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PubMed1995

Protection by lysosomal hydrolase inhibitors against cytotoxicity of 2-chloroethylethyl sulfide.

A possible participation of lysosomal hydrolases in the cytotoxicity of 2-chloroethylethyl sulfide in spleen lymphocytes was investigated using inhibitors of lysosomal phospholipases and proteases. Pepstatin (6 microM) and leupeptin (60 microM), inhibitors of lysosomal proteases, raised the viability of lymphocytes exposed to 2-chloroethylethyl sulfide from 63 to 87 and 88% of control, respectively. Serine protease inhibitors showed no significant effect on viability. Aminoglycoside inhibitors of lysosomal phospholipases were also found to prevent the decrease in viability of spleen lymphocytes exposed to 2-chloroethylethyl sulfide, and the effectiveness of these aminoglycosides (30 microM) was as follows: gentamicin > kanamycin > streptomycin, with viability increased to 89, 79 and 67%, respectively. In contrast to a co-operative action between leupeptin and gentamicin, the protection by pepstatin was reduced in the presence of gentamicin. Moreover, the order of the aminoglycosides in terms of the extent to which they antagonized the protective action of pepstatin was the same as their order of efficacy in preventing the cytotoxicity of CEES. It is suggested that inhibitors of lysosomal hydrolases reduce the cytotoxicity of 2-chloroethylethyl sulfide, presumably through lysosomal stabilization in spleen lymphocytes.

Alkylation↗
PubMed1980

Neutrophil chemotaxis and enzyme release competitive inhibition by a diazoacetamide pepsin inhibitor.

Treatment of human neutrophils with a reagent (diazoacetylnorleucine methyl ester plus copper ion) which covalently labels the active site of the acid proteases, pepsin and cathepsin D, inhibits neutrophil chemotaxis and enzyme release stimulated by the chemoattractants pepstatin and formylmethiony peptides. In contrast, chemotaxis and enzyme release in response to zymosan activated serum are not affected. Furthermore, diazoacetylnorleucine methy ester plus copper competes with [3H]formylmethionyl leucylphenylalanine for binding to neutrophils. Since pepstatin shares binding sites with formylmethionyl leucylphenylalanine, the present data suggest that diazoacetylnorleucine methyl ester plus copper reacts with the neutrophil receptor for pepstatin and formylmethionyl peptides, and thus may be useful in further characterization of this structure.

Acid Phosphatase↗
PubMed1993

Evidence for the role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from rat and mouse liver in whole-cell systems.

1. Sialyltransferase is a liver Golgi membrane-bound enzyme that is released from the liver under conditions of experimental inflammation. Previous work showed that the action of a cathepsin D-like proteinase was responsible for release of the enzyme from isolated Golgi membranes. This study shows that the same enzyme is responsible for release of sialyltransferase in whole-cell systems. 2. Gal beta 1-4GlcNAc alpha 2-6sialyltransferase (EC 2.4.99.1) was secreted from slices of rat and mouse liver into the incubation medium with larger amounts of activity being secreted from slices of liver from animals suffering from experimental inflammation. 3. The presence in the incubation medium of the cathepsin D proteinase inhibitor, pepstatin A, at 10(-4) M was sufficient to inhibit the release of sialyltransferase into the medium by about 60% after a 6 hr incubation. 4. The release of albumin and alpha 1 acid glycoprotein from rat liver slices, was not affected by the presence of pepstatin A, indicating that the proteinase inhibitor did not affect the synthesis and secretion of typical secretable proteins by the liver. 5. Intraperitoneal injections of pepstatin A into mice prior to preparation of liver slices also resulted in a significant reduction of the secretion of sialyltransferase into the incubation medium. 6. The results from these studies support the idea that a cathepsin D-like proteinase is responsible for the release of sialyltransferase into the extracellular space in whole cells in the rat and the mouse.

Albumins↗
PubMed2006

Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors.

Fonsecaea pedrosoi is the principal causative agent of chromoblastomycosis, which is a chronic, often debilitating, suppurative and granulomatous mycosis. Very little is known about the hydrolytic enzymes produced by this human fungal pathogen. In the present study, we have identified extracellular proteolytic activity from F. pedrosoi mycelial forms when grown in chemically defined conditions. Secretory aspartyl peptidase activity was measured during 15 days of fungal growth in vitro using bovine serum albumin (BSA) as the soluble substrate and extreme acidic pH (2.0). This activity was totally inhibited by pepstatin A, a classic aspartyl peptidase inhibitor. Conversely, metallo (o-phenanthroline), cysteine (E-64) and serine (PMSF) proteolytic inhibitors failed to restrain proteolytic activity. We also evaluated the effect of four distinct HIV aspartyl peptidase inhibitors on the secretory proteolytic activity of F. pedrosoi mycelia. Indinavir, ritonavir and nelfinavir powerfully inhibited extracellular aspartyl proteolytic activity by approximately 97, 96 and 87%, respectively, whereas saquinavir did not significantly interfere with BSA hydrolysis. Mycelial-derived secretory aspartyl peptidase activity cleaved other proteinaceous substrates, including human albumin, fibrinogen, fibronectin, laminin and type I collagen. As previously reported by our group, conidia also produce secretory aspartyl peptidase. In this sense, we investigated the effect of pepstatin A on F. pedrosoi development. Pepstatin A was able to inhibit the growth of conidium and its transformation into mycelium. Taken together, our results suggest a possible participation of aspartyl peptidases in the essential fungal processes, such as growth, differentiation, nutrition and cleavage of relevant host proteinaceous components.

Ascomycota↗
PubMed2001

Mitogen-activated protein kinase activity regulation role of angiotensin and endothelin systems in vascular smooth muscle cells.

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Angiotensin I↗
PubMed1999

Local renin-angiotensin system and mitogen-activated protein kinase activation in rat aorta.

We previously reported that endogenous angiotensin II is released to cause mitogen-activated protein (MAP) kinase stimulation in the media portion of the vasculature. In this study, we examined whether a functional renin-angiotensin system is indeed present within the media of the vasculature. In rat aortic strips, endothelium removal produced an increase of MAP kinase activity. The MAP kinase activation was inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of the MAP kinase activation by pepstatin A, captopril and the angiotensin receptor antagonist losartan was almost the same. Pepstatin A inhibited MAP kinase activation induced by renin but not by angiotensin I and angiotensin II. Captopril inhibited the MAP kinase activation induced by angiotensin I but not by angiotensin II. In nephrectomized rat aortic strips, endothelium removal also produced an increase in MAP kinase activity, but the MAP kinase activation was considerably small and minimally inhibited by losartan. Nephrectomy produced a marked decrease in plasma renin activity. These findings suggest that an apparently fully intact and functional renin-angiotensin system is present in the media of the rat vasculature and this system serves to increase MAP kinase activity. It appears that renin plays the determining role in the regulation of angiotensin generation also in the media and the major source of the renin is renin of kidney origin.

Angiotensin I↗
PubMed1992

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.

Amino Acid Sequence↗
PubMed1984

Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts.

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.

Cells, Cultured↗
PubMed1980

Regulation of mitochondrial biogenesis. Occurrence of non-functioning components of the mitochondrial respiratory chain in Saccharomyces cerevisiae grown in the presence of proteinase inhibitors: evidence for proteolytic control over assembly of the respiratory chain.

Yeast was grown in glucose- or galactose-containing media without or with proteinase inhibitors, phenylmethanesulphonyl fluoride and pepstatin. Culture growth was practically not affected by these compounds. Yeast growth on glucose in the presence of either phenylmethanesulphonyl fluoride or pepstatin entails accumulation of cytochromes c, c1, b and aa3 to a 25--30% excess above the control by the stationary phase, while cell respiration is unaffected. During growth on galactose the maximal cytochrome content (per unit weight of biomass) is reached in the mid-exponential phase and then decreases by 30--40% towards the stationary phase, while cell respiration remains constant. Addition of phenylmethanesulphonyl fluoride or pepstatin in the mid-exponential phase blocks the decrease in cytochrome levels and has no effect on cell respiration. Mitochondrial populations isolated from stationary-phase control and phenylmethanesulphonyl fluoride-grown cells glucose cultures display identical succinate oxidase and partial-respiratory-chain activities, despite the differences in cytochrome contents. However, the activities of individual respiratory complexes measured after maximal activation are nearly proportional to the amounts of corresponding components. The same situation holds true for mitochondrial populations from mid-exponential-phase, stationary-phase control and stationary-phase inhibitor-grown cells of galactose cultures. The findings suggest that the 'surplus' respiratory-chain components do not participate in electron flow because of the lack of interaction with adjacent carriers.

Cytochromes↗
PubMed1996

Specific delay in the degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis is derived from cellular proteolytic dysfunction rather than structural alteration of subunit c.

Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (NCL). To explore the mechanism of lysosomal storage of subunit c in patient cells, we investigated the mechanism of the lysosomal accumulation of subunit c both in cultured normal fibroblasts and in in vitro cell-free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumulation of subunit c and less accumulation of Mn(2+)-superoxide dismutase (SOD). In contrast, E-64-d stimulates greater lysosomal storage of Mn(2+)-SOD than of subunit c. Incubation of mitochondrial-lysosomal fractions from control and diseased cells at acidic pH leads to a much more rapid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn(2+)-SOD, beta subunit of ATP synthase, and subunit i.v. of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E-64-c, as in the cultured cell experiments. However, there are no differences in the lysosomal protease levels, including the levels of the pepstatin-sensitive aspartic protease cathepsin D between control and patient cells. The stable subunit c in mitochondrial-lysosomal fractions from patient cells is degraded on incubation with mitochondrial-lysosomal fractions from control cells. Exchange experiments using radiolabeled substrates and nonlabeled proteolytic sources from control and patient cells showed that proteolytic dysfunction, rather than structural alterations such as the posttranslational modification of subunit c, is responsible for the specific delay in the degradation of subunit c in the late infantile form of NCL.

Cathepsin B↗
PubMed2002

Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.

Amyloid Precursor Protein Secretases↗
PubMed1991

Direct proof for local generation and release of angiotensin II in peripheral human vascular tissue.

Previously we reported that immunoreactive angiotensin II (Ang II) release from isolated perfused human umbilical veins was inhibited by the angiotensin-converting enzyme inhibitor captopril. To further investigate the mechanism by which Ang II is generated in the blood vessels of humans, we examined the effects of various inhibitors of the renin-angiotensin system (captopril, delapril, N-acetyl-pepstatin, and human renin inhibitor KRI-1314) on Ang II release from perfused human umbilical cord veins in vitro. Isolated human umbilical veins were perfused with Krebs-Ringer solution, and immunoreactive Ang II released into the perfusate was measured directly by using a Sep-Pak C18 cartridge connected to the perfusion system. Both captopril and delapril diacid (10(-9) to 5 x 10(-6) mol/L), an active metabolite of delapril hydrochloride, suppressed the Ang II release in a dose-dependent fashion; the maximal percent suppression of Ang II release evoked by these inhibitors (5 x 10(-6) mol/L) was 56% and 64%, respectively, for captopril and delapril. Both N-acetyl-pepstatin (10(-9) to 10(-5) mol/L) and KRI-1314 (10(-9) to 10(-6) mol/L) suppressed Ang II release in a dose-related manner. At a 10(-6) mol/L concentration, KRI-1314 produced a 74% reduction in the basal rate of Ang II release, and a reduction threefold greater than the maximal reduction in basal Ang II release produced by N-acetyl-pepstatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed2006

Prodomain processing of recombinant plasmepsin II and IV, the aspartic proteases of Plasmodium falciparum, is auto- and trans-catalytic.

Prodomain processing of the four food vacuole plasmepsins (PMs), the malarial aspartic proteases, is prerequisite for their activity on hemoglobin degradation of the parasite Plasmodium falciparum. Although previous studies have suggested the involvement of a calpain-like PM convertase in the processing of PMs, the underlying mechanism of their processing remains to be clarified. Here, to investigate the mechanism by which food vacuole PM II and IV are processed, we used their wild-type and mutant proteins in which the catalytic Asp residue in two active-site motifs was mutated, as well as protease inhibitors. Autocatalytic processing of wild-type PM II and IV was inhibited only by an aspartic protease inhibitor pepstatin A. Unexpectedly, their proteolytic activities were inhibited not only by pepstatin A but also by calpain inhibitor ALLN. The active-site mutants of both PM II and IV showed neither autocatalytic processing nor proteolytic activities. However, the mutants of both PMs were efficiently processed upon incubation with their respective wild type proteins. Furthermore, the mutants of both PMs were processed upon incubation with each other's wild-type PM in both pepstatin A- and ALLN-sensitive manners. These results suggest that the processing of PM II and IV occurs via an intra- and inter-molecular autocatalytic event as well as via a transcatalytic event between them.

Animals↗
PubMed1989

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme.

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.

Biotransformation↗
PubMed1976

The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors.

Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.

Aminocaproates↗
PubMed1982

Monkey pepsinogens and pepsins. VI. One-step activation of Japanese monkey pepsinogen to pepsin.

When Japanese monkey pepsinogen was activated at pH 2.0 in the absence of pepstatin, the activation segment of the amino(N)-terminal 47 residues was released as a single intact polypeptide. This clearly shows that the pepsinogen was activated to pepsin directly. This direct activation was called a 'one-step' process. On the other hand, when pepsinogen was activated at pH 2.0 in the presence of pepstatin, an appreciable amount of pepsinogen was converted to an intermediate form between pepsinogen and pepsin, although a part of pepsinogen was activated directly to pepsin. The intermediate form was generated by releasing the N-terminal 25 residues of pepsinogen. This activation through the intermediate form is thought to be a 'two-step' or 'stepwise-activating' process involving a bimolecular reaction between pepstatin-bound pepsinogen and free pepsin.

Animals↗
PubMed1991

Evidence against a role for aspartyl proteases in intracellular processing of big endothelin.

CPAE endothelial cells were cultured in the presence of pepstatin, NH4Cl, or chloroquine in order to assess their effects on the secretion of endothelin-1 (ET-1). The first of these is an inhibitor of aspartyl proteases and the last two are known to neutralize acidic intracellular compartments. The pepstatin was encapsulated into liposomes to aid in its uptake, and uptake was confirmed by measuring the residual aspartyl protease activity in washed, lysed cells. Pepstatin had no effect (less than 5%) on the secretion of ET-1, 25 mM NH4Cl decreased secretion by 30-47%, and 25 microM chloroquine increased secretion by 37-79%. In contrast, each of these reagents is known to inhibit lysosomal degradation of intracellular proteins by 75-90%. Additionally, big ET was shown to be a very poor substrate, in terms of kcat/Km values, for aspartyl proteases. The rate constants were less than 10(4) M-1 s-1, which is approximately 1% of the value for the best substrates. The data, therefore, do not support a role for aspartyl proteases in the formation of ET-1. Similar to chloroquine, 0.5 microM monensin increased the secretion of ET-1 by 40-60%. Both of these reagents have previously been shown to increase the rate of constitutive secretion of peptides by affecting their partitioning between packaging into storage granules and constitutive secretion. The results would therefore provide supportive evidence for the existence of a storage form of ET-1 in endothelial cells.

Ammonium Chloride↗
PubMed1995

Role of pepsin in the development of indomethacin-induced antral ulceration in the rat.

AIMS: To examine the effects of a pepsin inhibitor, pepstatin-A, a long acting H2-receptor blocker, loxtidine, exogenous pepsin and exogenous acid against indomethacin-induced antral ulceration in the rat. RESULTS: Indomethacin (60 mg/kg s.c.) caused antral ulceration in fasted/re-fed rats over a period of 4 h. Ulceration was prevented in a dose-dependent manner by treatment with pepstatin-A (0.1-1 mg.kg hourly) or loxtidine (3 mg/kg) given orally. Acidified methylcellulose (1 mL hourly per os) enhanced damage and also prevented protection by loxtidine (3 mg/kg per os). The protection by pepstatin-A was not altered by treatment with acidified methylcellulose but was reversed by treatment with a 10-fold excess of pepsin. CONCLUSION: These studies suggest that mucosal degradation by pepsin, rather than direct damage by luminal acid, was the major factor in the development of indomethacin-induced antral ulceration in the rat.

Animals↗