasis is not related to the presence of contaminating endotoxin in the trypsin. In hamsters (n = 37), leucostasis did not occur when they were injected with heat-treated trypsin, but numerous granulocytes were sequestered in the vessels of hamsters receiving a fresh solution of trypsin. In these hamsters, the number of granulocytes harvested by lavage increased significantly (1.87 x 10(6) per ml, P < 0.001) compared with hamsters injected with either heat-denatured trypsin (0.89) or saline (0.86), or compared with controls (0.86). Emphysema was inconstantly observed in hamsters 6 or 12 weeks after injection with trypsin for 1 h. It was frequently (17/20) present and always (20/20) well developed (intercept + 180 per cent) in the 2-h perfused hamsters whose lungs were abnormally heterogeneous (index + 100 per cent) relative to the seven controls and to the nine saline-injected hamsters. The efficiency of trypsin in triggering emphysema (percentage of hamsters having abnormal values of intercept) was dependent on the time of perfusion. This form of experimental emphysema is thus considered to be due to an endotoxin-independent leucostasis.

Intravenously infused bovine trypsin, 75 mg kg-1 during 3 h, induced shock in rats which proved lethal. After 5 h, all the rats had died. In another group of rats receiving trypsin, the pancreatic secretory trypsin inhibitor, 75 mg kg-1, was infused during 5 h. These rats all survived. After about 1 h, alpha 1-macroglobulin was found to be saturated in both groups, while kininogen cleavage, coinciding with a decline in arterial blood pressure, only occurred in the untreated group. Trypsin complex formation with alpha 1-inhibitor 3 and alpha 1-proteinase inhibitor was more pronounced in untreated rats. It is concluded that the pancreatic secretory trypsin inhibitor may function as an effective trypsin inhibitor in plasma.

Monkey kidney epithelial cells, labeled with chromium and grown in culture, were killed in a synergistic manner when subtoxic amounts of ethanol were combined either with subtoxic amounts of glucose oxidase-generated hydrogen peroxide, or with mixtures of peroxide and with 2,2'-Azo-bis (2-amidinopropane)HCl (AAPH)-generated peroxyl radical. A further enhancement of cytotoxicity occurred when subtoxic amounts of trypsin were added to mixtures of all three agents. While ethanol alone caused shrinkage of the monolayers and cell rounding, no visible cytotoxic changes were observed. Hydrogen peroxide at the concentrations used (about 1 mM), caused only some cell rounding. On the other hand, cells exposed simultaneously to ethanol and to H2O2 developed extensive membrane damage characterized by the formation of large polar blebs, which is compatible with altered membrane permeability. The presence of trypsin markedly enhanced cellular cytotoxicity induced by mixtures of peroxide, peroxyl radical, and ethanol. This could markedly be depressed by catalase and by dimethylthiourea. The tissue culture model described might serve to further investigate the role played by synergy among oxidants and a variety of membrane-damaging agents, and by xenobiotics in tissue damage induced by inflammatory processes.

Experiments were undertaken to examine the chymotrypsin sensitivity and trypsin sensitivity of 13 avian reoviruses, and to determine if there was any correlation with pathogenicity of some chicken reoviruses. A wide variation in the degree of sensitivity of avian reoviruses to chymotrypsin and trypsin was observed. Overall, the infectivity of the 13 avian reoviruses for Vero cells was markedly reduced by treatment with 0.01% chymotrypsin (the lowest concentration tested) while 0.5% trypsin significantly reduced the infectivity of 9 of 13 strains. Comparison of four avian reoviruses, three resistant and one sensitive to trypsin, for pathogenicity in day old chicks following oral inoculation showed the strains that were resistant to trypsin to be more pathogenic. Tenosynovitis and virus persistence in intestines, liver, heart and hock joint tissues occurred only in chickens inoculated with the trypsin resistant strains. It is concluded that the degree of sensitivity to chymotrypsin and trypsin among avian reoviruses is heterogenous. Sensitivity to trypsin influenced the development of tenosynovitis based on microscopic lesions and virus persistence in tissues.

This study was undertaken to investigate the effects of a deficit in protease inhibitor (AT) induced by intravenously administered trypsin on the development of elastase-induced emphysema. Rats receiving a perfusion of trypsin (4.5 mg/kg body wt) intravenously (TIV rats) or one instillation of elastase (92 IU/subject) into the trachea (ELAS rats) were compared with rats receiving both trypsin and elastase (TIVELAS rats). Compared with 8 sham-injected rats, the serum AT activity of 14 TIV rats decreased slightly (5.5%) 150 min after the beginning of the perfusion. In six other TIV rats sacrificed early after the perfusion, a granulocyte sequestration with edema and vascular thrombi demonstrated early lung injury. Anatomical studies of lung and determination of the mean linear intercept (MLI) were carried out 56 days after the administration of the enzymes. Emphysema was confirmed by a significant (P less than .001) MLI increase (about 150 microns) in 22/24 TIV, 20/21 ELAS, and 21/21 TIVELAS rats in comparison with 40 control rats (78 microns). These similar results of the treated rats show that trypsin did not worsen elastase-induced emphysema and also indicate that trypsin given intravenously alone induces emphysema as does elastase when introduced into the airways. The AT activity decrease consequent to proteolysis by trypsin and pulmonary leucostasis may contribute to this trypsin-triggered emphysema.

The role of ethanol in precipitating acute pancreatitis has been studied in model experiments. The influence of ethanol on the survival of isolated pancreatic acinar cells (PAC) and a possible coaction with noxious agents (such as trypsin, chymotrypsin, temporary anoxia, and partial uncoupling of the oxidative phosphorylation by 2,4-dinitrophenol [DNP]) has been investigated. Isolated PACs withstood ethanol levels up to 180 mM without significant decrease of viability within 4 h incubation at 37 degrees C. A 90-min deprivation of oxygen was widely tolerated also. However, with a combination of both ethanol application and oxygen deprivation, a clearly forced cell damage was observed as was true with a combination of ethanol and chymotrypsin. The action of DNP or of extracellular trypsin on cell survival was not amplified by the addition of ethanol (180 mM). This study reveals two possible sites of action at which ethanol may contribute to the pathogenesis of acute pancreatitis.

The administration of trypsin 24 h before, admixed with, or 24 h after administration of an emphysema-inducing dose of porcine pancreatic elastase (PPE) to hamsters resulted in significantly enhanced destruction of lung tissue as evidenced by mean linear intercept values 4 weeks post PPE. The coadministration of trypsin with a nonemphysema-inducing dose of PPE resulted in a significant destructive lung lesion. Administration of trypsin 24 h before or admixed with human leukocyte elastase (HLE) resulted in a lesion that was significantly reduced relative to that produced by administration of HLE alone. When trypsin was administered 24 h after HLE, no effect on the lesion was observed. In vitro, coincubation of trypsin with PPE resulted in a slight enhancement of rate of hydrolysis of elastin, while coincubation of trypsin with HLE resulted in a significant reduction of the rate of hydrolysis. These results suggest that interaction with other proteases may offer an additional physiological control mechanism to prevent inappropriate tissue destruction.

Dextran of different molecular weight (Dx 40, Dx 60/70) has often been evaluated as adjunct treatment of experimental acute pancreatitis. A beneficial effect has been documented by a decrease in its lethality. However, the mechanism of action is poorly understood. A specific effect on the pancreatic microcirculation generally has not been documented and differentiation from unspecific improvement of pancreatic blood flow due to volume expansion has been difficult. This investigation was designed to quantify the effect of dextran on the impairment of pancreatic microcirculation during acute biliary pancreatitis by means of intravital microscopy. Dextran 60 (Dx 60, molecular weight 60,000) was chosen in light of the increase in vascular permeability in the early stage of pancreatitis as demonstrated previously in the same model. Isovolemic hemodilution, i.e., exchange of whole blood for Dx 60 was used as a mode of administration to achieve instantaneous onset of therapy without changes in intravascular volume. In the control group a progressive reduction of pancreatic capillary perfusion commenced 30 minutes after induction of acute pancreatitis, resulting in cessation of nutritive tissue perfusion after 3 hours. In the animals subjected to hemodilution, stabilization of the pancreatic microcirculation was accomplished throughout the observation period of 6 hours. Because volume-related effects could be excluded by the protocol and by monitoring central venous pressure and hematocrit, a specific effect of hemodilution with DX 60 on the pancreatic microcirculation is indicated by our results.

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.

The survival of isolated pancreatic acinar cells and of hepatocytes from rats was compared to mimic cell injury and death during the pathogenesis of acute pancreatitis in the presence of varying concentrations of trypsin, chymotrypsin and of the uncoupler 2.4-dinitrophenol (DNP). While hepatocytes were quickly damaged by trypsin they still excluded trypan blue though an excess amount of DNP was added. Pancreocytes revealed a reversed susceptibility to both noxae. The reduced sensitivity of pancreocytes to extracellular trypsin action could represent a special protectiveness against autodigestion and their increased susceptibility to an alteration of the cellular energy state stresses the importance of this factor to the survival of pancreatic tissue under normal and noxious conditions.

The elastase levels in rat serum were measured in experimental pancreatic disorders by radioimmunoassay. After the induction of acute pancreatitis either by trypsin or normal saline, the serum elastase levels increased significantly. The serum elastase levels in trypsin induced pancreatitis were significantly higher than in normal saline induced pancreatitis. The levels in ascitic fluid were also higher in trypsin induced pancreatitis than in normal saline induced pancreatitis. Microscopically, haemorrhagic pancreatitis was found in trypsin induced pancreatitis and edemotous pancreatitis was found in normal saline induced pancreatitis. The serum elastase levels decreased significantly in rats who were treated by ethionine for long period. Microscopically, atrophic pancreas was found in those rats. These results suggest that the measurement of pancreatic elastase in either blood or ascitic fluid by radioimmunoassay is a useful diagnostic method for pancreatic disorders, especially in acute pancreatitis.

A morphological study of the effects of different concentrations of trypsin on the normal rat bladder in vivo is described. A low concentration (5000 u/ml) of the pure enzyme caused extensive urothelial cell separation following intravesical instillation via a catheter for 30 min. Urothelial regeneration commenced within 12 h and the appearance of the bladder was normal within 3 months. However, the development of submucosal haemorrhage and ulceration, partly as a result of infection, indicates that further experimental work is required before trypsin can be evaluated clinically for removing in situ carcinoma or reducing tumour bulk. The enzyme might also be used to produce more cellular material for cytological examination.

Acute necrohemorrhagic pancreatitis was induced in rabbits by multiple interstitial trypsin injections in the body of the pancreas. The time course of regeneration was followed for 12 weeks. Chronic pancreatitis-like changes persisted for 4 weeks in all experimental animals, and the recovery was complete after 12 weeks. Reversible fibrosis and regressive acinar changes ("tubular complexes") were most severe in the region of the trypsin injections. Three-dimensional reconstruction of the pancreas showed an anastomosing tubular arrangement in the areas of "pseudochronic pancreatitis" but not in the normal pancreas.

Asperase is a relatively safe substance on peroral administration to mice and rats, moderately toxic upon intraperitoneal injection into rats. Subcutaneous injection of asperase brings about a 10-times lesser toxic effect as compared to intramuscular injection. As regards the toxicity asperase (single intraperitoneal and intravenous injections) does not differ from trypsin. Asperase toxicity is the consequence of an active protein contained by it.

Injection of saline as well as trypsin into the pancreatic duct of the rat may cause changes in the histology and surface tension of the lung, as well as histological and biochemical evidence of pancreatitis. Nontoxic chemicals also may be the source of pancreatitis and subsequent pulmonary dysfunction. In the research setting, pressure injection into the pancreatic duct may produce systemic effects.

We have addressed the problem of distinguishing angiogenesis induced in the chick chorioallantoic membrane by injury and inflammation from angiogenesis induced by primary stimulation. Focal, slow-release application of trypsin stimulated a localized spoke-wheel pattern of vascularity. In comparison, a range of doses up to a sublethal amount of trypsin applied generally, in liquid form, resulted in no change in DNA synthesis or vessel content, despite a transient influx of inflammatory cells. This contrasts with previous work with fibrin degradation products, histamine and heparin which each produce characteristic patterns of increased DNA synthesis leading to angiogenesis in the entire 'dropped' area of the chorioallantoic membrane. Such general application, therefore, avoids the danger of misinterpretation of focal, toxic effects.