Urinary Kallikrein and Non-Kallikrein Arginine Esterase Activities in Beagle Dogs

Transcription

1 Exp. Anim. 36(2), , 1987 Urinary Kallikrein and Non-Kallikrein Arginine Esterase Activities in Beagle Dogs Hitoshi OGAWA, Tohru FUKASE*, Yoshifumi MATSUDA**, Sumiyuki AKIHAMA** and Hiroshi ITAGAKI* Gifu Research Laboratory, The Center of Japan Biological Chemistry Co., Ltd., Fukue, Kaizu-cho, Kaizu-gun, Gifu , Japan, *Department of Parasitology, School of Veterinary Medicine, Azabu University, Fuchinobe, Sagamihara-shi, Kanagawa 229, and **First Department of Biochemistry, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154. (Received 1 July 1986/Accepted 8 October 1986) The activity of urinary kallikrein and non-kallikrein arginine esterase was measured in the 16hrs urine of 16 beagle dogs. Enzyme activity was assayed using D-valyl-Lleucyl-L-arginine-p-nitroanilide as a substrate at 30 Ž, ph 8.0, and the unit of measurement was defined as the activity which catalyzes the hydrolysis of 1 pmol of the substrate per minute. Kallikrein activity ranged from 3.87 to (28.48 average) mu/ml of urine, or from 1.05 to (4.56 average) U/16 hrs; whereas that of non-kallikrein arginine esterase ranged from 0.06 to (7.11 average) mu/ml, or from 0.01 to 9.78 (1.43 average) U/16 hrs. The ratio between the activity of these two enzymes was 1: : 2.98 (1: 0.42 average). Male dogs had a tendency to show a higher enzyme activity than females for urinary kallikrein and non-kallikrein arginine esterase. Kallikreins, which are included in the category of serine proteases, hydrolyze kininogens to release kinins, and the kinins develop such biological actions as vasodilatation, constriction of smooth muscles of the uterus and ileum, and appearance of pain. Kallikreins themselves also have esterase and amidase activities to the derivatives of some amino acids such as arginine and lysine. Urinary kallikrein that is excreted into the urine is generally thought to originate from the kidney [12, 14]. Assay of an urinary kallikrein activity has been considered an important clinical examination in medical science, and abnormality in the activity has heen reported in such diseases as primary aldosteronism [4, 5, 11, 15], essential hypertension [2, 4, 5, 15], and Bartter's syndrome [3, 16]. Non-kallikrein arginine esterase, which has no kinin-releasing activity but arginineester and -amido hydrolyzing activity, was recently reported in the urine of rats [13], dogs [8], and humans [6, 7]. Accordingly, the assay of urinary kallikrein activity by measuring its arginine-ester or -amido hydrolyzing activity must be reexamined. In the present preliminary examination, we measured the activity of kallikrein and non-kallikrein arginine esterase excreted into the urine of beagle dogs in order to apply the activity assay of these enzymes to clinical examinations in veterinary medicine and laboratory animal science.

2 118 Materials and Methods Dog urine: Urine samples used were collected from 16 beagle dogs during 16 hours (16: 00-08: 00). The dogs, 8 females and 8 males and 7.5 to 13.0 months old, had been individually kept in a metabolic cage set in an airconditioned room with a temperature of 23 }2 Ž and a light/dark cycle of 12 hrs/12 hrs (lights on from 07: 00), and had been daily fed with about 300 g of solid dry food, g LABO D STOCK h(nikon Nosan Kogyo Ltd., Yokohama, Japan) and water ad libitunl. The urine was stored frozen until use after cubic measurement was taken. Chemicals : Diethylaminoethyl (DEAE)- cellulose and D-valyl-L-leucyl-L-arginine-pnitroanilide (Val-Leu-Arg-pNA) were purchased from Nakarai Chemicals Ltd., Kyoto, Japan and Kabi Diagnostica, Stockholm, Sweden, respectively. All the other chemicals used were of analytical reagent grade. Isolation of kallikrein and non-kallikrein arginine esterase : Kallikrein and non-kallikrein arginine esterase were isolated by step-wise Fig. 1, Isolation procedure of urinary kallikrein and non-kallikrein arginine esterase

3 119 elution from the enzyme-adsorbed DEAE-cellulose, based on the method of Matsuda et al. (1984) [8]: 4 ml of each urine sample was diluted with 50 ml of deionized water to lower the conductivity. The diluted urine was then based on that the molar absorption coefficient of p-nitroaniline is 8.8x 10 at 410 nm. The unit of measurement for the enzymes was defined as the activity which catalyzes the hydrolysis of 1ƒÊmol of Val-Leu-Arg-pNA applied to a DEAF-cellulose column (1.0 ~12 cm) equilibrated with 0.02 M Tris-HC1 buffer per minute. at ph 7.5. After rinsed with the same buffer, Results enzymes were eluted first with 0.15 M NaCI in the buffer and secondly 1 M NaCI. The first eluted solution contained non-kallikrein arginine esterase and secondly eluted solution contained kallikrein (Fig. 1). Assay of enzyme activity: Activity of kallikrein and non-kallikrein arginine esterase was measured toward Val-Leu-Arg-pNA as a substrate by a modification of the method of Amundsen et al. (1979) [1]. That is, sample enzyme solution was incubated with substrate solution at 1.4 ~10-4 M substrate concentration at 30 Ž in 0.1 M Tris-HC1 buffer, ph 8.0. After that, an absorbance of the reacted solution was measured at 410 nm and the amount (mol) of substrate hydrolyzed was calculated The activity of kallikrein ranged from 3.87 to (28.48 average) mu/ml of urine, or from 1.05 to (4.56 average) U/16 hrs ; whereas that of non-kallikrein arginine esterase ranged from 0.06 to (7.11 average) mu/ml of urine, or from 0.01 to 9.78 (1.43 average) U/16 hrs. The ratio between the activity of these two enzymes was 1: : 2.98 (1: 0.42 average) (Table 1). Kallikrein and non-kallikrein arginine esterase had a tendency to be higher in male than in female dogs (Table 2) : Kallikrein activity was (22.33 average) mu/ ml or (3.24 average) U/16 hrs in females, whereas it was (34.63 Table 1. Activity of urinary kallikrein and non-kallikrein arginine esterase in beagle dogs Unit of measurement was defined as the enzyme activity which catalyzes the hydrolysis of 1 pmol of Val-Leu-Arg-pNA per minute.

4 120 Fig. 2. Relation between each enzyme activity and urine volume excreted during 16 hours : female, œ : male Fig. 3. Relation between each enzyme activity and dog age : female, œ : male

5 121 Table 2. Comparison of enzyme activity in female and male beagle dogs Values are represented as ranges with the averages in parentheses. average) mu/ml or (5.89 average) U/16 hrs in males. Non-kallikrein arginine esterase activity was (4.05 average) mu/ml or (0.75 average) U/16 hrs in females, whereas it was (10.16 average) mu/ml or (2.11 average) U/16 hrs in males. No remarkable relationship was recognized between the urine volume excreted during 16 hours and each enzyme's activity per ml of urine or per 16 hours (Fig. 2). No relationship was also noticed between each enzyme's activity and the age of the dogs at least within the age group examined, i. e. 7.5 to 13.0 months old (Fig. 3). Discussion Many researchers have studied urinary kallikrein from physiological and pathophysiological viewpoints. The enzyme, nevertheless, has not been sufficiently clarified regarding its physiological function or in relation toarenal physiology, although the enzyme seems to be closely related to the function of the kidney. In addition, it was recently reported that human urinary arginine esterase, which is very similar to canine urinary arginine esterase, will also prove to be related to renal function [9] similarly to urinary kallikrein. In order to elucidate the physiological function of these enzymes and their relation to pathophysiology, the normal range of each enzyme activity must first be determined. In the present examination, we determined the activity of urinary kallikrein and non-kallikrein arginine esterase of dogs. But the values of the activity, especially that of nonkallikrein arginine esterase, varied between samples, although the urine samples used were of beagle dogs kept in a well-controlled circumstances. Accordingly, the activity of these enzymes seems to be changeable over a broad range. Male dogs had a tendency to show higher activity, especially in non-kallikrein arginine esterase, than females. McPartland (1981) [10] described that the activity of urinary esterase A (non-kallikrein argining esterase) of rats is subject to sex differences in the animals. This also appears to be the case for non-kallikrein arginine esterase in dog urine. Concerning the ratio between kallikrein activity and non-kallikrein arginine esterase activity, it was 1: : 2.98 (1: 0.42 average). When a projecting value in dog No. 10 which seems to be exceptional is excluded, the average of the ratio become 1: At any rate, activity of kallikrein was much higher than that of non-kallikrein arginine esterase. Matsuda et al. (1984) [8], on the other hand, reported that, in dog urine, about 55% of the total arginine ester hydrolyzing activity is due to non-kallikrein arginine esterase. Therefore, the ratio of non-kallikrein arginine esterase to kallikrein was much lower in the present examination than that reported by Matsuda et al. (1984) [8]. This discordance in the activity ratio between these two enzymes may be due to the variability of nonkallikrein arginine esterase activity. In fact, non-kallikrein arginine esterase occasionally shows much higher activity than kallikrein (see dog No. 10 in Table 1). Beagle dogs are becoming increasingly important as laboratory animals, and a con-

6 122 siderable body of physiological data on them has been presented by many researchers. We tried to discern the normal activity values of urinary kallikrein and non-kallikrein arginine esterase, and clarified that these activities were variable, espcially that of non-kallikrein arginine esterase. Accordingly, more urine samples are necessary to determine more precisely the normal range of the activity and lurther to elucidate the physiological and pathophysiological meanings of these enzymes. References [1] Amundsen, E., Putter, J., Friberger, P., Knos, M., Larsbraten, M., and Claeson, G. (1979). Methods for the determination of glandular kallikrein by means of a chromogenic tripeptide substrate. In Advances in Experimental Medicine and Biology, Vol. 120A, pp , Fujii, S., Moriya, H., and Suzuki, T. (edit.), Plenum Press, New York. [2] Elliot, A. H., and Nuzum, F. R. (1934). The urinary excretion of a depressor substance (kallikrein of Frey and Kraut) in arterial hypertension. Endocrinology, 18, [3] Lechi, A., Covi, G., Lechi, C., Mantero, F., and Scuro, L. A. (1976). Urinary kallikrein excretion in Bartter's syndrome. J. C'lin. Endocrinol. Metab., 43, [4] Margolius, H. S., Geller, R., Pisano, J. J., and Sjoerdsma, A. (1971). Altered urinary kallikrein excretion in human hypertension. Lancet, No. 7733, [5] Margolius, H. S., Horwitz, D., Pisano, J. J., and Keiser, H. R. (1974). Urinary kallikrein excretion in hypertensive man. Circ. Res., 35, [6] Matsuda, Y., Akihama, S., Fujimoto, Y., Moriya, H., and Miyashita, A. (1983). Studies on human arginine esterase. Yakugaku Zasshi, 103, (in Japanese with English summary). [7] Matsuda, Y., Fujimoto, Y., Akihama, S., and Miyashita, A. (1986). Studies on acidic arginine esterase excreted in urine. II. Purification and some properties of human urinary arginine esterase. Chem. Pharm. Bull., 34, [8] Matsuda, Y., Fujimoto, Y., Akihama, S., and Moriya, H. (1984). Studies on acidic arginine esterase excreted in urine. I. Purification and characterization of dog urinary arginine esterase. Chem. Pharm. Bull., 32, [9] Matsuda, Y., Miyashita, A., Fujimoto, Y., and Akihama, S. (1986). Urinary arginine esterase activity in the patients with hypertension, chronic nephritis and primary aldosteronisru. Jim. J. Clin. Chem., 15, [10] McPartland, R. P., Sustarsic, D. L., and Rapp, J.P. (1981). Evidence for an androgen-dependent urinary arginine esterase in the rat: Separation from other urinary arginine esterase including karlikrein. Endocrinology, 108, [11] Miyashita, A. (1971). Urinary kallikrein determination and its physiological role in human kidney. Jpn. J. Urol., 62, (in Japanese with English abstract). [12] Nustad, K. (1970). The relationship between kidney and urinary kininogenase. Br. J. Pharmacol., 39, [13] Nustad, K., and Pierce, J. V. (1974). Purification of rat urinary kallikrein and their specific antibody. Biochemistry, 13, [14] Nustad, K., Pierce, J. V., and Kirsten Vaaje (1975). Synthesis of kallikreins by rat kidney slices. Br. J. Pharmacol., 53, [15] Seino, M., Abe, K., Otsuka, Y., Saito, T., Irokawa, N., Yasujinia, M., Chiba, S., and Yoshinaga, K. (1975). Urinary kallikrein excretion and sodium metabolism in hypertensive patients. Tohoku J. E.zp. Med., 116, [16] Yamada, K., Watanabe, M., Nishikawa, T., Mikami, K., Shirai, K., Tamura, Y., Yamamoto, M., Kumagai, A., Matsuda, Y., Moriya, H. (1978). Quantitative and qualitative estimation of urinary kallikrein in a patient with Bartter's syndrome. Endocrinol. Japon., 25,

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