Neurochemical Research, Vol. 4, No. 6, 1979
CYCLIC 3',5'-ADENOSINE MONOPHOSPHATE PHOSPHODIESTERASE (cAMP PDE) AND CYCLIC 3',5'-GUANOSINE MONOPHOSPHATE PHOSPHODIESTERASE (cGMP PDE) IN MICROVESSELS ISOLATED FROM BOVINE CORTEX V. STEFANOVICH Hoechst Aktiengesellschaft Frankfurt am Main Werk Albert 6200 Wiesbaden 12, Germany
Accepted March 26, 1979
It was established that microvessels of a bovine cortex exhibit significant cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic 3%5'guanosine monophosphate phosphodiesterase (cGMP PDE) activities. These activities are dependent on the presence of Mg ~+. Absence of Ca z~ was virtually without effect. When both Mg 2+ and Ca 2+ were absent, PDE activities increased compared with activities observed in the absence of Mg2+. Xanthines (caffeine, theobromine, and theophylline) were better inhibitors of cAMP PDE than ofcGMP PDE. Imidazole, in very high concentration (1 x 10 2 M) only, exhibited PDE stimulatory activity at high concentrations of both substrates. Otherwise, it exhibited PDE-inhibitory properties.
INTRODUCTION The study of the metabolism of blood vessels in healthy and diseased states is very important in view of the high incidence of atherosclerosis and other cardiovascular diseases. Large vessels, such as human and animal aortas, have been the subject of many, sometimes extensive, biochemical studies. Microvessels, defined as blood vessels with a diameter 681 0364-3190/79/1200-0681503.00/0 9 1979 Plenum Publishing Corporation
682
v. STEFANOVICH
below 300 ~m, have not been the subject of biochemical studies until recently, in 1974, Brendel, Meezan, and Carlson (1) developed a method for isolation of microvessels from bovine cortex by using a simple procedure of trapping the microvessels on nylon sieves. The same authors have shown that the metabolic activity of cortex microvessels is significantly higher (5 to 10 times) than the metabolic activity of large vessels such as aorta. It appears therefore that microvessels isolated from bovine cortex can be useful for studying various metabolic events in vascular tissue. In this report the results of our study of cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic 3',5'-guanosine monophosphate phosphodiesterase (cGMP PDE) are presented.
EXPERIMENTAL PROCEDURE Microvessels of bovine cortex were isolated mainly as described by Brendel et al. (1). Bovine brain was obtained from the City Slaughter House in Wiesbaden, Germany, immediately after slaughter. Cortex was homogenized for 10 sec in Early's salt solution buffered with HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; 1 : 1 v/v, pH 7.4) by using an ESGE homogenizer, type M 100. Further homogenization was achieved with 10 strokes of a loosely fitting Teflon pestle in a glass tube. The homogenate was again diluted with buffer (1 : 1 v/v) and poured over a 200-~m nylon sieve (Schweiz, Seidengazefabrik, Zfirich) and filtered under a slight vacuum. The filtrate was passed through the same filter again, and this was repeated 8-10 times. The collected microvessels were washed with buffer with energetic shaking and filtered. This procedure was repeated several times, until microvessels were freed of any nonvascular contamination. The purity of microvessels during isolation was monitored by a television set-up coupled with a light microscope. A typical preparation yielded 800 mg from 100 g (wet weight) of bovine cerebral cortex. In experiments where homogenate was used for determination of cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic Y,5'-guanosine monophosphate phosphodiesterase (cGMP PDE) activities, the microvessels were suspended in a buffer (1 : l w/v) and homogenized for 3 min in a glassto-glass homogenizer. Further homogenization was achieved by sonication for 60 sec (40 watts, Branson sonifier, model B 12, Branson Sonic Power Co., Danbury, Connecticut). After centrifugation for 30 rain at 30,000 g, the supernatant was used for determination of cAMP PDE and cGMP PDE activities by the method of Pichard and Cheung (2). Substrate concentration was 0.2 mM and 1.0/~M for cAMP, and 0.1 mM and 0.1 ~xMfor cGMP. Amberlite IRP-58 (Chemical Dynamic Corp., South Plainfield, New York) was used as an ion-exchange resin. Doses necessary for 50% inhibition (ID30) of PDE activity by different agents were determined graphically from inhibition data obtained for at least six different concentrations. Each value represents an average of four determinations. All chemicals were obtained in analytical purity from commercial sources. Imidazole was obtained from E. Merck, Darmstadt, Germany.
683
c A M P P D E A N D c G M P PDE IN B O V I N E M I C R O V E S S E L S
RESULTS Our first experiments indicated that microvessels of bovine cortex possess significant cAMP PDE and cGMP PDE activities (Table I). It was therefore of interest to investigate the influence of various substances, including normal cell constituents on the activities of cAMP PDE and cGMP PDE. In assessing the effect of calcium and magnesium ions on cAMP PDE and cGMP PDE activities, it should be noted that Pichard and Cheung's (2) reaction mixture used for determination of PDE activities contains 5 mM MgSO4 and 50 IxM CaC12. Without Ca 2+ only insignificant changes in cAMP PDE and cGMP PDE activities (Table I) were observed. Absence of Mg 2+, however, produced a significant decrease in activities of both cAMP PDE and cGMP PDE at both substrate concentrations. The most pronounced decrease in activity was observed in the case of high-affinity (1.0 ~zM of substrate) cAMP PDE. When both Ca 2+ and M g 2+ w e r e omitted from the reaction mixture, PDE activities for cAMP and cGMP increased significantly compared' with results obtained in the absence of magnesium. The influence of cyclic nucleotides on the inhibition of PDE's is presented in Table II. It can be seen in this table that cAMP is a reasonably good inhibitor for cGMP PDE. The same was observed for cAMP PDE in respect to cGMP. The least inhibited was low affinity cGMP PDE by cAMP.
TABLE I I N F L U E N C E OF C A 2+ AND M G 2+ ON ACTIVITIES OF PHOSPHODIESTERASES Substrate concentration 0.2 m M c A M P 1.0 ix]VI c A M P 0.1 m M c G M P 0.1 p.M c G M P
N o r m a l values 9.20 • i0 n (46.5%) 6.01 x 10 -2 (60.1%) 5.73 x 10 (57.3%) 5.50 • 10 3 (55.0%)
- C a 2+ 8.52 x 10 (42.6%) 5.70 x 10 -z (57.0%) 6.06 x 10 (60.6%) 6.50 • 10 .3 (65.0%)
cAMP AND cGMP
-Mr + 5.08 x 10 (25.4%) 1.42 x 10 .2 (14.2%) 4.07 x 10 (40.7%) 2.96 • 10 -3 (29.6%)
Ca 2+, Mg z+ 7.48 x 10 (37.4%) 2.77 x 10 -2 (27.7%) 5.44 • 10 (54.5%) 4.69 • 10 .3 (46.9%)
" Data e x p r e s s e d in quantities of hydrolyzed substrate nmol/min/mg protein. All data are averages of three e x p e r i m e n t s with four determinations each. Results in parenthesis s h o w percents of hydrolysis of substrate.
684
v. STEFANOVICH
T A B L E II INHIBITION OF c A M P PHOSPHODIESTERASE (cAMP PDE) AND c G M P P H O S P H O D I E S T E R A S E ( c G M P P D E ) BY CYCLIC N U C L E O T I D E a % of hydrolysis of substrate in normal condition c G M P concn, 5 1 5 1 5 1
x x x x x x
10 - 3 M 10 - 3 M 10-4M 10-4M 10 -5 M 10 -5 M IDso
cAMP 0.2 m M
cGMP 1.0 ~M
54.9 56.3 inhibition in % 102.9 86.2 82.0 61.0 44.1 25.2 6.5 x 10-SM 7.5
0.1 m M
56.4 69.3 inhibition in %
c A M P concn,
93.2 78.1 74.9 58.1 39.0 59.4 x 10-SM
5 1 5 1 5
x 10 - 3 M z 10 - 3 M x 10-4M x 10-4M x 10-SM 1 x 10 5
0.1 ~ M
81.5 56.3 57.6 36.1 28.2 15.9 2.9 x 10-4M 8.0
91.9 83.9 75.1 56.4 39.8 19.9 x 10 SM
a For experimental details see Experimental Procedure.
TABLE III INHIBITION OF cAMP P H O S P H O D I E S T E R A S E AND cGMP PHOSPHODIESTERASE ISOLATED FROM B O V I N E C O R T E X MICROVESSELS BY T H E O P H Y L L I N E , CAFFEINE, AND THEOBROMINE cAMP
Theophylline Caffeine Theobromine
cGMP
0.2 m M
1.0 ixM
0.1 m M
0.1 tzM
4.80 • 10 4 M . 5.25 • 10 4 M 6.90 x 10 -4 M
1.48 x 1 0 - 4 M 2.35 x 10 - 4 M 3.60 x 10 -4 M
9.25 x 1 0 - 4 M 1.60 • 1 0 - 3 M 1.38 x 10 -3 M
9.50 x 1 0 - 4 M 2.90 x 1 0 - 4 M 3.42 x 10 4 M
IDs0 value obtained as described under Experimental Procedure.
The inhibition of cAMP PDE and cGMP PDEs by three classical xanthines, theophylline (1,3-dimethyl xanthine), theobromine (3,7-dimethyl xanthine), and caffeine (1,3,7-trimethyl xanthine), is presented in Table III. It can be seen in this table that somewhat similar values were obtained for all three xanthines. All three xanthines were more effective inhibitors of cAMP PDE than of cGMP PDE. The lowest inhibitory activity observed was that of caffeine and theobromine on low-affinity cGMP PDE. The effect of imidazole on cAMP PDE and cGMP PDE is presented in Table IV. It can be seen in this table that under the conditions of our
685
cAMP PDE A N D cGMP PDE IN BOVINE MICROVESSELS
CYCLIC 3',5'-ADENOSINE MONOPHOSPHATE PHOSPHODIESTERASE (cAMP PDE) AND CYCLIC 3',5'-GUANOSINE MONOPHOSPHATE PHOSPHODIESTERASE (cGMP PDE) IN MICROVESSELS ISOLATED FROM BOVINE CORTEX V. STEFANOVICH Hoechst Aktiengesellschaft Frankfurt am Main Werk Albert 6200 Wiesbaden 12, Germany
Accepted March 26, 1979
It was established that microvessels of a bovine cortex exhibit significant cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic 3%5'guanosine monophosphate phosphodiesterase (cGMP PDE) activities. These activities are dependent on the presence of Mg ~+. Absence of Ca z~ was virtually without effect. When both Mg 2+ and Ca 2+ were absent, PDE activities increased compared with activities observed in the absence of Mg2+. Xanthines (caffeine, theobromine, and theophylline) were better inhibitors of cAMP PDE than ofcGMP PDE. Imidazole, in very high concentration (1 x 10 2 M) only, exhibited PDE stimulatory activity at high concentrations of both substrates. Otherwise, it exhibited PDE-inhibitory properties.
INTRODUCTION The study of the metabolism of blood vessels in healthy and diseased states is very important in view of the high incidence of atherosclerosis and other cardiovascular diseases. Large vessels, such as human and animal aortas, have been the subject of many, sometimes extensive, biochemical studies. Microvessels, defined as blood vessels with a diameter 681 0364-3190/79/1200-0681503.00/0 9 1979 Plenum Publishing Corporation
682
v. STEFANOVICH
below 300 ~m, have not been the subject of biochemical studies until recently, in 1974, Brendel, Meezan, and Carlson (1) developed a method for isolation of microvessels from bovine cortex by using a simple procedure of trapping the microvessels on nylon sieves. The same authors have shown that the metabolic activity of cortex microvessels is significantly higher (5 to 10 times) than the metabolic activity of large vessels such as aorta. It appears therefore that microvessels isolated from bovine cortex can be useful for studying various metabolic events in vascular tissue. In this report the results of our study of cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic 3',5'-guanosine monophosphate phosphodiesterase (cGMP PDE) are presented.
EXPERIMENTAL PROCEDURE Microvessels of bovine cortex were isolated mainly as described by Brendel et al. (1). Bovine brain was obtained from the City Slaughter House in Wiesbaden, Germany, immediately after slaughter. Cortex was homogenized for 10 sec in Early's salt solution buffered with HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; 1 : 1 v/v, pH 7.4) by using an ESGE homogenizer, type M 100. Further homogenization was achieved with 10 strokes of a loosely fitting Teflon pestle in a glass tube. The homogenate was again diluted with buffer (1 : 1 v/v) and poured over a 200-~m nylon sieve (Schweiz, Seidengazefabrik, Zfirich) and filtered under a slight vacuum. The filtrate was passed through the same filter again, and this was repeated 8-10 times. The collected microvessels were washed with buffer with energetic shaking and filtered. This procedure was repeated several times, until microvessels were freed of any nonvascular contamination. The purity of microvessels during isolation was monitored by a television set-up coupled with a light microscope. A typical preparation yielded 800 mg from 100 g (wet weight) of bovine cerebral cortex. In experiments where homogenate was used for determination of cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic Y,5'-guanosine monophosphate phosphodiesterase (cGMP PDE) activities, the microvessels were suspended in a buffer (1 : l w/v) and homogenized for 3 min in a glassto-glass homogenizer. Further homogenization was achieved by sonication for 60 sec (40 watts, Branson sonifier, model B 12, Branson Sonic Power Co., Danbury, Connecticut). After centrifugation for 30 rain at 30,000 g, the supernatant was used for determination of cAMP PDE and cGMP PDE activities by the method of Pichard and Cheung (2). Substrate concentration was 0.2 mM and 1.0/~M for cAMP, and 0.1 mM and 0.1 ~xMfor cGMP. Amberlite IRP-58 (Chemical Dynamic Corp., South Plainfield, New York) was used as an ion-exchange resin. Doses necessary for 50% inhibition (ID30) of PDE activity by different agents were determined graphically from inhibition data obtained for at least six different concentrations. Each value represents an average of four determinations. All chemicals were obtained in analytical purity from commercial sources. Imidazole was obtained from E. Merck, Darmstadt, Germany.
683
c A M P P D E A N D c G M P PDE IN B O V I N E M I C R O V E S S E L S
RESULTS Our first experiments indicated that microvessels of bovine cortex possess significant cAMP PDE and cGMP PDE activities (Table I). It was therefore of interest to investigate the influence of various substances, including normal cell constituents on the activities of cAMP PDE and cGMP PDE. In assessing the effect of calcium and magnesium ions on cAMP PDE and cGMP PDE activities, it should be noted that Pichard and Cheung's (2) reaction mixture used for determination of PDE activities contains 5 mM MgSO4 and 50 IxM CaC12. Without Ca 2+ only insignificant changes in cAMP PDE and cGMP PDE activities (Table I) were observed. Absence of Mg 2+, however, produced a significant decrease in activities of both cAMP PDE and cGMP PDE at both substrate concentrations. The most pronounced decrease in activity was observed in the case of high-affinity (1.0 ~zM of substrate) cAMP PDE. When both Ca 2+ and M g 2+ w e r e omitted from the reaction mixture, PDE activities for cAMP and cGMP increased significantly compared' with results obtained in the absence of magnesium. The influence of cyclic nucleotides on the inhibition of PDE's is presented in Table II. It can be seen in this table that cAMP is a reasonably good inhibitor for cGMP PDE. The same was observed for cAMP PDE in respect to cGMP. The least inhibited was low affinity cGMP PDE by cAMP.
TABLE I I N F L U E N C E OF C A 2+ AND M G 2+ ON ACTIVITIES OF PHOSPHODIESTERASES Substrate concentration 0.2 m M c A M P 1.0 ix]VI c A M P 0.1 m M c G M P 0.1 p.M c G M P
N o r m a l values 9.20 • i0 n (46.5%) 6.01 x 10 -2 (60.1%) 5.73 x 10 (57.3%) 5.50 • 10 3 (55.0%)
- C a 2+ 8.52 x 10 (42.6%) 5.70 x 10 -z (57.0%) 6.06 x 10 (60.6%) 6.50 • 10 .3 (65.0%)
cAMP AND cGMP
-Mr + 5.08 x 10 (25.4%) 1.42 x 10 .2 (14.2%) 4.07 x 10 (40.7%) 2.96 • 10 -3 (29.6%)
Ca 2+, Mg z+ 7.48 x 10 (37.4%) 2.77 x 10 -2 (27.7%) 5.44 • 10 (54.5%) 4.69 • 10 .3 (46.9%)
" Data e x p r e s s e d in quantities of hydrolyzed substrate nmol/min/mg protein. All data are averages of three e x p e r i m e n t s with four determinations each. Results in parenthesis s h o w percents of hydrolysis of substrate.
684
v. STEFANOVICH
T A B L E II INHIBITION OF c A M P PHOSPHODIESTERASE (cAMP PDE) AND c G M P P H O S P H O D I E S T E R A S E ( c G M P P D E ) BY CYCLIC N U C L E O T I D E a % of hydrolysis of substrate in normal condition c G M P concn, 5 1 5 1 5 1
x x x x x x
10 - 3 M 10 - 3 M 10-4M 10-4M 10 -5 M 10 -5 M IDso
cAMP 0.2 m M
cGMP 1.0 ~M
54.9 56.3 inhibition in % 102.9 86.2 82.0 61.0 44.1 25.2 6.5 x 10-SM 7.5
0.1 m M
56.4 69.3 inhibition in %
c A M P concn,
93.2 78.1 74.9 58.1 39.0 59.4 x 10-SM
5 1 5 1 5
x 10 - 3 M z 10 - 3 M x 10-4M x 10-4M x 10-SM 1 x 10 5
0.1 ~ M
81.5 56.3 57.6 36.1 28.2 15.9 2.9 x 10-4M 8.0
91.9 83.9 75.1 56.4 39.8 19.9 x 10 SM
a For experimental details see Experimental Procedure.
TABLE III INHIBITION OF cAMP P H O S P H O D I E S T E R A S E AND cGMP PHOSPHODIESTERASE ISOLATED FROM B O V I N E C O R T E X MICROVESSELS BY T H E O P H Y L L I N E , CAFFEINE, AND THEOBROMINE cAMP
Theophylline Caffeine Theobromine
cGMP
0.2 m M
1.0 ixM
0.1 m M
0.1 tzM
4.80 • 10 4 M . 5.25 • 10 4 M 6.90 x 10 -4 M
1.48 x 1 0 - 4 M 2.35 x 10 - 4 M 3.60 x 10 -4 M
9.25 x 1 0 - 4 M 1.60 • 1 0 - 3 M 1.38 x 10 -3 M
9.50 x 1 0 - 4 M 2.90 x 1 0 - 4 M 3.42 x 10 4 M
IDs0 value obtained as described under Experimental Procedure.
The inhibition of cAMP PDE and cGMP PDEs by three classical xanthines, theophylline (1,3-dimethyl xanthine), theobromine (3,7-dimethyl xanthine), and caffeine (1,3,7-trimethyl xanthine), is presented in Table III. It can be seen in this table that somewhat similar values were obtained for all three xanthines. All three xanthines were more effective inhibitors of cAMP PDE than of cGMP PDE. The lowest inhibitory activity observed was that of caffeine and theobromine on low-affinity cGMP PDE. The effect of imidazole on cAMP PDE and cGMP PDE is presented in Table IV. It can be seen in this table that under the conditions of our
685
cAMP PDE A N D cGMP PDE IN BOVINE MICROVESSELS
