Methods have been described to obtain a kininase free substrate from a turpentine-induced rat pleural exudate (24 h post-turpentine) and for the determination of kininase activity and total kininogen in such exudates. With the methods used, a mean of 7.4 min was necessary for 0.25 ml of exudate to inactivate 50% of a given amount (1 µg) of added brady-kinin. Total kininogen in exudate with leukocytes and without leukocytes were 1.97 µg/ml and 1.80 µg/ml, respectively, calculated as bradykinin. It was concluded that most of the kininogen in pleural exudate is present in the cell-free supernatant. Evidence was put forward that the rat uterus-stimulating agents that develop in the exudate upon activation with acetone are kinins not significantly contaminated with other smooth muscle-stimulating agents such as, for instance, histamine and serotonin. The conclusion was drawn that pleural exudate obtained 24 h post-turpentine in the rat is a transudate of rat plasma and that the kinin system could play a role, locally, in bringing about formation of the exudate.
Methods were developed for determining the kininogen fractions, kininase and prekallikrein. The plasma prekallikrein was activated by 20 % (v/v) acetone for about 17 hours (20–24°). Urine kallikrein was prepared by dialysis of urine against running tap water for about 24 hours. Kininase activity was eliminated in plasma, plasma kallikrein and urine kallikrein by incubation at 37° with EDTA‐2Na (1.0 × 10 2 M) for about 24 hours. Kinin assays were carried out on the isolated rat uterus. Released kinin was calculated as μg bradykinin/ml plasma. The total kininogen, whether determined by activation with acetone (16 % v/v for not less than 5 hours) and subsequent incubation with plasma kallikrein, by incubation with plasma kallikrein and then urine kallikrein, or by incubation with acetone (20 % v/v for 17 hours) and subsequent evaporation of the acetone, was found to be the same, 2.0 μg/ml plasma as an average value of 7 plasma batches corresponding to a total of 90 rats (S. D. = 0.09). The average values of kinin released by incubation with plasma kallikrein and by urine kallikrein were 1.5 μg/ml and 1.4 μg/ml plasma respectively with S. D. values of 0.11 and 0.06 respectively. The procedures for kininase and for prekallikrein determinations corresponded closely to previously published methods for estimation of the same parameters in human plasma ( RINVIK, DYRUD & BRISEID 1966 ; BRISEID, DYRUD & ARNTZEN 1968 ).
Indirect evidence has been provided for the presence of 3 kininogen fractions: The average amounts of kinin released by rat plasma kallikrein (1.5 μg/ml plasma, S. E. M. = 0.03) and by rat urine kallikrein (1.4 μg/ml plasma, S. E. M. = 0.03) in 7 plasma batches corresponding to a total of 90 rats, when added up, significantly exceeded the total kininogen (2.0 μg/ml plasma, S. E. M. = 0.04). Methods and materials were as described by BRISEID, DYRUD & ÖIE (1970). It is suggested that plasma kallikrein released kinin from 2 kininogen fractions, S1″ and S1″, and that urine kallikrein released kinin from 2 kininogen fractions, S1″ and S2. Repeated incubation with each of the kininogenase preparations used did not increase the yield of kinin. Soybean trypsin inhibitor did not reduce the amount of kinin released by urine kallikrein; the plasma kallikrein, however, was strongly inhibited. In control experiments leucine aminopeptidase transformed kallidin to bradykinin, but did not increase the kinin activity of the urine kallikrein incubates.
The decrease in the kininogen content of the plasma is dependent on the quantity of intravenously injected carrageenin or glass. A dose response correlationis also evident in animals treated with kaolin, although the curve is comparatively flat.
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