Kazuyuki Kizuki

Regression of Glomerular Injury by Kallikrein Infusion in Dahl Salt-Sensitive Rats Is a Bradykinin B2-Receptor-Mediated Event

Aims: We investigated whether kallikrein infusion attenuates renal injury in Dahl salt-sensitive rats with hypertension and assessed the role of bradykinin-nitric oxide axis in the renal protection using HOE-140, the bradykinin type-2 (B2) receptor specific antagonist. Methods: Subdepressor dose of purified rat urinary kallikrein (RUK) (400 ng/day) was continuously infused through the jugular vein by an osmotic mini-pump for 4 weeks in Dahl salt-sensitive (Dahl S) rats fed a high-salt (2% NaCl) diet. Results: Blood pressure increased in a time-dependent manner in Dahl S rats fed a high-salt diet. The RUK infusion did not influence the elevation of blood pressure in Dahl S rats. However, the RUK infusion significantly decreased urinary protein excretion, and increased glomerular filtration rate, as compared with untreated high-salt Dahl S rats. Morphological investigation disclosed that the RUK infusion significantly attenuated glomerulosclerosis and arterial and tubular injuries in the kidney of hypertensive Dahl S rats. The RUK infusion produced an increase in urinary excretions of nitric oxide and cyclic guanosine monophosphate. In addition, the RUK infusion enhanced the generation of nitric oxide from the kidney slices. The functional and morphological effects of the RUK infusion on the kidney were completely lessened by co-administration of the bradykinin B2-receptor antagonist, HOE-140. Conclusion: Long-term infusion of subdepressor dose of rat urinary kallikrein attenuates functionally and morphologically the progression of renal injury in Dahl rats susceptible to salt-induced hypertension, and that the protection is mediated by stimulation of bradykinin B2 receptor.

Immunohistochemical demonstration of tissue kallikrein in the neurons of rat brain

The distribution of tissue kallikrein (EC.3.4.21.35) in the rat brain was investigated by an enzyme-linked immunosorbent assay (ELISA) and immunohistochemical technique using antiserum against rat urinary kallikrein. More than 75% of the total amount of kallikrein in the extracts of the cerebral cortex and brain stem was determined by the ELISA to be in the form of prokallikrein, suggesting that the greater part of the enzyme exists in the form of pro-enzyme in the central nervous system. Furthermore, immunohistochemical examination revealed that, although the kallikrein-positive cells were widespread and scattered in the brain, the immunoreactive substances preferentially locate in the neuronal cell bodies and their processes in both the cerebral cortex and brainstem.

Observation of tissue prokallikrein activation by some serine proteases, arginine esterases in rat submandibular gland.

Two serine proteases, arginine esterases (esterases I and II) which showed the activity of tissue prokallikrein activation were identified in rat submandibular gland. These enzymes were separated from the homogenate of rat submandibular gland by two successive DEAE-cellulose chromatographies and were further purified and characterized. Esterases I and II were found to be identical with tonin and esterase B-like enzyme, respectively. Both enzymes activated rat urinary prokallikrein at near neutral pH. Esterase B-like enzyme activated rat urinary prokallikrein better than tonin.

Potential role of kallikrein in diurnal rhythms and perivascular distribution in rat pineal glands

Kallikrein hydrolyzes various biologically active peptides, other than kininogens, such as vasoactive intestinal polypeptide (VIP), in vitro. Since kallikrein and VIP have been immunohistochemically shown to be present in the perivascular areas of the pineal gland, this study was designed to determine their topographic proximity in these glands, using immunohistochemical and immunoelectron microscopic double staining methods. Furthermore, since this gland is well-known to have a circadian rhythm, the kallikrein content was measured every 4 h, using a synthetic substrate, Pro-Phe-Arg-MCA, and an enzyme-linked immunosorbent assay (ELISA) to determine whether kallikrein has a circadian rhythm. The immunoreactivities of kallikrein and VIP were highly localized in the perivascular extracellular spaces and were virtually identical in distribution. The kallikrein content changed every 4 h and was high under light and low under dark conditions. The change was more evident when the synthetic substrate was used, and this rhythm was subtle on ELISA. VIP is also said to have a circadian rhythm in the pineal glands, being low under light and high under dark conditions, i.e., opposite to that of kallikrein. Since kallikrein degrades VIP in vitro, it is reasonable to speculate that pineal gland kallikrein is involved in the processing of VIP and possibly other biologically active peptides in the perivascular areas with a discernible circadian rhythm.

Biochemical and immunohistochemical demonstration of tissue kallikrein in the neuronal nuclei of the developing rat brains

Kallikrein content and cellular localization in the prenatal, newborn and adult rat brains were determined by the enzyme-linked immunosorbent assay and immunohistochemistry. The content was the highest in the prenatal rats and highly predominant in the neuronal nuclei during the prenatal to newborn periods, whereas the immunoreactive kallikrein was mainly located around neuronal cell bodies and their processes in the adult rats. The preferential nuclear localization in the prenatal rat brains was further confirmed by the immunoblotting technique after the SDS-polyacrylamide gel electrophoresis of the lysate of the nuclei fractionated from the prenatal rat brains. The meaning(s) of this kallikrein localization in the neuronal nuclei at the prenatal and newborn stages is unknown. However, we would like to conclude that this enzyme plays an important role in the morphogenesis of brain by acting on the substance(s) in the neuronal nuclei at the developing stage.

Rat Testicular Angiotensin I Converting Enzyme: Purification and Comparison with Rat Pulmonary Enzyme

Enzymological properties of rat testicular angiotensin I converting enzyme (RT-ACE) were compared with those of rat pulmonary angiotensin I converting enzyme (RP-ACE). The molecule of RT-ACE was different from that of RP-ACE with respect to the molecular weight, i.e., the molecular weight of RT-ACE was estimated to be 104 kilo-dalton (kd) and that of RP-ACE (150 kd) on SDS-polyacrylamide gel electrophoresis. On the other hand, the enzymochemical properties of RT-ACE were very similar to those of RP-ACE, with regard to activation by NaCl, optimum pH, Km value for N*-hippuryl-His-Leu-OH hydrolysis and sensitivities to various inhibitors. Therefore, it was speculated that the portions contributing to the appearance of catalytic activity would be similar between RT-ACE and RP-ACE.

Tissue kallikrein in chronic atrophic gastritis.

We measured amount of tissue kallikrein of gastric mucosa in 39 patients of chronic gastritis by sandwich type enzyme linked immunosorbent assay (S-ELISA) . Amount of tissue kallikrein was 9.1±4.4 ng/g tissue (mean±S.E.) in gastric mucosa of gastric glands, and was about twenty five times as much as it in the parts of atrophic gastric mucosa of them. The quantity of kallikrein was in proportion to age and the extent of atrophic gastritis. It was speculated that tissue kallikrein would participate in inflammatory atrophic change of gastric mucosa in chronic gastritis.

Studies on carbohydrate structure and immunological properties of human urinary kallikreins.

Some studies on carbohydrates structure and immunological properties of human urinary kallikreins purified from the human urine of healthy men (HUK) were investigated. A general technique for fractionating asparagine-linked oligosaccharides of HUK was applied. This involves serial chromatographies on concanavalin A-Sepharose 4B and other lectins bound Agarose. Asparagine-linked main oligosaccharides of both active and inactive types of HUK were mixture of tri- or tetra-antennary complex oligosaccharide having GlcNAC beta 1-4Man beta residue (bisecting GlcNAc) (type I-2), bi-antennary complex oligosaccharide having core Fuc alpha 1-6GlcNAc residue (type III-3), tri- or tetra-antennary complex oligosaccharides having Gal beta 1-4GlcNAc beta 1-4 (Gal beta 1-4GlcNAc beta 1-2)Man alpha-residue (type I-1-A-a) etc. Besides these structural studies, the method to probe the heterogeneous profiles of HUK due to the different structures of carbohydrate chains bound, was devised without the complete purification of urinary kallikrein even with individual subject of not only active but also inactive forms of the kallikrein.

Kinin-inactivating Enzyme from the Mushroom Tricholoma conglobatum. VI. Actions on Angiotensins I and II

A potent kinin-inactivating enzyme from the mushroom Tricholoma conglobatum, Shimeji kininase, liberated angiotensin II and the dipeptide, H-His-Leu-OH, from angiotensin I. Thus, this enzyme was considered to have both kininase and angiotensin I converting activities like kininase II (angiotensin I converting enzyme, EC 3.4.15.1), which is widely distributed in mammals of various species. On the other hand, this enzyme had angiotensinase activity in addition to angiotensin I converting activity. However, the rate of angiotensin II hydrolysis was very slow as compared with that of kinin hydrolysis ; the molar ratio of angiotensin II hydrolysis to bradykinin hydrolysis was about 1 : 70.

Mushroom kininase: Its application to the kallikrein-kinin system

The Japanese mushroom, Tricholoma conglobatum (Shimeji in Japanese), contains an extremely potent kininase. This kininase was purified 320-fold by ammonium sulfate fractionation and various chromatographic steps, the final preparation being homogeneous in disc electrophoresis. It splits the bradykinin molecule at the amino side of Phe(5) and of Phe(8) and the former bond was more easily cleaved than the latter [1]. The present paper deals with the stability of this kininase in the blood, its effect on plasma prekallikrein and its anti-inflammatory action. The kininase was incubated with rat plasma for 3 h at 30 ~ and this plasma-treated kininase was added to synthetic bradykinin together with 8-hydroxyquinoline which almost completely inhibits the kininase(s) in rat plasma but not the mushroom kininase. It was found that the kininase still held the activity to degrade bradykinin. Furthermore, 30 U of the mushroom kininase was injected i.v. into the rat and blood