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Experimental arteriosclerosis and the plasma kininogen and low molecular weight kininogen antigen concentration in rabbits
Exp. Path. 27, 221-226 (1985) Department of Cell Pathophysiology of the Postgraduate Medical School, Warsaw, Poland
Experimental arteriosclerosis and the plasma kininogen and low molecular weight kininogen antigen concentration in rabbits By J.
KLENlEWSKI,
E.
HAGEL-LEWICKA
and Z.
LEWICKI
With 5 figures (Received May 25, 1983)
Address for correspondence,' Dr. J6ZEF KLENIEWSKI, Department of Cell Pathophysiology, PostGraduate Medical School, Marymoncka 99, Warsaw, Poland
Key w 0 r d s: arteriosclerosis; cholesterol; kininogen; kininase activity; kininogen antigen concentration; rabbit
Summary A decrease in trypsin-releasable kinin and in low molecular weight kininogen antigen concentrations was observed in plasma of rabbits with cholesterol-induced arteriosclerosis. In rabbits receiving normal diet for 30 days after 30 days of cholesterol almost complete normalization of the morphological status was observed, which was accompanied by normalization of plasma kinin concentration and by higher than normal concentration of kininogen antigen measured. These changes were not observed in animals receiving normal diet for 30 days after 60 days of cholesterol administration. No data were obtained suggesting a massive kinin release during induction of arteriosclerosis by experimental hypercholesteremia. The possibility of intense kinin formation during a period of repair of the morphological changes was discussed.
Introduction The morphology of arteriosclerosis and numerous biochemical disturbances in the blood resulting from this process are relatively well known, though the mechanism of the development of the latter has not been elucidated until now. The kallikrein-kininogen-kinin-kininase system represents one of those which may be either disturbed by arteriosclerosis or participate in the development of this process, as kinins besides their ability to cause migration of polymorphonuclears, contraction of smooth muscles and pain feeling exert also a high activity in dilating the blood vessels and increasing their permeability (1). The purpose of this work was to investigate the effect of experimental cholesterol arteriosclerosis on plasma kininogens in rabbits.
Materials and Methods White rabbits of Popielno strain, 2.0-2.5 kg of initial body weight were used. The animals were allocated to 4 groups, each of them consisting of 5 rabbits. The animals were fed with a standard granulated food and for drinking they were given: Group 1: cow milk for 90 days, Group 2: cow milk with cholesterol for 30 days, Groups 3 and 4: cow milk with cholesterol for 30 and 60 days respectively, followed by milk without addition of cholesterol for further 30 days. The concentration of cholesterol in milk was adjusted to a dose of roughly 1.0 g/kg body weight! day. This work was supported by Grant No. W. 10.S.
221
The concentration of cholesterol in the serum of the animals of group 1 was 92 ± 12 mg% and remained constant during the duration of experiment, and in rabbits receiving this substance for 30 days or longer it varied from 350 mg% to 1200 mg%. The animals of groups 1 and 4 were killed after 90 days and those of group 2 and 3 after 30 and 60 days respectively. The rabbits were put to sleep by intravenous administration of Vetbutal and thereafter bled by cardiopuncture. For morphological examination fragments of the descending part of arcus aortae were taken. The material for histological examination was fixed in 4 % buffered formalin and embedded in paraffin. The sections were stained with hematoxylin and eosin and using the PAS method. Blood samples were taken by puncture of central ear artery, than given into the vessels eontaining 1 vol. of 3.8 % sodium citrate per 9 vol. of the blood, before and every 30th day of the experiment. The plasma was separated by 10 min centrifugation of the blood at 3000 x g. Kinins were liberated by 30 min incubation at 37°C with trypsin (0.2 mg/ml) of plasma samples heated previously for 2 h at 61°C. The reaction was terminated by addition of soya bean trypsin inhibitor (0.23 mg/ml). The concentration of kinin liberated was estimated using a rat uterine horn (2) and synthetic bradykinin as a standard. The antigenic concentration of plasma low molecular weight kininogen was determined by electroimmunodiffusion (6) in 1 % agarose containing 0.25 % addition of sheep antiserum against this protein. Pooled plasma of 15 healthy rabbits was used as a standard for the measurement of plasma kininogen antigen concentration. Rabbit plasma low molecular weight kininogen was isolated and the antiserum against it was produced as described (3). The concentration of cholesterol in serum was estimated according to ZLATKIS et al. (8). Protein concentration was measured using the Folin-phenol reaction (7) and bovine serum albumin as a standard. The reagents used were the same as in former experiments (4, 5). Statistical analysis was performed using Student's t test; mean and standard deviation values were given. Statistical differences expressed in P were calculated from t-values.
Fig. 1 a. Fragment of the aorta of a rabbit from the control group. HE, x 400. Fig. 1 b. Fragment of the aorta of a rabbit fed with cholesterol for 30 days. HE, x 400.
222
Exp. Path. 27 (1985) 4
•l. •
Fig. 2. The effect of 30 days cholesterol administration on plasma kinin and low molecular weight kininogen antigen concentration. Fig. 3. The effect of 30 days normal diet on plasm a kinin and low molecular weight kininogen antigen concentration in rabbits receiving previously chol.esterol for 30 days.
Results No changes in aortas were found in morphological examinations of rabbits of th e control group (fig. 1 a). In rabbits fed with cholesterol for 30 days, fl at or elevated focal tarnishing of the intima and flat parietal thrombi were seen. The thickening of th e intima was formed by foamy cells, situated within th e enlarged sub endotheli al area and in so me cases between the first and second elastic lamina. Within cells of the endothelium and in the subendothelial area oedema was obs erved (fig. 1 b). In plasma of rabbits of th e co ntrol group no changes of the concentration of both kinins released "in vitro" and of low mol ec ular weight kininogen antigen were found during the experiment. In fig. 2, values of the co ncentration of kinins released by trypsin and of low molecular weight kininogen antigen in plasma of 15 rabbits receiving cholesterol diet for 30 days (belonging to groups 2, 3 and 4) are presented. A statistically significant and co mparable decrease in the concentration of kininogen antigen and kinin in plasma of these animals was noted during a given tim e of observation. The values of both parameters measured separately in 5 rabbits of group 3 were lower after 30 days of cholesterol administration than at the beginning of the experiment (fig. 3). The concentration of trypsin releasable kinin was more or less norm al and the low molecular weight kininogen anti gen concentration in plasma of these rabbits was even higher than normal when the anim als were fed with normal diet for 30 days after 30 days of cholest erol administration. As it is seen from fig. 4 the values of plasma kinin and of kininogen antigen concentration in 5 rabbits of group 4 after 60 days of cholesterol administration were lower than normal and more or less equal to that after 30 days of cholesterol diet. Feeding of these anim als with normal diet for additional 30 days did not cause norm alization of the values of both parameters measured. Fig. 5 shows mean valu es of plasma kininase activity in rabbits of an expflrimflnt.a.1 gTnllp. Normal kininase activity was found in plasma of rabbits fed with normal diet for 30 days after 30 days of cholesterol administration. Lower th an normal kininase activity was stated in plasma of rabbits fed with cholest erol for 30 days and in those animals which received Exp. Path. 27 (1985) 4
223
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CJ 1M tIJtt ~ (J/ dItJiatltfIl did .. attN GO d.!'P (J/ ~ . • j() daJ/s fII normal dil'/ GOd
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Fig. 4. The concentration of low molecular weight kininogen antigen and of kinin in plasma of rabbits fed for 30 days with normal diet after 60 days administration of cholesterol.
,
~
~
""' fII incubaUOtl (m II) PtO¥rlQ r/tobbCt.s ; 0 htalthlJ; . ,~ forJlJIJalJS with dJoltsteroi ditto A nMrif!C} normal dltt for 30 days alttr JOdal,Js
o
dIolt$~
• nc ·lIing normol dit for JOdaljs afttr 6fJ datp of cho~sterol
Fig. 5. Kininase activity of plasma in rabbits of different experimental groups. 1.9 ml of bradykinin in 0.15 M NaCI were mixed with 0.1 ml of the plasma, the mixture was incubated at 37°C and at given time intervals 0.1 ml was tested for bradykinin activity.
normal diet for 30 days after 60 days of cholesterol administration. Statistically highly significant differences were found (P < 0.002) when the values of bradykinin concentration in mixture with normal plasma at the 4th and 8th min of the experiment were compared to those found in a mixture of this peptide with plasmas of the latter groups of animals.
Discussion In the plasma of rabbits as well as of human beiings the low molecular weight kininogen represents about 85 % of both kininogens (3). In rabbits fed a cholesterol diet the reduction in low molecular weight kininogen antigen concentration exceeds 25 % and indicates a real
224
Exp. Path. 27 (1985) 4
decrease in the level of this protein under experimental conditions. The parallel decrease in the concentration of the plasma kinin source suggests the possibility of decreased synthesis of both plasma kininogens in effect of changes caused by hypercholesteremia. Devoid of kinins human kininogen preserves its antigenic properties (4, 5, 8). On the other hand, kinins released in the circulation are rapidly destroyed by kininases. If antigenic properties of rabbit kininogens are, like human ones, resistant to the action of kininogenases and if under experimental hypercholesteremia a profuse liberation of kinins occurred, a major decrease in the concentration of trypsin-releasable kinin without significant changes in th e concentration of kininogen antigen in the plasma should result. Such a phenomenon has not been observed in present investigations and the percentage of decrease in trypsin· releasable plasma kinin concentration was similar to reduction of the main plasma kininogen antigen concentration. This seems to indicate that a massive, at least, kinin release does not occur in arteriosclerosis induced by experimental hypercholesteremia. The mechanism of the decrease in plasma kininogen antigen concentration is still unclear because this reduction might result from morphological changes due to arteriosclerotic process, or could be of pure biochem ical nature. In this regard the results obtained in rabbits receiving normal diet after 30 or 60 days administration of cholesterol are of interest. A complete normalization of plasma kinin concentration in rabbits resting for 30 days after the same periode of cholesterol diet was found. In the same experimental condition the concentration of kininogen antigen measured was found to be even higher than normal. Thus in these animals the concentration of th e main plasma kininogen, as measured with an immunological method, was higher than measured indirectly by estimation of trypsin-released kinins, even when the presence of high molecular weight kininogen in the plasma as a source of kinins was ignored. This indicates the presence of kinin-free kininogen derivatives in the plasma of rabbits fed with normal diet for 30 days after 30 days of cholesterol administration, which points to an intensive kinin release in these rabbits. In rabbits, which prior to administration of normal diet lasting for 30 days were given cholesterol for 60 days, no normalization of the concentration of trypsin-releasable kinin and of kininogen antigen in the plasma was observed. In the light of these facts it is of interest that normalization of both kinin and kininogen antigen concentration found in rabbits of group 3 was accompanied by almost complete normalization of the morphological status. On the other hand, in rabbits of group 4, in which no distinct remission of morphological arteriosclerotic changes after 30 days of normal diet was found, no normalization of trypsin-releasable kinin and of kininogen antigen concentration was observed, too. The site of kininogen synth esis remains unknown. The present results seem to indicate that this process may occur in tissues which are damaged by arteriosclerotic changes. The results of the present study point to a relationship between experimental arteriosclerosis and kininogen metabolism, however the question of participation of the kinin system in development of arteriosclerosis remains unknown. The decrease in plasma kininogen concentration found in rabbits in th e course of cholesterol arteriosclerosis may suggest that the effectiveness of the kinin system was similarly decreased. Such supposition seems to be not sufficiently justified, as particularly in the same animals, in which cholesterol administration caused reduction of plasma kininogens, a decrease in kininase activity of the plasma was found. As a consequence the kinins liberated even in subnormal concentration could be more biologically effective due to decreased activity of enzymes responsible for their decomposition.
Literature 1. ERDOS, E. G., Hypotensive peptides: bradykinin, kallidin and eledoisin. Advan. Pharmacol. 4,
2.
15
1-90 (1966). DE JALON, P. G., Y. M. BAYO and M. G. DE JALON, Sensible y nuevo metods de valoretion de adrendina en utero osseaus de rata. Farmacoterap. Actual. 2, 313 (1945).
Exp. Path. 27 (1986) 4
220
3. KLENIEWSKI, J., and E. HAGEL-LEWICKA, Rabbit plasma low molecular weight kininogen: isolation from plasma and some physicochemical properties. Brit. J. Pharmacol. (in press). 4. - V. H. DONALDSON and C. J. WAGNER, Some molecular and functional changes in high molecular weight kininogen induced by plasmin and trypsin. Thromb. Res. 25, 387-399 (1982). 5. - Plasma high molecular weight kininogen concentration in health and in chosen impairments of haemostasis: evidence that plasmin uncovers a new antigenic site in high molecular weight kininogen. Thromb. Haemostas. 42, 1046-1055 (1979). 6. LAURELL, C. B., Quantitative estimation of proteins by electrophoresis in agarose containing antibodies. Anal. Biochem. 15, 45-51 (1966). 7. LOWRY, O. H., N. J. ROSENBROUGH, A. L. FARlt and R. J. RANDALL, Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193, 265-275 (1951). 8. ZLATKIS, A., B. ZAK and A. J. BOYLE, Colorimetric determination of total cholesterol in serum. J. Lab. CUn. Med. 41, 486-491 (1953).
226
Exp. Path. 27 (1985) 4
Experimental arteriosclerosis and the plasma kininogen and low molecular weight kininogen antigen concentration in rabbits By J.
KLENlEWSKI,
E.
HAGEL-LEWICKA
and Z.
LEWICKI
With 5 figures (Received May 25, 1983)
Address for correspondence,' Dr. J6ZEF KLENIEWSKI, Department of Cell Pathophysiology, PostGraduate Medical School, Marymoncka 99, Warsaw, Poland
Key w 0 r d s: arteriosclerosis; cholesterol; kininogen; kininase activity; kininogen antigen concentration; rabbit
Summary A decrease in trypsin-releasable kinin and in low molecular weight kininogen antigen concentrations was observed in plasma of rabbits with cholesterol-induced arteriosclerosis. In rabbits receiving normal diet for 30 days after 30 days of cholesterol almost complete normalization of the morphological status was observed, which was accompanied by normalization of plasma kinin concentration and by higher than normal concentration of kininogen antigen measured. These changes were not observed in animals receiving normal diet for 30 days after 60 days of cholesterol administration. No data were obtained suggesting a massive kinin release during induction of arteriosclerosis by experimental hypercholesteremia. The possibility of intense kinin formation during a period of repair of the morphological changes was discussed.
Introduction The morphology of arteriosclerosis and numerous biochemical disturbances in the blood resulting from this process are relatively well known, though the mechanism of the development of the latter has not been elucidated until now. The kallikrein-kininogen-kinin-kininase system represents one of those which may be either disturbed by arteriosclerosis or participate in the development of this process, as kinins besides their ability to cause migration of polymorphonuclears, contraction of smooth muscles and pain feeling exert also a high activity in dilating the blood vessels and increasing their permeability (1). The purpose of this work was to investigate the effect of experimental cholesterol arteriosclerosis on plasma kininogens in rabbits.
Materials and Methods White rabbits of Popielno strain, 2.0-2.5 kg of initial body weight were used. The animals were allocated to 4 groups, each of them consisting of 5 rabbits. The animals were fed with a standard granulated food and for drinking they were given: Group 1: cow milk for 90 days, Group 2: cow milk with cholesterol for 30 days, Groups 3 and 4: cow milk with cholesterol for 30 and 60 days respectively, followed by milk without addition of cholesterol for further 30 days. The concentration of cholesterol in milk was adjusted to a dose of roughly 1.0 g/kg body weight! day. This work was supported by Grant No. W. 10.S.
221
The concentration of cholesterol in the serum of the animals of group 1 was 92 ± 12 mg% and remained constant during the duration of experiment, and in rabbits receiving this substance for 30 days or longer it varied from 350 mg% to 1200 mg%. The animals of groups 1 and 4 were killed after 90 days and those of group 2 and 3 after 30 and 60 days respectively. The rabbits were put to sleep by intravenous administration of Vetbutal and thereafter bled by cardiopuncture. For morphological examination fragments of the descending part of arcus aortae were taken. The material for histological examination was fixed in 4 % buffered formalin and embedded in paraffin. The sections were stained with hematoxylin and eosin and using the PAS method. Blood samples were taken by puncture of central ear artery, than given into the vessels eontaining 1 vol. of 3.8 % sodium citrate per 9 vol. of the blood, before and every 30th day of the experiment. The plasma was separated by 10 min centrifugation of the blood at 3000 x g. Kinins were liberated by 30 min incubation at 37°C with trypsin (0.2 mg/ml) of plasma samples heated previously for 2 h at 61°C. The reaction was terminated by addition of soya bean trypsin inhibitor (0.23 mg/ml). The concentration of kinin liberated was estimated using a rat uterine horn (2) and synthetic bradykinin as a standard. The antigenic concentration of plasma low molecular weight kininogen was determined by electroimmunodiffusion (6) in 1 % agarose containing 0.25 % addition of sheep antiserum against this protein. Pooled plasma of 15 healthy rabbits was used as a standard for the measurement of plasma kininogen antigen concentration. Rabbit plasma low molecular weight kininogen was isolated and the antiserum against it was produced as described (3). The concentration of cholesterol in serum was estimated according to ZLATKIS et al. (8). Protein concentration was measured using the Folin-phenol reaction (7) and bovine serum albumin as a standard. The reagents used were the same as in former experiments (4, 5). Statistical analysis was performed using Student's t test; mean and standard deviation values were given. Statistical differences expressed in P were calculated from t-values.
Fig. 1 a. Fragment of the aorta of a rabbit from the control group. HE, x 400. Fig. 1 b. Fragment of the aorta of a rabbit fed with cholesterol for 30 days. HE, x 400.
222
Exp. Path. 27 (1985) 4
•l. •
Fig. 2. The effect of 30 days cholesterol administration on plasma kinin and low molecular weight kininogen antigen concentration. Fig. 3. The effect of 30 days normal diet on plasm a kinin and low molecular weight kininogen antigen concentration in rabbits receiving previously chol.esterol for 30 days.
Results No changes in aortas were found in morphological examinations of rabbits of th e control group (fig. 1 a). In rabbits fed with cholesterol for 30 days, fl at or elevated focal tarnishing of the intima and flat parietal thrombi were seen. The thickening of th e intima was formed by foamy cells, situated within th e enlarged sub endotheli al area and in so me cases between the first and second elastic lamina. Within cells of the endothelium and in the subendothelial area oedema was obs erved (fig. 1 b). In plasma of rabbits of th e co ntrol group no changes of the concentration of both kinins released "in vitro" and of low mol ec ular weight kininogen antigen were found during the experiment. In fig. 2, values of the co ncentration of kinins released by trypsin and of low molecular weight kininogen antigen in plasma of 15 rabbits receiving cholesterol diet for 30 days (belonging to groups 2, 3 and 4) are presented. A statistically significant and co mparable decrease in the concentration of kininogen antigen and kinin in plasma of these animals was noted during a given tim e of observation. The values of both parameters measured separately in 5 rabbits of group 3 were lower after 30 days of cholesterol administration than at the beginning of the experiment (fig. 3). The concentration of trypsin releasable kinin was more or less norm al and the low molecular weight kininogen anti gen concentration in plasma of these rabbits was even higher than normal when the anim als were fed with normal diet for 30 days after 30 days of cholest erol administration. As it is seen from fig. 4 the values of plasma kinin and of kininogen antigen concentration in 5 rabbits of group 4 after 60 days of cholesterol administration were lower than normal and more or less equal to that after 30 days of cholesterol diet. Feeding of these anim als with normal diet for additional 30 days did not cause norm alization of the values of both parameters measured. Fig. 5 shows mean valu es of plasma kininase activity in rabbits of an expflrimflnt.a.1 gTnllp. Normal kininase activity was found in plasma of rabbits fed with normal diet for 30 days after 30 days of cholesterol administration. Lower th an normal kininase activity was stated in plasma of rabbits fed with cholest erol for 30 days and in those animals which received Exp. Path. 27 (1985) 4
223
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....
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s
~V 8c:i
<:0
c:s
::::-
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Ill.
Ill.
-...;
,& -I :~
,J
CJ 1M tIJtt ~ (J/ dItJiatltfIl did .. attN GO d.!'P (J/ ~ . • j() daJ/s fII normal dil'/ GOd
m
01 ~
Fig. 4. The concentration of low molecular weight kininogen antigen and of kinin in plasma of rabbits fed for 30 days with normal diet after 60 days administration of cholesterol.
,
~
~
""' fII incubaUOtl (m II) PtO¥rlQ r/tobbCt.s ; 0 htalthlJ; . ,~ forJlJIJalJS with dJoltsteroi ditto A nMrif!C} normal dltt for 30 days alttr JOdal,Js
o
dIolt$~
• nc ·lIing normol dit for JOdaljs afttr 6fJ datp of cho~sterol
Fig. 5. Kininase activity of plasma in rabbits of different experimental groups. 1.9 ml of bradykinin in 0.15 M NaCI were mixed with 0.1 ml of the plasma, the mixture was incubated at 37°C and at given time intervals 0.1 ml was tested for bradykinin activity.
normal diet for 30 days after 60 days of cholesterol administration. Statistically highly significant differences were found (P < 0.002) when the values of bradykinin concentration in mixture with normal plasma at the 4th and 8th min of the experiment were compared to those found in a mixture of this peptide with plasmas of the latter groups of animals.
Discussion In the plasma of rabbits as well as of human beiings the low molecular weight kininogen represents about 85 % of both kininogens (3). In rabbits fed a cholesterol diet the reduction in low molecular weight kininogen antigen concentration exceeds 25 % and indicates a real
224
Exp. Path. 27 (1985) 4
decrease in the level of this protein under experimental conditions. The parallel decrease in the concentration of the plasma kinin source suggests the possibility of decreased synthesis of both plasma kininogens in effect of changes caused by hypercholesteremia. Devoid of kinins human kininogen preserves its antigenic properties (4, 5, 8). On the other hand, kinins released in the circulation are rapidly destroyed by kininases. If antigenic properties of rabbit kininogens are, like human ones, resistant to the action of kininogenases and if under experimental hypercholesteremia a profuse liberation of kinins occurred, a major decrease in the concentration of trypsin-releasable kinin without significant changes in th e concentration of kininogen antigen in the plasma should result. Such a phenomenon has not been observed in present investigations and the percentage of decrease in trypsin· releasable plasma kinin concentration was similar to reduction of the main plasma kininogen antigen concentration. This seems to indicate that a massive, at least, kinin release does not occur in arteriosclerosis induced by experimental hypercholesteremia. The mechanism of the decrease in plasma kininogen antigen concentration is still unclear because this reduction might result from morphological changes due to arteriosclerotic process, or could be of pure biochem ical nature. In this regard the results obtained in rabbits receiving normal diet after 30 or 60 days administration of cholesterol are of interest. A complete normalization of plasma kinin concentration in rabbits resting for 30 days after the same periode of cholesterol diet was found. In the same experimental condition the concentration of kininogen antigen measured was found to be even higher than normal. Thus in these animals the concentration of th e main plasma kininogen, as measured with an immunological method, was higher than measured indirectly by estimation of trypsin-released kinins, even when the presence of high molecular weight kininogen in the plasma as a source of kinins was ignored. This indicates the presence of kinin-free kininogen derivatives in the plasma of rabbits fed with normal diet for 30 days after 30 days of cholesterol administration, which points to an intensive kinin release in these rabbits. In rabbits, which prior to administration of normal diet lasting for 30 days were given cholesterol for 60 days, no normalization of the concentration of trypsin-releasable kinin and of kininogen antigen in the plasma was observed. In the light of these facts it is of interest that normalization of both kinin and kininogen antigen concentration found in rabbits of group 3 was accompanied by almost complete normalization of the morphological status. On the other hand, in rabbits of group 4, in which no distinct remission of morphological arteriosclerotic changes after 30 days of normal diet was found, no normalization of trypsin-releasable kinin and of kininogen antigen concentration was observed, too. The site of kininogen synth esis remains unknown. The present results seem to indicate that this process may occur in tissues which are damaged by arteriosclerotic changes. The results of the present study point to a relationship between experimental arteriosclerosis and kininogen metabolism, however the question of participation of the kinin system in development of arteriosclerosis remains unknown. The decrease in plasma kininogen concentration found in rabbits in th e course of cholesterol arteriosclerosis may suggest that the effectiveness of the kinin system was similarly decreased. Such supposition seems to be not sufficiently justified, as particularly in the same animals, in which cholesterol administration caused reduction of plasma kininogens, a decrease in kininase activity of the plasma was found. As a consequence the kinins liberated even in subnormal concentration could be more biologically effective due to decreased activity of enzymes responsible for their decomposition.
Literature 1. ERDOS, E. G., Hypotensive peptides: bradykinin, kallidin and eledoisin. Advan. Pharmacol. 4,
2.
15
1-90 (1966). DE JALON, P. G., Y. M. BAYO and M. G. DE JALON, Sensible y nuevo metods de valoretion de adrendina en utero osseaus de rata. Farmacoterap. Actual. 2, 313 (1945).
Exp. Path. 27 (1986) 4
220
3. KLENIEWSKI, J., and E. HAGEL-LEWICKA, Rabbit plasma low molecular weight kininogen: isolation from plasma and some physicochemical properties. Brit. J. Pharmacol. (in press). 4. - V. H. DONALDSON and C. J. WAGNER, Some molecular and functional changes in high molecular weight kininogen induced by plasmin and trypsin. Thromb. Res. 25, 387-399 (1982). 5. - Plasma high molecular weight kininogen concentration in health and in chosen impairments of haemostasis: evidence that plasmin uncovers a new antigenic site in high molecular weight kininogen. Thromb. Haemostas. 42, 1046-1055 (1979). 6. LAURELL, C. B., Quantitative estimation of proteins by electrophoresis in agarose containing antibodies. Anal. Biochem. 15, 45-51 (1966). 7. LOWRY, O. H., N. J. ROSENBROUGH, A. L. FARlt and R. J. RANDALL, Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193, 265-275 (1951). 8. ZLATKIS, A., B. ZAK and A. J. BOYLE, Colorimetric determination of total cholesterol in serum. J. Lab. CUn. Med. 41, 486-491 (1953).
226
Exp. Path. 27 (1985) 4