Neutral Proteinase Inhibitor Research Articles

Influence of the Treatment Used in Inflammatory Bowel Disease on the Protease Activities.

IntroductionThere is growing evidence that intestinal proteases have a role in the pathogenesis of gastrointestinal inflammatory diseases. Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), has an additional source of proteases represented by infiltrated and activated inflammatory cells. The aim of our study was to determine proteolytic system activity in patients with CD and UC. We limited the number of proteases tested by determining proteases active in acidic, neutral and alkaline pH.Materials and MethodsThe study included 40 patients with IBD – 20 CD patients and 20 UC patients. The control group consisted of 20 healthy subjects. Among the 20 CD patients, 17 were treated with aminosalicylates, 14 with azathioprine, and 4 with corticosteroids, while 8 patients were undergoing biological treatment. Among the 20 UC patients, 19 were treated with aminosalicylates, 8 with azathioprine, and 3 with corticosteroids. The total protein concentration was assayed by the Lowry method. The optimal pH was assayed in pH from 2.2 to 12.8, separated by 0.2 intervals. Proteolytic activities were determined against different substrates (gelatine, haemoglobin, ovalbumin, albumin, cytochrome C, and casein), and haemoglobin was the optimal substrate. Protease activities were determined according to Anson method. Determination of the activities of natural inhibitors of acidic, neutral and alkaline proteases is based on the Lee and Lin method.ResultsDecreases were observed in the activities of acid proteases (pH 5), alkaline proteases (pH 7), and neutral proteases (pH 7.6 and 8.6) in the groups of CD patients in remission in comparison with the active phase. In the group of patients with biologically treated CD patients, acid protease activity (pH 5.0) was lower than in CD patients not receiving biological treatment. Activities of neutral (pH 7.0) and alkaline (pH 7.6 and 8.6) proteases in the plasma of patients with UC in remission were lower in comparison to the active phase. Activities of acid (pH 5.0) and alkaline (8.6) protease inhibitors were higher in CD patients in the active phase in comparison to remission. In UC patients with exacerbation of the disease, the activity of alkaline (pH 8.6) protease inhibitors was increased compared to remission.Conclusion1. Our research may suggest that the immunomodulatory treatment used in IBD, aimed at reducing the level of leukocytes and reduction of inflammation, may contribute to a reduction in protease activity. 2. The decrease of protease activities in patients with CD and UC in remission may be a marker suggesting the patients’ response to the treatment.

Протеолитические ферменты и их белковые ингибиторы из зерна тритикале

Протеолитические ферменты играют значительную роль в процессах, протекающих при хранении и переработке растительного сырья. На современном этапе приоритетами любой пищевой технологии является сохранение нативных свойств биологически ценных компонентов сырья, обладающих лечебно-профилактическими свойствами, и модификация пищевого сырья с целью получения компонентов с определенными технологическими и функциональными свойствами. В настоящее время наибольшее распространение получили методы ферментативной модификации зернового сырья с использованием микробных ферментных препаратов. В то же время исследование собственных протеолитических ферментов, их выделение и очистка, с целью применения для модификации эндогенных белков, к которым они проявляют наибольшее сродство, представляется весьма интересной задачей, в плане использования всего биопотенциала зерна и “экологичности” получаемых продуктов. Такое исследование неразрывно сопряжено с изучением белковых ингибиторов протеиназ, которые представляют исключительный интерес как фактор регуляции процесса протеолиза. В статье приведены экспериментальные данные по выделению, частичной очистке и характеристике протеолитических ферментов зерна тритикале, действующих в кислой, нейтральной и щелочной областях рН. Показано, что кислые и нейтральные протеиназы зерна тритикале являются ферментами тиолового типа, Щелочные протеиназы относятся к сериновым протеиназам. Методом гель-хроматографии определена их молекулярная масса: 75000, 250000 и 50000 Да соответственно. Предложена схема выделения и очистки белковых ингибиторов эндогенных протеиназ из зерна тритикале. Показана их способность подавлять активность собственных протеиназ и трипсина. Проведено разделение ингибиторов трипсина и щелочных протеиназ тритикале (с молекулярной массой 30000 ÷ 35000 Да) и ингибиторов кислых и нейтральных протеиназ тритикале (с молекулярной массой 15000 ÷ 20000 Да). Частично очищенных препараты протеиназ и их белковых ингибиторов, полученные по предложенным схемам могут быть использованы для ферментативной модификации растительных белков и регуляции процессов протеолиза в различных пищевых технологиях, использующих зерновое сырье.

Fast and Cost-Effective Biochemical Spectrophotometric Analysis of Solution of Insect "Blood" and Body Surface Elution.

Using insect hemolymph (“blood”) and insect body surface elutions, researchers can perform rapid and cheap biochemical analyses to determine the insect’s immunology status. The authors of this publication describe a detailed methodology for a quick marking of the concentration of total proteins and evaluation of the proteolytic system activity (acid, neutral, and alkaline proteases and protease inhibitors), as well as a methodology for quick “liver” tests in insects: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and urea and glucose concentration analyses. The meaning and examples of an interpretation of the results of the presented methodology for biochemical parameter determination are described for the example of honey bees.

Varroa treatment with bromfenvinphos markedly suppresses honeybee biochemical defence levels

AbstractWe examined the influence of bromfenvinphos, a commonly used acaricide, on activities of many metabolic enzymes affecting the biochemical defences/physiology of the western honeybee, Apis mellifera L. (Hymenoptera: Apidae), as well as on some metabolic compound concentrations, percentage of global DNA methylation, and Nosema spp. infection levels. Bromfenvinphos‐treated workers had decreased haemolymph volumes and higher protein concentrations on their cuticle but lower protein concentrations in the haemolymph. They had higher global DNA methylation levels independent of the age‐related variants. Bromfenvinphos decreased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), acidic, neutral, and alkaline protease inhibitors and enzymatic physiological markers (AST, ALT, ALP), and concentrations of urea, uric acid, creatinine, cholesterol, glucose, Mg2+, and Ca2+ in worker haemolymph, depending on the age of the bees. Protease activities were higher only in the haemolymph of young bromfenvinphos‐treated bees in comparison with untreated bees. This compound decreased the activities of alkaline proteases and neutral protease inhibitors on the cuticle. Unexpectedly, in the treated bees, the activities of acidic and neutral proteases, and acidic and alkaline protease inhibitors, were higher in the young bees and lower in the older workers in comparison to the untreated group. The bromfenvinphos‐treated workers were more heavily infested with Nosema spp. Thus, bromfenvinphos not only supressed many levels of biochemical defences, and therefore stress‐resistance‐related biochemical pathways but also visibly increased the Nosema spp. infection levels.

Influence of Amitraz and Oxalic Acid on the Cuticle Proteolytic System of Apis mellifera L. Workers.

This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments.

Bilateral lavage with diluted surfactant improves lung function after unilateral lung contusion in pigs*

This study evaluates the effects of bronchoalveolar lavage with diluted surfactant on unilateral lung contusion-induced lung dysfunction. Randomized prospective animal study. An animal laboratory. Twenty adult pigs, weighing 25-35 kg. Animals were randomly assigned to controls and surfactant treatment. Bilateral lavage with surfactant treatment began 30 mins after unilateral lung contusion. Then 25 mg/kg of body weight diluted Curosurf (5 mg/mL) was applied in a volume of 5 mL/kg of body weight. Observation time was 8 hrs postinjury. The Pao2/Fio2 ratio fell from 500 to 250 and then recovered gradually in controls and surfactant-treated pigs. After another 4 hrs, the Pao2/Fio2 ratio deteriorated again in controls, but not in surfactant-treated animals. Total compliance fell by 50% after injury but was completely restored by surfactant treatment. Lung contusion increased the median number of neutrophils in bronchoalveolar lavage fluid from 2% to 30% of total cells and peaked >60% at 480 mins in the contused lungs of control pigs. Surfactant-treated pigs had 40% neutrophils at 480 mins without reaching significant difference to controls. The leukocyte neutral proteinase inhibitor increased to 500 ng/mL at 30 mins postinjury in the contused lungs and increased to 2000 ng/mL after surfactant treatment. Bilateral bronchoalveolar lavage with diluted surfactant can effectively improve lung function after experimental unilateral lung contusion in pigs.

1,2,5-Thiadiazolidin-3-one 1,1-dioxide-based heterocyclic sulfides are potent inhibitors of human tryptase

We describe herein the design, synthesis, and in vitro biochemical evaluation of a series of potent, time-dependent inhibitors of the mast cell-derived serine protease tryptase. The inhibitors were readily obtained by attaching various heterocyclic thiols, as well as a basic primary specificity residue P 1, to the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold. The inhibitors were found to be devoid of any inhibitory activity toward a neutral (elastase) or cysteine (papain) protease, however they were also fairly efficient inhibitors of bovine trypsin. The differential inhibition observed with trypsin suggests that enzyme selectivity can be optimized by exploiting differences in the S′ subsites of the two enzymes. The results described herein demonstrate the versatility of the heterocyclic scaffold in fashioning mechanism-based inhibitors of neutral, basic, and acidic (chymo)trypsin-like serine proteases.

Modulation of ERG25 expression by LDL in vascular cells.

Plasma low density lipoproteins (LDL) play a key role in the pathogenesis of atherosclerosis. LDL modify gene expression in vascular cells leading to disturbances in the functional state of the vessel wall. Expression levels of C-4 sterol methyl oxidase gene (ERG25), sterol regulatory element binding protein (SREBP)-1 and -2 were evaluated in porcine aortic endothelial cells (PAEC), porcine and human smooth muscle cells (SMC) and in the vascular wall from normolipemic and hyperlipemic pigs by RT-PCR. SREBP-1 protein levels were assessed by Western blot and SREBP-SRE binding by EMSA. SREBP-2 was overexpressed by transient transfection with lipofectin. We have identified expression of the ERG25 in vascular cells and analyzed its regulation by LDL. ERG25, an enzyme involved in cholesterol biosynthesis, is expressed in vascular endothelial and SMC from porcine and human origin and is downregulated by LDL in a time- and dose-dependent manner. Downregulation of ERG25 by LDL was abolished by an inhibitor of neutral cysteine proteases (N-acetyl-leucyl-leucyl-norleucinal) that abrogates SREBP catabolism. LDL downregulated SREBP-2 mRNA levels but not SREBP-1 expression in these cells and both ERG25 and SREBP-2 gene expression was significantly decreased in the vascular wall of diet-induced hypercholesterolemic swine. Finally, in cell transfection experiments SREBP-2 overexpression blocks ERG25 downregulation caused by LDL. Our results indicate that LDL modulate ERG25 expression in the vascular wall and suggest the involvement of SREBP-2 in this mechanism.

Factors affecting premature chromosome condensation of cumulus cell nuclei injected into rat oocytes.

To date, production of cloned rats by somatic cell nuclear transfer (NT) has not yet been successful. Inducing premature chromosome condensation (PCC) of injected cell nuclei in recipient cytoplasm is considered essential for successful mouse cloning by the Honolulu method. In the present study, some factors affecting PCC of rat cumulus cell nuclei injected into rat oocytes were examined. Wistar female rats (young: 4 to 5-week-old, mature: > or =10-week-old) were superovulated by injections of eCG and hCG, and oocytes recovered 14 or 17 h after hCG injection were received with cumulus cell nuclei using piezo-driven micromanipulator. When the oocytes were recovered 14 h post-hCG injection from young rats and the nuclear injection into oocytes was completed within 45 min, PCC was observed in 44-49% of NT oocytes. In the case of oocytes from mature rats, PCC occurred in 11-19% of the NT oocytes. Oocytes recovered 17 h post-hCG injection did not support PCC of the injected nuclei (0-7%) regardless of the donor age. Treatment of oocytes with a neutral cysteine protease inhibitor, N-acetylleucylleucylnorleucinal, slightly increased the incidence of PCC (48 vs 37%). Comparison of rat strains for oocyte donors indicated that proportions of NT oocytes undergoing PCC in Wistar and LEW oocytes (41-46%) were higher than those in Donryu and F344 oocytes (17-25%). Thus, ability of rat oocytes to promote PCC of the injected nuclei is dependent on the characteristics of oocytes, such as age or strain of donor rats, and timing of oocyte recovery.

Mast Cell Activation Triggers a Urothelial Inflammatory Response Mediated by Tumor Necrosis Factor-α

Mast cells have been implicated in bladder inflammation and pathogenesis. To determine if mast cell secretion products can modulate urothelial inflammatory responses we developed an in vitro model of mast cell-urothelial cell interactions. Cultures of the immortalized urothelial cell line TEU-2 were incubated in the conditioned medium of mast cell cultures. The urothelial inflammatory response to mast cell secretion products was then determined by quantifying nuclear factor kappaB activity, the expression of endogenous nuclear factor kappaB dependent genes and the protein expression of inflammation markers. Conditioned medium from RBL-2H3 mast cells induced a 4-fold increase in TEU-2 nuclear factor kappaB activity that was independent of the activation state of the mast cells. In contrast, ribonuclease protection assays revealed that the nuclear factor kappaB dependent transcripts tumor necrosis factor-alpha (TNF-alpha), interleukin (IL) 8 and 1beta, and intracellular adhesion molecule 1 (ICAM-1) were induced by mast cell conditioned medium in a manner that strictly depended on mast cell activation (antigen challenge of IgE sensitized RBL-2H3 cells). The dependence on mast cell activation was confirmed by the observation that IL-8 secretion and ICAM-1 protein expression in TEU-2 cultures were induced only by conditioned medium of stimulated RBL-2H3 cells The induction of TEU-2 IL-8 secretion and ICAM-1 expression by mast cell conditioned medium could be blocked by an anti-TNF-alpha antibody or the cysteine protease inhibitor N-acetyl-leucinyl-leucinyl-norleucinal. Our data support the hypothesis that mast cells may participate in bladder inflammation. Furthermore, TNF-alpha acting via the nuclear factor kappaB signaling pathway may be a mediator of the urothelial response to mast cell secretion products.

A Novel Double-headed Proteinaceous Inhibitor for Metalloproteinase and Serine Proteinase

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.

Effect of protease inhibitors on angiotensin-converting enzyme activity in human T-lymphocytes

The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with o-phtaldialdehyde. The effect of various concentrations (10 −9 to 10 −3 mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphoyctes.

Bradykinin B2-receptor antagonism attenuates fatal cardiocirculatory breakdown induced by severe experimental pancreatitis.

To investigate the impact of the long-acting bradykinin B2 receptor antagonist HOE 140 (Icatibant) on survival time in a model of severe porcine pancreatitis. Randomized, controlled intervention trial. Thirty domestic pigs of either gender anesthetized by intravenous application of piritramide, midazolam, and pancuronium and mechanically ventilated. Pancreatitis was induced by an injection of sodium taurocholate (5%, 1 mL/kg body weight [BW]) and enterokinase (10 U/kg BW). Control animals (group 1, n = 10) underwent the spontaneous course of the disease. In two treatment groups, Icatibant was administered either in a low (100 nmol/kg BW; group 2, n = 10) or in a high dosage (5000 nmol/kg BW; group 3, n = 10). Mean survival time was significantly prolonged by Icatibant (controls, 6.6 hrs; group 2, 9.8 hrs; p = .022; group 3, 10.9 hrs; p = .007). Six hours postinduction, the decline of total peripheral resistance (52% of baseline) and cardiac index (92% of baseline) in controls was significantly improved by Icatibant, both in the low (16% and 44%; p < .05) and high (6% and 45%; p < .05) dosage. The concentrations of free, nonreceptor-bound kinin in plasma 6 hrs postinduction were significantly lower in controls than in groups 2 and 3 animals (111+/-50 vs. 208+/-40 and 237+/-52 fmol/mL, respectively). Six hours postinduction, the pretreatment with Icatibant was associated with significantly higher plasma concentrations of phospholipase A2 (controls, +1194%; group 2, +2000%; group 3, +2285% of baseline values) and interleukin-1 receptor antagonist (controls, 1900+/-800; group 2, 3100+/-800; group 3, 3600+/-800 pg/mL). In contrast, the increase of urinary trypsinogen activation peptides indicating local pancreatic damage (589+/-114 nmol/L in controls) was substantially attenuated by pretreatment with Icatibant (group 2, 467+/-102, NS; 352+/-91 nmol/L in group 3; p = .022 vs. controls). Systemic inflammatory reactions, however, as quantified by C-reactive protein and the extracellularly discharged neutrophil cytosolic inhibitor leukocyte neutral proteinase inhibitor were not influenced by the bradykinin B2-receptor antagonist. Pretreatment with the bradykinin B2 receptor antagonist Icatibant resulted in prolonged survival time and in delayed impairment of major macrocirculatory and pulmonary variables. Icatibant resulted in elevated concentrations of free, circulating kinin. This was associated with increased concentrations of phospholipase A2 and interleukin-1 receptor antagonist, suggesting that circulating kinins strengthen the activation of some mediator cascades, the association of which with the kinin metabolism requires further experimental clarification. Other variables indicating a systemic inflammatory response (C-reactive protein, leukocyte neutral proteinase inhibitor) remained unaffected by Icatibant. Bradykinin antagonism distinctly ameliorated the local pancreatic damage, indicated by increased urinary concentrations of trypsinogen activation peptides. It is concluded that the kinin metabolism plays an important role in the pathophysiology of systemic complications after severe experimental pancreatitis.

Vasopressin-induced disruption of actin cytoskeletal organization and canalicular function in isolated rat hepatocyte couplets: possible involvement of protein kinase C.

The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca2+-buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca2+-activated neutral protease inhibitor, leupeptin, nor the antioxidants, alpha-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca2+-ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca2+-dependent) PKC appear to be involved because intracellular Ca2+ is required for VP to induce its harmful effects.

Local and systemic reactions after lung contusion: an experimental study in the pig.

The present study was designed to investigate the consequences of isolated unilateral lung contusion on local alveolar and systemic inflammatory responses in an animal model in the pig. Isolated unilateral lung contusion was induced by bolt shot in eight mechanically ventilated animals under general anesthesia (sham: n=4). Plasma and bronchoalveolar lavage fluid were collected during a period of 8 h following lung contusion. Leukocytes, leukocyte neutral protease inhibitor (LNPI), terminal complement complex (TCC), thrombin-antithrombin-complex (TAT) as well as pulmonary microvascular permeability and surfactant function were determined. Within 30 min, lung contusion was found to cause a significant local and systemic increase in TCC and TAT concentrations and a systemic increase in LNPI concentrations. The latter was accompanied by a sequestration of leukocytes in the contused lung. Complement activation and leukocyte sequestration in the contused lung progressively increased during the investigation period. Although surfactant function decreased in the entire lung 30 min after contusion, TCC, TAT, and leukocyte sequestration was unchanged in the contralateral lung. The first indication of an involvement of the contralateral lung was obtained by an increase in leukocyte sequestration 8 h after lung contusion. Unilateral lung contusion initiates an early systemic activation of humoral and cellular defense systems. Involvement of the contralateral lung appears to be a secondary event caused by a systemic inflammatory reaction.

Matrix Metalloproteinases Produced by Rat IL-2-Activated NK Cells

We have previously documented that adoptively transferred IL-2-activated NK (A-NK) cells can accumulate within cancer metastases. Electron microscopic studies of pulmonary metastases have revealed that adoptively transferred A-NK cells that accumulate within metastases bind to endothelial cells and are able to traverse basement membranes. We have now extended these morphologic studies. We report that rat A-NK cells produce two matrix metalloproteinases: MMP-2 and MMP-9, as determined by SDS-PAGE gelatin zymography. These activities are inhibited following incubation with BB-94 (batimastat), a specific inhibitor of matrix metalloproteinases but not with 3,4-dichloroisocoumarin, an inhibitor of neutral serine proteases. The identity of MMP-2 was confirmed by Western blots using a polyclonal Ab against human MMP-2, whereas reverse transcriptase-PCR analysis of mRNA extracts of A-NK cells has confirmed the presence of MMP-9. In addition, we report for the first time that A-NK cells can migrate through a model basement membrane-like extracellular matrix. Moreover, the ability of A-NK cells to migrate through this model basement membrane was partially inhibited by BB-94; however, BB-94 has no effect on A-NK cell-mediated cytotoxicity, suggesting that matrix metalloproteinases do not contribute to cytolytic function of A-NK cells. In sum, our studies show that A-NK cells employ BB-94-inhibitable matrix metalloproteinases to degrade extracellular matrices. This suggests that matrix metalloproteinases may play a role in the accumulation of A-NK cells within cancer metastases.

The role of elevations in intracellular [Ca2+] in the development of low frequency fatigue in mouse single muscle fibres.

1. Intracellular free calcium concentration ([Ca2+]i) and force were measured in isolated single skeletal muscle fibres from mice. The aim was to determine the extent to which elevations in [Ca2+]i during various stimulation protocols affected subsequent muscle performance. 2. A protocol of repeated tetanic stimulation which elevated [Ca2+]i and caused a large decline in force (fatigue) had a [Ca2+]-time integral of 36.4 +/- 8.1 microM s. A protocol of repeated tetani at a lower duty cycle (stimulation) caused only a small decline in force (9-16%) but elevated the [Ca2+]-time integral to 16.7 +/- 2.8 and 24.9 +/- 1.6 microM s in the absence and presence of 10 mM caffeine, respectively. Caffeine alone raised the [Ca2+]-time integral to 20.3 +/- 3.4 microM s. 3. Following the fatigue protocol there was a proportionately greater loss of force at low stimulation frequencies (30 and 50 Hz) compared with high frequencies (100 Hz) which persisted for up to an hour. This pattern of force loss could be attributed to a uniform reduction in [Ca2+]i at all frequencies. Similar effects were observed after elevating [Ca2+]i with the caffeine + stimulation protocol but were not observed after stimulation or caffeine alone. The higher [Ca2+]-time integrals during the fatigue and caffeine + stimulation protocols suggest that some threshold for [Ca2+]i must be reached before these effects are observed. 4. The reductions in low frequency force induced by the fatigue and caffeine + stimulation protocols were not due to decreased Ca2+ sensitivity or to decreases in maximum force-generating capacity of the contractile proteins and therefore are due to a failure of Ca2+ release. 5. The Ca(2+)-activated neutral protease (calpain) inhibitor calpeptin was not effective in preventing the effects of caffeine + stimulation indicating that the reduction in Ca2+ release was not due to calpain-mediated hydrolysis of the Ca2+ release channel. 6. Our findings indicate that low frequency fatigue results from increases in [Ca2+]i during fatigue and that these elevations in [Ca2+]i activate some process which leads to failure of excitation-contraction (E-C) coupling and Ca2+ release.

Rundown and reactivation of ATP-sensitive potassium channels (KATP) in mouse skeletal muscle

Dissociated single fibers from the mouse flexor digitorum brevis (FDB) muscle were used in patch clamp experiments to investigate the mechanisms of activation and inactivation of KATP in mammalian skeletal muscle. Spontaneous rundown of channel activity, in many excised patches, occurred gradually over a period of 10-20 min. Application of 1.0 mM free-Ca2+ to the cytoplasmic side of the patch caused irreversible inactivation of KATP within 15 sec. Ca(2+)-induced rundown was not prevented by the presence of 1.0 microM okadaic acid or 2.0 mg ml-1 of an inhibitor of calcium-activated neutral proteases, a result consistent with the conclusion that phosphatases or calcium-activated neutral proteases were not involved in the rundown process. Application of 1.0 mM Mg.ATP to Ca(2+)-inactivated KATP caused inhibition of residual activity but little or no reactivation of the channels upon washout of ATP, even in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase (10 U ml-1). Mg.ATP also failed to reactivate KATP, even after only partial spontaneous rundown, despite the presence of channels that could be activated by the potassium channel opener BRL 38227. Nucleotide diphosphates (500 microM; CDP, UDP, GDP and IDP) caused immediate and reversible opening of Ca(2+)-inactivated KATP. Reactivation of KATP by ADP (100 microM) increased further upon removal of the nucleotide. In contrast to KATP from cardiac and pancreatic cells, there was no evidence for phosphorylation of KATP from the surface sarcolemma of dissociated single fibers from mouse skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of intracellular calcium in superoxide-induced hepatocyte injury

The mechanisms of hepatocyte injury caused by exogenous superoxide were investigated with the use of cultured rat hepatocytes. Cell viability, cytosolic free calcium concentration and cell surface structure were observed. Superoxide was produced by adding hypoxanthine and xanthine oxidase to the buffer. Cytosolic free calcium concentration was calculated by means of ratio imaging of fura 2 fluorescence with multiparameter digitized microscopy. In the buffer containing 1.27 mmol/L of calcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the addition of hypoxanthine and xanthine oxidase, small blebs were recognized on the cell surface with a scanning electron microscope; then a gradual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentration had risen to about 700 nmol/L. Depriving the buffer of calcium (< 10 μmol/L) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the basal level (200 nmol/L). When ethylene glycol-bis (β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free calcium concentration decreased. Superoxide dismutase combined with catalase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpeptin, a Ca2+-dependent neutral protease inhibitor, did not affect the rise in cytosolic free calcium concentration but prevented cell injury. We concluded that cell injury caused by superoxide is initiated by influx of calcium and that the resultant activation of Ca2+-dependent protease may play an important role in bleb formation and cell death. (HEPATOLOGY 1994;19:1223–1228.)

Role of intracellular calcium in superoxide-induced hepatocyte injury

The mechanisms of hepatocyte injury caused by exogenous superoxide were investigated with the use of cultured rat hepatocytes. Cell viability, cytosolic free calcium concentration and cell surface structure were observed. Superoxide was produced by adding hypoxanthine and xanthine oxidase to the buffer. Cytosolic free calcium concentration was calculated by means of ratio imaging of fura 2 fluorescence with multiparameter digitized microscopy. In the buffer containing 1.27 mmol/L of calcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the addition of hypoxanthine and xanthine oxidase, small blebs were recognized on the cell surface with a scanning electron microscope; then a gradual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentration had risen to about 700 nmol/L. Depriving the buffer of calcium (< 10 mumol/L) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the basal level (200 nmol/L). When ethylene glycol-bis (beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free calcium concentration decreased. Superoxide dismutase combined with catalase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpeptin, a Ca(2+)-dependent neutral protease inhibitor, did not affect the rise in cytosolic free calcium concentration but prevented cell injury.(ABSTRACT TRUNCATED AT 250 WORDS)