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It has been reported that only one-third of normotensive subjects and half of hypertensive patients are salt-sensitive. Many causes of salt-sensitivity have been proposed. Our suggestion is that a reduced urinary kallikrein level may be one cause, since mutant kininogen-deficient rats, which cannot generate kinin in the urine, are salt-sensitive. Renal kallikrein is secreted by the connecting tubule cells of the kidney, which are located just distal to the macula densa or the tubuloglomerular feedback system. Excess amounts of sodium taken overflow into the distal tubules and are reabsorbed in the collecting ducts. Kinins generated inhibit sodium reabsorption in the collecting ducts. Both blacks and whites with essential hypertension excrete less urinary kallikrein than do their normotensive counterparts, but the mean value in “normotensive blacks” were not different from that in “hypertensive whites”. African-Americans consume less potassium than whites. Potassium and ATP-sensitive potassium channel blockers are releasers of renal kallikrein. In a small-scale study, sodium loading caused more increase in the systolic blood pressure in urinary low-kallikrein group than in urinary high-kallikrein group. Largescale clinical studies, under strict control of potassium intake, are needed to elucidate the relationship between salt-sensitivity and urinary kallikrein levels.

Abstract:Objective: The present study was designed to examine the involvement of bradykinin in thermal and mechanical hyperalgesia induced by chronic constriction nerve injury (CCI) using B₁ and B₂ receptor antagonists and mutant kininogen-deficient rats. Methods: Sprague-Dawley (SD) rats and Brown Norway (B/N-) rats given CCI treatment on day 0, were used as a model of neuropathic pain. Either a kinin B₁ antagonist des-Arg⁹-[Leu⁸]-bradykinin or the receptor B₂ antagonist HOE-140 was constantly infused into the left jugular vein of SD rats on days 15 to 22 after CCI. Vehicle-treated rats and sham-operated rats without nerve injury were also prepared as controls. In all rats, we observed pain behavior, and measured the latency period of paw withdrawal from the thermal stimuli and, with von Frey filaments, the mechanical pain threshold, before surgery and on days 14 and 22 after CCI. B/N-Katholiek rats, which congenitally lack plasma kininogen and release no kinin, were also tested for hyperalgesic parameters. Expression of kinin receptor mRNA in the dorsal root ganglia was detected by RT-PCR. Results: Most of the rats (88%)showed some pain behavior, which was reduced to 67% by a B₁ antagonist and to 57% by a B₂ antagonist infused between days 15 to 22. Thermal hyperalgesia was significantly reduced from 7.25 ± 0.41 sec (mean ± SEM) to 8.36 ± 0.41 sec in paw withdrawal latency on day 22 by a B₁ antagonist and from 7.24 ± 0.19 sec to 8.23 ± 0.21 sec by a B₂ antagonist (P < 0.05). Mechanical hyperalgesia was also ameliorated from 0.02 ± 0.007 g force to 0.16 ± 0.08 g force in pain threshold by a B₁ antagonist and from 0.03 ± 0.007 g force to 0.10 ± 0.003 g force on day 22 by a B₂ antagonist. Moreover, deficient B/N-Katholiek rats showed a low incidence of thermal and mechanical hyperalgesia on day 14. Expression of both B₁ and B₂ receptor mRNAs was detected in the lumbar dorsal ganglia ipsilateral to the site of the ner ve injury. Conclusion: These data suggests that kinin were at least partly involved in yielding nociceptor hypersensitivity up to day 14 after CCI. Bradykinin and its B₁ and B₂ receptors were involved in the maintenance of hyperalgesia.

We investigated the profound involvement of chymase, an alternative angiotensin II-generating enzyme, in angiogenesis using a hamster sponge implant model. In vivo transfection of human pro-chymase cDNA or a direct injection of purified chymase into the sponges implanted resulted in marked increment of hemoglobin contents in the sponge granuloma tissues, demonstrating that chymase has an ability to elicit angiogenesis and is a potent angiogenic factor. Daily injection of basic fibroblast growth factor into the sponges implanted also induced angiogenesis, which was suppressed by the treatment with chymostatin, an inhibitor of chymase, or TCV-116, an antagonist of angiotensin II (Ang II) type 1 receptor. Expression of chymase mRNA and production of Ang II in the granuloma tissues were enhanced by the stimulation with basic fibroblast growth factor. Chymase activity in the sponge granulomas increased in parallel with the rise in hemoglobin contents, and mast cells observed in the granuloma tissues were positively stained with anti-chymase antibody. Exogenous administration not only of Ang II but of angiotensin I (Ang I) directly into the sponges could enhance angiogenesis. Chymostatin inhibited the angiogenesis induced by Ang I but not Ang II, suggesting the presence of a chymase-like Ang II-generating activity in the sponge granulomas. Our results may suggest a potential ability of chymase to promote angiogenesis through the local chymase-dependent and angiotensin-converting enzyme-dependent Ang II generating system in pathophysiological angiogenesis.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.

An endo-beta-glucuronidase acting on chondroitin sulfate was isolated from rabbit liver and purified about 550-fold, using a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephacryl S-300, affinity chromatography through heparin-Sepharose CL-6B and preparative polyacrylamide gel electrophoresis. The pH optimum of this enzyme was 4.0 and the Km value 7 X 10(-3) M for chondroitin sulfate (Mr 40,000). The isoelectric point of the enzyme was found to be at pH 5.4. The molecular weight, estimated by gel filtration through Sephacryl S-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 35,000. This enzyme, which was found in the liver, kidney, spleen, and lung, hydrolyzed the glucuronyl galactose linkage of the linkage region of chondroitin sulfate possessing a very small peptide segment. The enzyme did not hydrolyze proteoglycan. It was concluded that an endo-beta-glucuronidase is involved in the catabolism of proteoglycan chondroitin sulfates.

Abstract. Cyclooxygenase (COX)-2 was induced in an acute exudative inflammatory model (rat carrageenin-induced pleurisy) in which prostaglandin E₂, 6-keto-prostaglandin F_1α, and thromboxane B₂ were generated in the pleural fluid. Selective COX-2 inhibitors, such as NS-398, inhibited the plasma exudation and generation of prostaglandin E₂, but not that of thromboxane B₂ and 6-keto-prostaglandin F_1α, in the pleural fluid. In proliferative inflammatory models, COX-2 was induced, and selective COX-2 inhibitors suppressed granuloma formation, particularly, microvessel formation. COX-2 was induced during angiogenesis in a sponge model implanted into skin of rat, and the COX-2 inhibitor suppressed the angiogenesis. As induction of COX-2 was reported in osteoblasts, COX-2 was involved in most characteristic responses of acute exudative inflammation, granuloma formation, bone resorption, and pain in rheumatoid arthritis. The prevention of these COX-2 responses provides a rationale for the effectiveness of COX-2 inhibitors in the treatment of rheumatoid arthritis.

The reconstruction of glycosaminoglycan chains using the transglycosylation reaction of testicular hyaluronidase was investigated. First, the optimal conditions for the transglycosylation reaction catalyzed by the enzyme were determined by incubation with the enzyme, using hyaluronic acid (M(r) = 800,000) as a donor and pyridylaminated hyaluronic acid hexasaccharide having glucuronic acid at the nonreducing terminal as an acceptor. The carbohydrate chains as reaction products were determined by high performance liquid chromatography and mass spectrometry. The optimal pH for hydrolysis by the enzyme was found to be about 5.0, whereas that for the transglycosylation reaction was about 7.0. Sodium chloride in the reaction medium inhibited the transglycosylation reaction. Under the optimal conditions, the carbohydrate chains were sequentially transferred along with disaccharide units to the nonreducing terminal of the acceptor and elongated up to docosasaccharide from the acceptor, pyridylaminated hexasaccharide. Using a combination of hyaluronic acid, chondroitin, and chondroitin 4- and 6-sulfate as an acceptor and a donor, it was possible to reconstruct hybrid chains, which were natural or unnatural types of glycosaminoglycan chains. Therefore, it is highly likely that application of the transglycosylation reaction using testicular hyaluronidase would facilitate artificial reconstruction of glycosaminoglycans having some physiological functions.

We have developed an ELISA for BK-(1–5) (Arg1-Pro2-Pro3-Gly4-Phe5). In rat carrageenin-induced pleurisy, in which a plasma exudation peak was observed 5 h after carrageenin, BK levels in the exudates were negligible (<60 pg/rat). BK-(1–7) (des-Phe8-Arg9-BK) was detectable (900–400 pg/rat) over the entire course of the inflammation. However, a larger amount of BK-(1–5) was detectable in association with the increase in plasma exudation, showing a peak (8800±1200 pg/rat) 3 h after carrageenin. Bromelain (10 mg/kg, i.v.) and soy bean trypsin inhibitor (0.3 mg/rat, intra-pleural) significantly reduced BK-(1–5) levels (by 60–93%, 3, 7 and 19 h after carrageenin) and plasma exudation rates (by 61–74%, 3 and 7 h after carrageenin). Dexamethasone (0.3 mg/kg, i.p.) reduced BK-(1–5) levels (by 78%) and decreased plasma exudation (by 70%) 3 h after carrageenin. In nasal allergy patients, antigen challenge of nasal mucosa elevated BK-(1–5) levels and active kallikrein levels in nasal washes. These results verify that BK-(1–5) determined by ELISA is a good indicator for release of kinins in vivo.

Administration of human recombinant granulocyte colony-stimulating factor (G-CSF, 100 μg/kg/day, s.c) to rats for 4 days significantly increased circulating neutrophil counts (by 1130%), together with an increase in mononuclear leukocyte counts (by 119%). Infiltrated pleural neutrophil counts in G-CSF-treated rats (G-CSF-r) 5h after the intrapleural injection of zymosanactivated serum were significantly higher (by 155%) than those in control rats (Vehicle-r). In carrageenin-induced pleurisy, counts of infiltrated pleural neutrophils in G-CSF-r 5 and 7h after carrageenin were significantly higher (by 119% and 116%) than those in Vehicle-r. G-CSF treatment increased the volume of pleural exudate and the plasma exudation rate by 122% and 226%, compared to values in Vehicle-r 5h after carrageenin. Cobra venom factor (75 μg/kg, i.v.) significantly reduced pleural neutrophil migration in G-CSF-r (by 53%) and Vehicle-r (by 49%). Bromelain (10 mg/kg, i.v.) and aspirin (100 mg/kg, p.o.) reduced pleural neutrophil migration and reduced exudate volume and plasma exudation. Intrapleural bradykinin-(1–5) and prostaglandin E2 levels were significantly higher in G-CSF-r than in Vehicle-r. The increased neutrophil migration in G-CSF-r may be atributed to enhanced activation of the complement system facilitated by increased plasma exudation due to bradykinin and prostaglandins.

Abstract: In anesthetized, mechanically ventilated guinea pigs, infusion of captopril (1 mg/kg/h), an angiotensin converting enzyme inhibitor, significantly enhanced bronchoconstriction induced by intravenous injection of bradykinin (BK; 0.1–30 nmol/kg). Pretreatment of guinea pigs with capsaicin (100 mgrg/kg) slightly suppressed the bronchoconstriction by BK alone and almost all of the enhancement of BK-induced bronchoconstriction by captopril was suppressed. Intravenous injection of substance P (SP; 0.1–100 nmol/kg), neurokinin A (NKA; 0.1–30 nmol/kg) and neurokinin B (NKB; 0.1–30 nmol/kg) also induced dose-dependent bronchoconstriction but captopril treatment enhanced only the bronchoconstriction induced by SP. BK degradation in bronchoalveolar lavage fluid (BALF) in vitro was significantly suppressed by captopril (p<0.05). Captopril infusion to guinea pigs significantly increased the levels of BK, SP, and NKA in BALF after BK injection (p<0.05). FK224, an NK1 and NK2 receptor antagonist and SR 48968, an NK2 receptor antagonist, significantly suppressed the broncoconstriction induced by BK alone (p<0.01 and p<0.05, respectively) as well as the enhancement by captopril (p<0.01). It can be concluded that the enhancement of BK-induced bronchoconstriction by captopril was attributable to inhibition of the degradation of BK itself and thereby enhanced release of NKA and partly of SP from sensory nerves by BK.

Various oligosaccharides from hyaluronic acid, which have glucuronic acid or N- acetylglucosamine at the nonreducing terminal, were prepared by digestion with a combination of testicular hyaluronidase and beta-glucuronidase. These oligo saccharides were analyzed by negative-mode ion-spray mass spectrometry (MS) with an atmospheric pressure ion source. Introduction of collisionally activated dissociation tandem mass spectrometry (CAD-MS/MS) produced ions derived from cleavage of the glycosidic bonds, allowing the structure to be analyzed. The CAD-MS/MS spectrum showed an intense and characteristic fragment ion at m/z 193 for oligosaccharides having glucuronic acid at the nonreducing terminal. On the other hand, this ion was not observed in the spectra of oligosaccharides having N- acetylglucosamine at the nonreducing terminal. Therefore, the fragmentation pattern revealed by CAD-MS/MS provides useful information for distinguishing glucuronic acid and N- acetylglucosamine at the nonreducing terminal of oligosaccharides derived from hyaluronic acid and other glycosaminoglycans. This ion-spray CAD-MS/MS technique was also applied successfully to the characterization of glycosaminoglycans reconstructed by glycotechnology.

A large-capacity lithium-ion battery of 1 kWh (300 Ah) class was fabricated by using LiCoO2and natural graphite as the cathode and anode materials, respectively, and LiPF6as the electrolyte to develop industrially usable batteries for energy source. Our battery delivered an energy density of 60 Wh/kg (133 Wh/l) with weight and volume of a container included with a cycle life of 245 cycles, which was longer compared with that of our former battery using LiBF4as the electrolyte [5,6]. These improvements were achieved by changing the following factors: (i) use of LiPF6as the electrolyte; (ii) optimizing the mixing ratio of the solvent (EC/DEC); (iii) employing a plate spring for the buffer of electrode expansion and shrinkage; (iv) minimizing residual moisture.