Pancreatic secretory trypsin inhibitor I reduces the severity of chronic pancreatitis in mice overexpressing interleukin-1ß in the pancreas.

IL-1ß is believed to play a pathogenic role in the development of pancreatitis. Expression of human IL-1ß in pancreatic acinar cells produces chronic pancreatitis, characterized by extensive intrapancreatic inflammation, atrophy, and fibrosis. To determine if activation of trypsinogen is important in the pathogenesis of chronic pancreatitis in this model, we crossed IL-1ß transgenic [Tg(IL1ß)] mice with mice expressing a trypsin inhibitor that is normally produced in rat pancreatic acinar cells [pancreatic secretory trypsin inhibitor (PTSI) I]. We previously demonstrated that transgenic expression of PSTI-I [Tg(Psti1)] increased pancreatic trypsin inhibitor activity by 190%. Tg(IL1ß) mice were found to have marked pancreatic inflammation, characterized by histological changes, including acinar cell loss, inflammatory cell infiltration, and fibrosis, as well as elevated myeloperoxidase activity and elevated pancreatic trypsin activity, as early as 6 wk of age. In contrast to Tg(IL1ß) mice, pancreatitis was significantly less severe in dual-transgenic [Tg(IL1ß)-Tg(Psti1)] mice expressing IL-1ß and PSTI-I in pancreatic acinar cells. These findings indicate that overexpression of PSTI-I reduces the severity of pancreatitis and that pancreatic trypsin activity contributes to the pathogenesis of an inflammatory model of chronic pancreatitis.

Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor.

Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45x10(-9) m for furin and of 5.60x10(-7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N-terminal and C-terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1' being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1' residue Ser was mutated to D-Ser or N-methyl-Ser. The N-methyl-Ser mutant gave rise to a Ki value of 4.70x10(-8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.

Urinary trypsin inhibitor suppresses vascular smooth muscle contraction by inhibition of Ca2+ influx.

Urinary trypsin inhibitor (UTI) and its precursor form inter-alpha trypsin inhibitor (ITI) are present in plasma. To determine the action of UTI on blood vessels, we performed isometric vascular muscle contraction tests, microcirculation studies and measurement of cytosolic free Ca2+ in vascular smooth muscle cells. An isometric vascular muscle contraction test showed that the contractions stimulated by endothelin-1 or norepinephrine were suppressed in the presence of UTI, and that the contractions were not inhibited in the presence of ITI. The microcirculation study showed that the contraction of mesenteric arterioles of WKY rats induced by norepinephrine were inhibited by treatment of UTI, and that they did not alter by treatment of ITI. Pre-incubation of UTI, but not ITI, with vascular smooth muscle cells inhibited the increase of cytosolic free Ca2+ induced by endothelin-1 or norepinephrine. Cell-binding study by biotinylated UTI showed that vascular smooth muscle cells have specific binding site for UTI, but not for ITI. We propose that circulating UTI converted from ITI has a regulatory effect on local vascular tone by regulation of Ca2+ influx into smooth muscle cells.

Identification of trypsin inhibitor in bovine pituitary extracts as a survival factor for adult rat hepatocytes in primary culture.

Adult rat hepatocytes in primary culture cannot survive more than 2 days in the absence of calf serum. An extract of bovine pituitary gland had a similar effect to calf serum on the survival of hepatocytes, but its specific activity was 70 times that of calf serum. The survival factor was purified from bovine pituitary gland, and obtained in a homogeneous state judging by sodium dodecylsulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography. Its purification was achieved by acid extraction of the pituitary gland, gel filtrations on Sephadex G-75 and Ultrogel AcA 202, ion-exchanger chromatography on CM-cellulose, and then reverse phase high performance liquid chromatography. The purified factor was effective at 10 ng/ml and maximally effective at 100 ng/ml. The overall recovery of its activity was 30%, and the specific activity of the purified factor was 3,100 times that in the acid extract. The molecular weight (Mr 8,000), estimated by Sephadex G-200 filtration, and the strong basic character (pI 10.5) of the purified survival factor suggested that it may be the trypsin inhibitor found in various tissues. Indeed, the amino acid composition of the pure material was identical with that of bovine pancreatic trypsin inhibitor (bPTI). The purified survival factor had the same inhibitory activity on trypsin as commercial bPTI and, conversely, commercial bPTI greatly enhanced survival of rat hepatocytes. Thus, the survival factor in bovine pituitary gland was identified as bPTI.

Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice.

OBJECTIVE: To investigate the mechanisms whereby urinary trypsin inhibitor prevents lipopolysaccharide-induced preterm delivery in mice. METHODS: On day 15 of pregnancy, C3H/HeNCrg female mice impregnated by Crg:B6D2F1 male mice were treated intraperitoneally with lipopolysaccharide (50 micrograms/kg, twice at a 3-hour interval) to induce preterm delivery. Urinary trypsin inhibitor (2.5 x 10(4), 7.5 x 10(4), or 25 x 10(4) units/kg, ten times at 1-hour intervals) or saline solution was administered intraperitoneally to the animals. RESULTS: The incidence of preterm delivery was significantly decreased on a dose-related basis by urinary trypsin inhibitor treatment. Urinary trypsin inhibitor prevented the morphologic and functional changes in fetal membranes and cervical ripening preceding the onset of preterm delivery. Urinary trypsin inhibitor also suppressed the increase in plasma and amniotic fluid concentrations of interleukin-1 alpha, interleukin-6, and tumor necrosis factor-alpha after the lipopolysaccharide dosing in this animal model for preterm delivery. CONCLUSION: Urinary trypsin inhibitor prevents the pathogenicity of preterm delivery through the suppression of cytokine production.

Mechanisms of disease: Advances in understanding the mechanisms leading to chronic pancreatitis.

Chronic pancreatitis remains a challenging and frustrating clinical problem. In the past few years, however, advances in genetic and immunologic research have spawned new insights and approaches to chronic pancreatitis. Genetic and environmental risk assessment may help identify individuals who are likely to develop severe chronic pancreatitis early in the disease course, and allow targeted attention to reduce confounding risks and slow or prevent this problem in the future.

The sympathoadrenal system mediates the blood pressure and cardiac effects of human coagulation factor XII-related "new pressor protein".

OBJECTIVE: To investigate the major cardiovascular effects of human plasma "new pressor protein" (NPP) and how the adrenal medulla contributes to these effects. METHODS: NPP was injected into bioassay rats intravenously, and the effects on blood pressure and cardiac function were investigated. Acute adrenal medullectomy (2MDX), alpha- and beta-adrenergic blockade and plasma catecholamine levels were also used to evaluate the role of the sympathoadrenal system in mediating the NPP effects. RESULTS: NPP significantly raised systolic blood pressure (SBP) and mean arterial pressure but not diastolic blood pressure (DBP), with no significant change in total peripheral resistance. Heart rate, cardiac output and stroke volume rose by 16%, 53% and 36%, respectively. Plasma catecholamines increased massively, notably adrenaline, raising the adrenaline to noradrenaline ratio from about 4:1 to 18:1. 2MDX attenuated the increments of SBP and heart rate by more than 90% and more than 70%, respectively, implicating the adrenal medulla. Beta-adrenergic blockade (propranolol) potentiated the NPP-induced increase of SBP and DBP, but not that of heart rate. Combined alpha- and beta-adrenergic blockade (phentolamine and propranolol) blocked the rise in SBP, DBP and heart rate. CONCLUSIONS: NPP's hypertensive action is attributable mainly to increases in systolic blood pressure, heart rate and cardiac output (an increase in heart rate and stroke volume) with massive release of adrenal medullary catecholamines. Such effects suggest a novel axis between coagulation factor XII and the sympathoadrenal system, the cardiovascular effects of which are controlled by combined alpha- and beta-adrenergic blockade, but not by angiotensin-converting enzyme inhibition. Clinical relevance depends on whether NPP is formed in vivo in thrombotic states.

Trypsin inactivation by intact Hymenolepis diminuta (Cestoda): some characteristics of the inactivated enzyme.

In the presence of intact Hymenolepis diminuta, trypsin was inactivated; intact worms had no apparent effect on subtilisin, pepsin, or papain. Inactivation of trypsin was demonstrable using azoalbumin as a substrate, but the inactivated enzyme retained full catalytic activity against benzoyl-DL-arginine-p-nitroanilide, p-tosyl-L-arginine methyl ester (low molecular weight synthetic trypsin substrates) and p-nitro-p-guanidinobenzoate (an active site titrant). Inactivation was not reversible under conditions of heating, freezing and thawing, or prolonged dialysis of the enzyme. Analyses of inactivated 3H-trypsin by cationic and SDS-polyacrylamide gel electrophoresis, and gel chromatography failed to indicate the presence of a high molecular weight trypsin inhibitor associated with the inactivated enzyme; no low molecular weight, dissociable inhibitor was demonstrable following thermal denaturation of the inactivated enzyme. Analyses of trypsin after incubation in the presence of pulse-labeled worms also failed to demonstrate the presence of any inhibitor of worm origin associated with the inactivated enzyme. The data suggest that inactivation is the result of a small structural or conformational change in the enzyme molecule, a change which partially (rather than totally) inactivates the enzyme towards protein substrates.

[Proteins of the inter-alpha trypsin inhibitor (ITI) family. A major role in the biology of the extracellular matrix]. / Les protéines de la famille de l'ITI (inter-alpha trypsine inhibiteur). Un rôle majeur dans la biologie de la matrice extra-cellulaire.

A glycoprotein with a high inhibitory activity against trypsin was isolated in 1961 from human plasma and named inter-alpha trypsin inhibitor (ITI). Since then, several other proteins that share antigenic and structural similarities with ITI have been identified and classified as members of the ITI protein family. These glycoproteins built up from different combinations of four polypeptides HC1, HC2, HC3 and bikunin are encoded by four genes H1, H2, H3, L on three chromosomes. Bikunin has two proteinase inhibitor domains and belongs to the Kunitz-type protease inhibitor family; it displays an inhibitory activity against trypsin, leukocyte elastase and plasmin. The heavy chains do not have any protease inhibitory properties but have the capacity to interact in vitro and in vivo with hyaluronic acid. This binding promotes the stability of the extra-cellular matrix. Consequently, the ITI protein family is suspected of playing a key role in the extra-cellular matrix biology. Isolation of free heavy chains in bronchial secretions and the recent emphasis about the bikunin role in tumoral invasion should enhance the interest about ITI protein family in the pathophysiology of chronic bronchopulmonary diseases or lung cancer progression.

Protective effects of urinary trypsin inhibitor on vascular permeability following subarachnoid hemorrhage in a rat model.

AIMS: Inflammation and apoptosis play important roles in increasing vascular permeability following subarachnoid hemorrhage (SAH). The objective of this study was to evaluate whether urinary trypsin inhibitor (UTI), a serine protease inhibitor, attenuates vascular permeability by its antiinflammatory and antiapoptotic effects after experimental SAH. METHODS: Subarachnoid hemorrhage models were established in adult male Sprague-Dawley rats by endovascular perforation. UTI was administered by intraperitoneal injection immediately following SAH. Brain edema was assessed by magnetic resonance imaging (MRI) at 24 h after SAH. Neurological deficits, brain water content, vascular permeability, malondialdehyde (MDA) concentration, and myeloperoxidase (MPO) activity were evaluated. Immunohistochemical staining and Western blot were used to explore the underlying protective mechanism of UTI. RESULTS: Urinary trypsin inhibitor 50,000 U/kg significantly attenuated brain edema and neurological deficits and reduced vascular permeability at 24 h after SAH. MDA concentration and MPO activity in hippocampus were significantly decreased with UTI treatment. Furthermore, the levels of phosphorylated JNK, NF-κB (p65), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and proapoptotic protein p53, caspase-3 were elevated in the microvascular endothelial cells of the hippocampus after SAH, which were alleviated with UTI treatment. CONCLUSION: Urinary trypsin inhibitor reduced vascular permeability after SAH through its antiinflammatory and antiapptotic effects via blocking the activity of JNK, NF-κB, and p53.

cDNA cloning, structural, and functional analyses of venom phospholipases A2 and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi.

Snake venom phospholipases A2 (PLA2s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA2s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA2 cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA2 (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA2 (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA2 and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA2 isoforms. Remarkably, the most abundant PLA2 isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom.

Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues.

In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.