The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice.

BACKGROUND AND PURPOSE: Recent findings suggest the importance of mast cells in the pathogenesis of rheumatoid arthritis and their potential as a therapeutic target. Tranilast is an anti-allergic compound with a potent membrane-stabilizing effect on mast cells and a wide range of anti-inflammatory effects, thus may be advantageous in the treatment of arthritis. Here, we have evaluated the effects of tranilast on the progression of collagen-induced arthritis in mice. EXPERIMENTAL APPROACH: Tranilast (400 mg.kg(-1).day(-1)) was orally administered for 8 weeks to mice with established collagen-induced arthritis. Arthritis was assessed by clinical signs and X-ray scores. In paw tissue, the numbers of mast cells and osteoclasts were measured by histological analysis, and several inflammatory factors were assessed by RT-PCR and Western blot analysis.* KEY RESULTS: TNF-alpha-positive mast cells were present extensively throughout the inflamed synovium of vehicle-treated arthritic mice, with some mast cells in close proximity to osteoclasts in areas of marked bone and cartilage destruction. Tranilast significantly reduced clinical and X-ray scores of arthritis and decreased numbers of TNF-alpha-positive mast cells and mRNA levels of TNF-alpha, chymase (mouse mast cell protease 4), tryptase (mouse mast cell protease 6), stem cell factor, interleukin-6, cathepsin-K, receptor activator of nuclear factor-kappaB, and of receptor activator of nuclear factor-kappaB-ligand, but increased interleukin-10 mRNA level in paws of arthritic mice. Osteoclast numbers were decreased by treatment with tranilast. CONCLUSIONS AND IMPLICATIONS: Tranilast possesses significant anti-rheumatic efficacy and, probably, this therapeutic effect is partly mediated by inhibition of mast cell activation and osteoclastogenesis.

Isoform-selective upregulation of mast cell chymase in the development of skin fibrosis in scleroderma model mice.

The involvement of connective-tissue-type mast cells and chymase, a protease unique to their secretory granules, has been implicated in fibrotic diseases. To elucidate the role of chymase in fibroproliferative inflammation, in this study we examined the enzymatic activity and mRNA expression of chymase in the sclerotic skin of tight-skin mice; syngeneic Pallid mice served as the control. Dorsal skin specimens from mice aged 5, 10, and 20 wk were evaluated by morphometric and biochemical analyses. At ages 10 and 20 wk, the hydroxyproline concentration in tight-skin dermis was higher than that in Pallid. At any age, the subcutaneous fibrous layer was thicker in tight-skin than in Pallid. In accordance with these fibrous changes, both connective-tissue-type mast cell counts and chymase activity were higher in tight-skin skin than in Pallid skin up to 20 wk of age. Age-matched (10-wk-old) tight-skin and Pallid were quantified for their mRNA of connective-tissue-type mast-cell-specific chymase, mouse mast cell protease-4, by the competitive reverse transcriptase polymerase chain reaction technique, which revealed its higher level in tight-skin than Pallid. In contrast, the mRNA level of mouse mast cell protease-5, the chymase isoform of undifferentiated mast cells, in tight-skin skin was only a tenth that of mouse mast cell protease-4 and no different from the mouse mast cell protease-5 mRNA level of Pallid mice. An in situ hybridization study confirmed the higher expression of mouse mast cell protease-4 by connective-tissue-type mast cells in tight-skin skin than Pallid skin. These results strongly support the contention that the connective-tissue-type mast cell chymase plays a crucial role in fibroproliferative remodeling of the skin.

Substituted 3-phenylsulfonylquinazoline-2,4-dione derivatives as novel nonpeptide inhibitors of human heart chymase.

A series of 3-phenylsulfonylquinazoline-2,4-dione derivatives have been synthesized and evaluated for their ability to inhibit human heart chymase. The structure-activity relationship studies on these compounds gave the following results. The phenyl moiety of quinazoline participates in a hydrophobic interaction where an optimum size is required. In this moiety, 7-chloroquinazoline is the best moiety for inhibiting chymase, chymotrypsin and cathepsin G. A 3-phenylsulfonyl moiety substituted with hydrophobic electron-withdrawing groups at the 4-position potentiated the activity. Anthranil moiety also enhanced the activity. Pyridylmethyl and N-pyridylacetamide at the 1-position gave an IC50 in the order of 10(-8)M. Molecular modeling studies on the interaction of 7-chloro-3-(4-chlorophenylsulfonyl) quinazoline-2,4(1H, 3H)-dione (4) with the active site of human heart chymase suggested that the phenyl moiety of quinazoline interacts with the hydrophobic P1 pocket, the 3-phenylsulfonyl moiety resides in the S1'-S2' subsites, the moiety at the 1-position locates in the S2-S3 subsites and the 4-carbonyl and 3-sulfonyl group interact with the oxyanion hole and the His57 side-chain of chymase, respectively.

Elevation of striatal interleukin-6 and serum corticosterone contents in MPTP-treated mice.

1. Changes in the content of striatal interleukins (IL-1 beta and IL-6) and serum corticosterone in relation to deterioration of the dopaminergic system induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; a dopaminergic neurotoxin; 20 mg/kg i.p., four administrations/12 h) in C57BL/6J mice were investigated. 2. Striatal dopamine, IL-1 beta, IL-6 and serum corticosterone were measured on days 1 and 7 post-MPTP. 3. Dopamine depletion was more severe on day 7 than on day 1 post-treatment. 4. Increases in IL-6 were observed on days 1 and 7 post-MPTP. The increase in striatal IL-6 content varied with the extent of dopamine depletion, although the IL-1 beta concentration remained unchanged compared with control values on days 1 and 7 post-treatment. 5. Serum corticosterone was not different from control on day 1 post-MPTP. However, marked increases in the serum corticosterone were observed on day 7 post-treatment. 6. These results suggest that changes in striatal IL-6 and serum corticosterone are closely associated with the severity of MPTP-induced dopaminergic degeneration.

Sex- and age-related changes in urinary contents of alpha 1-microglobulin and ulinastatin in mice.

1. Using two murine strains, ICR/Jcl and senescence-accelerated-prone inbred mouse 1 (SAMP1), age-associated changes in urine indices (e.g. urine volume, creatinine contents, contents of alpha 1-microglobulin (alpha 1M) and ulinastatin (UT) and the relation of urinary contents of alpha 1M and UT were investigated. 2. Sex-related differences in the indices were not observed in ICR nor in SAMP1 mice. 3. Urine indices per 24 h of ICR mice at 6 and 14 months of age were higher than those at 3 months of age, although indices of SAMP1 mice did not change with ageing. Urinary contents of alpha 1M and UT in ICR mice at 6 and 14 months of age were higher than those in SAMP1 mice. However, contents of alpha 1M and UT expressed as the contents per creatinine did not differ between these two strains. 4. In the relation between urinary contents of alpha 1M and UT, a positive correlation was displayed both in ICR and SAMP1 mice, and the regression slope did not significantly differ with ageing in these two strains. 5. These results suggest that ageing per se is not a factor which affects the relation of urinary contents of alpha 1M and UT.

Differential suppression of pressure-overload cardiac and aortic hypertrophy in rats by angiotensin-converting enzyme inhibitors.

Role of tissue angiotensin-converting enzyme (ACE) in the development of pressure-overload cardiovascular hypertrophy was examined in rats by comparing the inhibitory effect of trandolapril (high efficiency on tissue ACE) with that of enalapril (low efficiency) at equally antihypertensive doses. Rats with abdominal aorta banded or sham-operated were orally treated with trandolapril (0.5 mg/kg per day), enalapril (20 mg/kg per day) or vehicle for 8 weeks after the surgical maneuvers. In vehicle-treated rats, the banding raised the intra-aortic systolic pressure by 58%, diastolic pressure by 31%, maximum velocity of pressure rise by 65%, left ventricular (LV) weight by 41%, LV hydroxyproline concentration by 56%, aortic mass by 46%, LV ACE activity by 45%, and aortic ACE activity by 265%. Although both drugs equally reduced the aortic systolic pressure to approx. 70% and diastolic pressure to approx. 80% that of banded rats receiving vehicle, trandolapril partially prevented the LV hypertrophy, whereas enalapril yielded nonsignificant suppression. Trandolapril completely prevented the LV increments in hydroxyproline and ACE activity, whereas enalapril partially inhibited the LV hydroxyproline increase with little inhibition of LV ACE activity. In contrast, both inhibitors almost completely prevented the aortic hypertrophy, with the ACE activity of the aorta being potently inhibited. These results suggest that tissue ACE is the principal factor for pressure-induced aortic hypertrophy and an important yet non-essential factor for LV hypertrophy.

Increases in mast cells and chymase in fibroproliferative paws of collagen-induced arthritic mice.

OBJECTIVE AND DESIGN: To investigate whether mast cells (MCs) and chymase, the major protease of murine MCs, were involved in a chronic fibroproliferative disorder of the paws associated with type II collagen (CII)-induced arthritis. MATERIALS: Eighteen DBA/1J mice were divided into 3 groups and were used to study fibroproliferative changes in paws elicited by immunization. TREATMENT: Arthritis was induced by immunization with CII, which was intradermally injected as an emulsion made with adjuvant. A booster shot was done 3 weeks after the initial shot. A group with no treatment and that received adjuvant alone served as control. METHODS: Twelve weeks after the booster shot, inflammation of the paws was evaluated for pathological and biochemical indices. Chymase activity was determined with a chromogenic peptide substrate. RESULTS: In CII-immunized group, collagen bundles accumulated around the destructed joints. In accordance with the pathological findings, MC density in the affected paws was increased (154.8+/-13.3/mm2; p<0.05 vs. control) and chymase activity was also increased (29.5+/-2.8 mU/mg protein; p<0.01 vs. control). CONCLUSIONS: The present results demonstrate increases in MCs and chymase in fibroproliferative paws of collagen-induced arthritic mice.

Impairment of the correlation between urinary contents of alpha-1-microglobulin and ulinastatin is induced by intracerebroventricularly administered interleukin-6 in mice.

We have found previously that the correlation between urinary contents of alpha-1-microglobulin (alpha1M) and ulinastatin (UT) depends on the type of neuropsychiatric disease. Since interleukin (IL)-1beta and IL-6 are closely involved in pathophysiological aspects of various neuropsychiatric diseases, effects of intracerebroventricularly (i.c.v.) administered IL-1beta and IL-6 on the correlation between urinary contents of these two glycoproteins were examined in mice, a species in which alpha1M and UT and also the correlation between the urinary contents thereof are expressed similarly to humans. Indices (volume, contents of creatinine, alpha1M and UT, and alpha1M/UT ratio) in urine collected after i.c.v. administrations of 2 and 20 ng of either IL-1beta or IL-6 were not statistically different from those of the vehicle-treated (control) groups. Neither IL-1beta (2 and 20 ng) nor the lower dose of IL-6 (2 ng) affected the positive correlation between urinary contents of alpha1M and UT. However, a higher dose of IL-6 (20 ng) nullified the positive correlation for 2 days after administration. Recovery to a positive correlation was thereafter displayed. These findings suggest that central IL-6 plays an important role in correlating urinary contents of alpha1M and UT without affecting the renal functions.

Tranilast suppresses vascular chymase expression and neointima formation in balloon-injured dog carotid artery.

BACKGROUND: Activation of vascular chymase plays a major role in myointimal hypertrophy after vascular injury by augmenting the production of angiotensin (ANG) II. Because chymase is synthesized mainly in mast cells, we assumed that the chymase-dependent ANG II formation could be downregulated by tranilast, a mast cell-stabilizing antiallergic agent. We have assessed inhibitory effects of tranilast on neointima formation after balloon injury in the carotid artery of dogs, which share a similar ANG II-forming chymase with humans, and further explored the pathophysiological significance of vascular chymase. METHODS AND RESULTS: Either tranilast (50 mg/kg BID) or vehicle was orally administered to beagles for 2 weeks before and 4 weeks after balloon injury. Four weeks after the injury, remarkable neointima was formed in the carotid arteries of vehicle-treated dogs. Chymase mRNA levels and chymaselike activity of vehicle-treated injured arteries were increased 10.2- and 4.8-fold, respectively, those of uninjured arteries. Angiotensin-converting enzyme (ACE) activity was slightly increased in the injured arteries, whereas ACE mRNA levels were not. Tranilast treatment completely prevented the increase in chymaselike activity, reduced the chymase mRNA levels by 43%, and decreased the carotid intima/media ratio by 63%. In vehicle-treated injured arteries, mast cell count in the adventitia showed a great increase, which was completely prevented by the tranilast treatment. Vascular ACE activity and mRNA levels were unaffected by tranilast. CONCLUSIONS: Tranilast suppressed chymase gene expression, which was specifically activated in the injured arteries, and prevented neointima formation. Suppression of the chymase-dependent ANG II-forming pathway may contribute to the beneficial effects of tranilast.

[Effects of orally available prodrug of cromoglycic acid on collagen-induced arthritis mice].

Cromoglicate lisetil (CL) is an orally deliverable prodrug of cromoglycic acid, having diethyl promoieties and a lysyl promoiety for its optimum drug-delivery. We examined the effects of CL on bovine type II collagen (CII)-induced arthritis (CIA) of male DBA/1J mice, an experimental model for human rheumatoid arthritis, and its action mechanism. CL (100 mg/kg/day) was given by gavage to CII-immunized mice once daily for 6 weeks, starting when arthritic symptoms became evident. Symptomatic scores of arthritis obviously elevated in non-treated CIA mice at week 6.5 after initial immunization and continued elevated thereafter throughout the experiment, the elevation which was reduced by CL. CL also improved radiographic score of phalangeal destruction and pathohistological indexes at the end of treatment period. Serum anti-CII antibody titer was increased in non-treated CIA mice and the elevation was reduced by CL treatment. Mast cells (MCs) number in arthritic region was increased in non-treated CIA mice but not by CL treatment. In conclusion, oral CL treatment proved beneficial in CIA mice. Observed correlation between the CL effect on CIA and that on MCs number suggests the potential contribution of MCs to accelerate chronic arthritic processes and may further implicate potential action mechanism of CL, which may act by regulating MC functions for chronic inflammation.

Skin mast cell promotion of matrix remodeling in burn wound healing in mice: relevance of chymase.

Inflammation, granulation, and collagen accumulation, which are observed in the wound healing process, occasionally lead to hypertrophic scarring. Several in vitro reports have suggested that skin mast cells (MCs) and their major protease, chymase, participate in the healing process as well as in fibrotic skin diseases. The present study examined the potential involvement of MCs and MC chymase in the healing of burns in mouse dorsal skin. The size of the burn wounds, density of the capillaries, collagen accumulation, MC number, and chymase activity were measured before and 1, 3, 7, and 14 days after burning. The healing process corresponded strongly with MC density and chymase activity in both acute and subacute phases. The maximum decrease in MC number and chymase activity occurred on day 3 when tissue loss due to necrosis was maximal. From day 7 to 14, the burn wounds retracted rapidly accompanied by increases in capillaries and collagen fibers, in correspondence with fast increments in MC numbers and chymase activity at the wound edges. The present results combined with previous in vitro results strongly support the contention that skin MC chymase plays a role in the normal wound healing process, and presumably in dermal fibrotic disorders.

Differential expression and internal feedback regulation of 1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening.

We investigated the feedback regulation of ethylene biosynthesis in tomato (Lycopersicon esculentum) fruit with respect to the transition from system 1 to system 2 ethylene production. The abundance of LE-ACS2, LE-ACS4, and NR mRNAs increased in the ripening fruit concomitant with a burst in ethylene production. These increases in mRNAs with ripening were prevented to a large extent by treatment with 1-methylcyclopropene (MCP), an ethylene action inhibitor. Transcripts for the LE-ACS6 gene, which accumulated in preclimacteric fruit but not in untreated ripening fruit, did accumulate in ripening fruit treated with MCP. Treatment of young fruit with propylene prevented the accumulation of transcripts for this gene. LE-ACS1A, LE-ACS3, and TAE1 genes were expressed constitutively in the fruit throughout development and ripening irrespective of whether the fruit was treated with MCP or propylene. The transcripts for LE-ACO1 and LE-ACO4 genes already existed in preclimacteric fruit and increased greatly when ripening commenced. These increases in LE-ACO mRNA with ripening were also prevented by treatment with MCP. The results suggest that in tomato fruit the preclimacteric system 1 ethylene is possibly mediated via constitutively expressed LE-ACS1A and LE-ACS3 and negatively feedback-regulated LE-ACS6 genes with preexisting LE-ACO1 and LE-ACO4 mRNAs. At the onset of the climacteric stage, it shifts to system 2 ethylene, with a large accumulation of LE-ACS2, LE-ACS4, LE-ACO1, and LE-ACO4 mRNAs as a result of a positive feedback regulation. This transition from system 1 to system 2 ethylene production might be related to the accumulated level of NR mRNA.

[Angiotensin II formation by chymase in the cardiovascular tissue].

Angiotensin-converting enzyme (ACE) inhibitors attenuated the contractile responses to angiotensin (Ang) I of arterial strips of humans, monkeys, and dogs, as can be expected. Unexpectedly, however, the response was not abolished by sufficient doses of ACE inhibitors, the facts suggesting the Ang I conversion by a non-ACE enzyme(s). HPLC analysis of the incubation product of Ang I with vascular tissues revealed that Ang II was yet formed despite complete ACE inhibition, and the ACE inhibitor-insensitive Ang II formation was blocked by chymostatin. The disclosed Ang II-forming enzyme was identified as chymase, which was later found in abundance in the human heart. Another notable discovery by us is the species difference in chymase processing of Ang I: chymases of primates, dog, and hamster convert Ang I to Ang II, while chymases of rat, rabbit, and probably mouse do not. Accumulating evidence indicating that Ang II is not merely a vasopressor agent but also a growth-promoting factor, which leads to tissue hypertrophy and fibrosis, together with the results our studies lead us to propose the tissue-remodeling roles of chymase-formed Ang II in various cardiovascular diseases: dog neointimal proliferation after angioplasty, hamster cardiomyopathy, etc., in which chymase mRNA is increased concordantly with tissue remodeling. The fact that Ang II receptor antagonists, not ACE inhibitors, suppress the tissue remodeling supports our argument that Ang II is formed predominantly by chymase in diseased tissues. Orally active chymase inhibitors, evolving in our study, should help explore the actual roles of chymase as well as the rational treatment of tissue-remodeling disorders.

Age-related changes in aortic sensitivity to noradrenaline and acetylcholine in rats.

1. The purpose of the present study was to determine the relationship between plasma and tissue lipid levels and the effects of age on vascular responses to noradrenaline (NA) and acetylcholine (ACh). 2. Studies were performed in young and aged rats and the response of endothelium-intact and -denuded aortic rings to NA and to ACh was measured. The plasma concentration of cholesterol (total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL)) and 17 beta-oestradiol was determined, as was the aortic tissue content of phospholipids, cGMP and cholesterol (total, free and esterified). 3. Levels of all types of cholesterol in plasma and aorta increased with age; cholesterol levels in plasma correlated with those in the aorta; levels of phospholipid in the aorta did not increase with age but correlated with those of LDL cholesterol in plasma; levels of 17 beta-oestradiol did not change, but those of cGMP increased with age. 4. In endothelium-intact rings, the maximum tension developed by exposure to NA did not change, but the EC50 of NA increased with age and correlated with total cholesterol in the plasma and with the levels of all types of cholesterol in the aorta. In rings precontracted with NA, age decreased the maximum relaxation induced by ACh. The EC50 of ACh decreased with age and was inversely correlated with levels of cholesterol in the plasma and aorta. Treatment with NA increased cGMP levels in aged rats. Removal of the endothelium abolished the response to ACh and heightened the sensitivity to NA in young and aged rats. 5. Aortic endothelial cells seem to inhibit amine-induced contraction, while age-related changes in the levels of cholesterol in aortic tissue affect the sensitivity of the tissue to NA and ACh.

Cloning of the gene and cDNA for hamster chymase 2, and expression of chymase 1, chymase 2 and angiotensin-converting enzyme in the terminal stage of cardiomyopathic hearts.

Chymase is responsible for the formation of angiotensin II, which plays crucial roles in the pathogenesis of cardiovascular diseases. In the present study we determined the gene organization of a novel hamster chymase (hamster chymase 2) and analysed the expression of chymase 1, chymase 2 and angiotensin-converting enzyme (ACE) in hamster hearts at the terminal stage of cardiomyopathy. The gene encoding hamster chymase 2 is 3.2 kb in length and has five exons and four intervening sequences. The overall organization of this gene is similar to that of several other serine proteases. The deduced amino acid sequence revealed the existence of a preproenzyme composed of a signal peptide with 19 amino acids, a propeptide with two amino acids and a catalytic domain with 226 amino acids. The predicted full sequence of the catalytic domain was revealed to be very similar to the sequences of mouse mast-cell protease 5 (86%), rat mast-cell protease III (85%) and human chymase (70%) and less similar to hamster chymase 1 (56%). The expression of chymase 1 in heart was higher than that of chymase 2. The cardiac chymase-like activity, as well as the mRNA levels of chymase 1 and 2 of BIO 14.6 cardiomyopathic hamsters at the age of 60 weeks were increased 3.4-, 2.8- and 5.1-fold respectively compared with age-matched BIO F1B control hamsters. The cardiac ACE activity and the ACE mRNA level of cardiomyopathic hamsters were also increased 4.1- and 2.4-fold compared with those of age-matched controls. These results suggest that up-regulation of both ACE and chymases participates in the pathophysiology of the terminal stage of cardiomyopathy.

Acetylcholine-induced systemic vasodilation resistant to NG-nitro-L-arginine in an anaesthetized rats.

1. The effects of NG-nitro-L-arginine (L-NNA; 20 mg/kg bodyweight (BW), i.v.) and metyrapone (300 mg/kg BW, s.c.) on acetylcholine (ACh)-induced depressor responses were investigated in anaesthetized rats. 2. Acetylcholine (0.05, 0.5, 5 micrograms/kg BW, i.v.) dose-dependently evoked a sharp fall in mean blood pressure (BP) followed by a slow recovery under control conditions. 3. Basal BP level was elevated when rats were treated with L-NNA, indicating endogenous nitric oxide (NO) participated in BP regulation. However, pretreatment with L-NNA did not attenuate but rather augmented the ACh-induced maximum vasodilation. In contrast, the time for recovery of mean BP to the pre-ACh administration level was shortened by L-NNA. These observations suggested that ACh-induced vasodilation consisted of two phases: a sharp and transient fall (phase 1) that was resistant to L-NNA followed by a longer depressor response (phase 2) that was suppressed by L-NNA. 4. To examine whether augmentation of phase 1 by L-NNA resulted from the elevation of basal BP, an appropriate dose of phenylephrine was infused to obtain similar BP elevation. Phenylephrine infusion augmented the phase 1 in a similar manner to L-NNA pretreatment but showed little effect on phase 2, supporting the selective inhibition of phase 2 by L-NNA. 5. The s.c. pretreatment with metyrapone for 3 days failed to attenuate phase 1. Thus, the involvement of endothelium-derived hyperpolarizing factor that could be formed by a metyrapone-sensitive oxidase in phase 1 was unlikely. 6. These results suggest that some factor(s), which is not inhibitable by L-NNA or metyrapone, may induce the phase 1 depressor response to ACh while NO is responsible for the phase 2 response. The mechanism inducing the phase 1 response remains to be identified.

Nullification of a positive correlation between urinary contents of alpha1-microglobulin and ulinastatin with intracerebroventricularly administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) in mice.

Striatal dopamine contents in C57BL/6J mice were reduced at 24 h after intracerebroventricular (ICV) administration of 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+) in a dose-dependent manner. A dose of 1.8 microg MPP+ significantly (P < 0.05) suppressed the dopamine contents, whereas a similar dose of MPTP did not. A definite positive correlation between urinary contents of alpha1-microglobulin (alpha1M) and ulinastatin (UT) existed in normal mice. However, this correlation was nullified by ICV administration of 18 and 36 microg MPTP or 1.8 and 18 microg MPP+. With 1.8 microg MPTP, a positive correlation between urinary contents of alpha1M and UT was displayed. The urine volume, creatinine content, glomerular filtration rate, alpha1M and UT contents, and alpha1M/UT ratio of urine collected for 24 h post-ICV administration of MPTP or MPP+, were not statistically different from those of control mice. Our findings suggest that the central effects of MPP+, a neurotoxic metabolite of MPTP, nullify the positive correlation between urinary contents of alpha1M and UT without affecting renal functions.

Ischemia induces a reduction in the content of ulinastatin-like substance in the murine hippocampus.

Effects of ischemia on the content of a ulinastatin (UT)-like substance in the murine cerebral cortex and hippocampus were studied. At 24 h post-ischemia, a significant (p < 0.05) decrease in the content of UT-like substance in the hippocampus but not the cerebral cortex and a concurrent increase in the activity of micro-calpain were observed. In in vitro experiments, a decrease was registered in the content of UT-like substance in the hippocampus in the presence of calcium. This decrease was inhibited by both EDTA and calpastatin treatments. These results implicate the destruction of UT-like substance by micro-calpain in the ischemic hippocampus.

Three-dimensional molecular modeling explains why catalytic function for angiotensin-I is different between human and rat chymases.

Although angiotensin (ANG)-I is a substrate sensitive to chymase, the cleavage site differs among the chymase families. While human chymase (HC) hydrolyses the Phe8-His9 bond of ANG-I to ANG-II, rat chymase (RMCP-I) degrades the Tyr4-Ile5 bond of ANG-I to the inactive fragments. To clarify this different catalysis for ANG-I at the atomic level, three-dimensional structures of HC and RMCP-I were constructed by the molecular dynamic simulation. The energy-refined models clearly showed the significant difference in the electrostatic potential of the solvent surface. From the modeling study of their complex structures with ANG-I, the functional difference between both enzymes was clearly related with the electrostatic difference, especially at the C-terminal substrate-binding site.

Chymase: its pathophysiological roles and inhibitors.

Chymase is a chymotrypsin-type serine protease mainly localized in mast cells (MCs). Human, primate, and dog chymase generate angiotensin II (Ang II) from Ang I, while mouse and rat chymases degrade Ang II. It is suggested that chymase generating Ang II might be an alternative Ang II-forming enzyme to angiotensin-converting enzyme (ACE) in the renin-angiotensin system in tissues, but not in blood, and cause hypertrophy and remodeling of cardiovascular tissues. Chymase also degrades extracellular matrix, and processes procollagenase, inflammatory cytokines and other bioactive peptides. As a result, chymase plays important roles in inflammatory tissues through its proteolytic activities to cause tissue remodeling, that is, a chymase inhibitor may have the ability to prevent diseases caused by the above inflammatory reactions. The investigation of chymase inhibitors by pharmaceutical companies has yielded peptide and peptide mimetic inhibitors. We also found potent non-peptide low molecular inhibitors. However, the in vivo functions of chymase have not been verified so far by applying a chymase inhibitor to in vivo pathological models. In this article, we overview the pathophysiological roles of chymase and chymase inhibitors proposed to date, and discuss the structure-activity relationships of substituted 3-phenylsulfonyl-1-phenylimidazolidine-2,4-dione derivatives.