Ca/CaM·STIMUIATED AND cGMP·SPECIFIC PHOSPHODIESTERASES IN VASCULAR AND NON·VASCULAR TISSUES H.S. Ahn, M. Foster, M. Cable, B.J.R. Pitts and E.J. Sybertz Department of Pharmacology Schering-Plough Research Division Bloomfield, NJ 07003 INTRODUCTION A large body of evidence indicates that guanosine 3',5'-cyclic monophosphate (cGMP) plays an essential role in vasorelaxant actions of various agents such as atrial natriuretic factor (ANF), nitrogen oxide containing compounds (e.g., nitroprusside) and endothelium dependent vasodilators (e.g., acetylcholine) 0). These agents elevate cGMP by stimulating either soluble or particulate guanylate cyclase (1). In addition to guanylate cyclase, cyclic nucleotide phosphodiesterase (PDE) appears to play an important role in regulating cGMP levels and thus vasorelaxation. Although many PDEs have been identified (2), vascular tissues are known to contain two to three forms of PDE1) calcium/calmodulin-stimulated PDE (CaM-PDE or type I PDE), 2) cGMPspecific PDE (cG-PDE or type III), and 3) cAMP-PDE or type IV PDE (4-6). Recently, selective PDE inhibitors became available and have been used to characterize different forms of PDEs. In vascular tissues, vinpocetine and 8-methoxymethyl isobutylmethylxanthine (8-MeOMeMIX) were reported to be selective inhibitors of CaM-PDE (3,7), and dipyridamole and M&B 22948 selective cG-PDE inhibitors (5,6,8). Although both CaM-PDE and cG-PDE were present and accounted for most cGMP hydrolysis in aorta (9), the relative importance of these PDEs in cGMP hydrolysis is not known. Also, it is not clear whether or not vascular tissue CaM-PDE differs from non-vascular CaM-PDE. The purpose of this study was to determine the relative activity ofCaM-PDE and cG-PDE in various tissues and cultured cells using selective inhibitors and a conformation-specific anti-calmodulin monoclonal antibody (0) which selectively binds CaM-PDE. In addition, CaM-PDE was purified from porcine aortic extracts and was compared to a similarly purified bovine brain enzyme. Results of this study show similar properties of aortic and brain CaM-PDEs but a large difference in relative activity of CaM-PDE and cG-PDE in various tissues and cell types.
Cellular and Molecular Mechanisms in Hypertension Edited by R.H. Cox. Plenum Press. New York. 1991
191
METHODS
PDEAssays PDE activity was measured by a radio enzymatic assay as previously described (11). Enzyme or tissue extract was incubated with 1-2 JlM of unlabelled substrate and tritiated substrate (about 60,000 cpm of 3H-cGMP or 3H-cAMP) in 50 mM Tris-HCI buffer, pH 7.5, containing 2 mM MgCI2, 0.1 J.!.M CaM and 0.2 mM CaCl2 at 30°C for 15 min. Culture ofBovi.ne Aortic Endothelial Cells and Rat Aortic Smooth Muscle Cells
Both cell cultures were propagated from frozen cells at early (2 to 4) passages. Bovine aortic endothelial cells were cultivated in RPM! 1640 medium (Gibco, NY) supplemented with 20% fetal calf serum (Hyclone, UT), penicillin (100 units), streptomycin (100 Jlg/ml) and 2 mM of Lglutamine at 37°C in a humidified atmosphere of 95% air-5% C02. Rat aortic smooth muscle cells were cultured in Dulbecco's modified essential medium supplemented with 10% fetal calf serum and penicillinstreptomycin as above. Preparation of Cell and Tissue Extracts Confluent bovine aortic endothelial or rat aortic smooth muscle cells were washed once with Dulbecco's phosphate buffered saline and scraped in 2 ml of a homogenization buffer from 3-100 mm plates. The harvested cells were homogenized using an all glass Kontes dual homogenizer. The homogenization buffer was 50 mM Tris-HCI, pH 7.5, containing 5 mM MgCI2, 0.1 mM of phenylmethylsulfonyl fluoride (PMSF), 10 J.!.M leupeptin and 0.2 mg/ml BSA. Various porcine tissues were homogenized in 3 to 10 volume (v/w) of the homogenization buffer. The cell and tissue homogenates were centrifuged at 2000 g for 20 min at 5°C. These supernatants were used in immunoadsorption assays. Immunoadsorption of CaM-PDE in Isolated CaM·PDE Preparation, Tissue and Cultured Cell Extracts A solid phase CaM-PDE monoclonal antibody (ACC-1 linked to Pansorbin cell) was prepared and immunoadsorption assays carried out as previously described (11). Aliquots (200-300 Jll) of an isolated CaM-PDE or the 2000 g supernatant of tissue and cell extracts were incubated with the native or boiled solid phase ACC-1 (1 Jlg) or C1 in the presence of CaM (0.5 Jlg/0.2 mL) and CaCl2 (0.1 mM) at 5°C for 2 to 3 hr, and were centrifuged at 10,000 g. The supernatants and resuspended pellets were assayed for PDE activity using 2 JlM cGMP as substrate. Purification of CaM·PDE CaM-PDE was purified from porcine aorta media layer by successive chromatography on DEAE-sephacel column and CaM affigel 15 column. The minced porcine aortic media was homogenized in 3 vol (v/w) of 50 mM Tris-acetate buffer (pH 6.0) containing 3.75 mM 2-mercaptoethanol, 0.1 mM PMSF and 10 J.!.M leupeptin (PDE buffer) using a Brinkman PT-10 polytron (2 x 15 sec bursts at the maximal speed). The homogenate was centrifuged at 100,000 g for 60 min. The resulting supernatant was applied to a DEAEsephacel column equilibrated with the PDE isolation buffer. PDE peaks 192
TABLEl IMMUNOADSORPTION OF cGMP-HYDROLYZING ACTIVITY BY SOLID PHASE CaM MONOCLONAL ANTIBODY IN EXTRACTS OF VARIOUS TISSUES AND CELIB Tissue or Cell
Conditions of Antibody
SUEematant Pellet cGMP-hydrolyzing activity (pmol/tube/min)
Pig aorta
boiled native
13.60±0.03 3.92 ± 0.17a
Pig coronary artery
boiled native
7.BO ± 0.22
Pig lung
boiled native
Pig heart
0.46±0.07 11.20 ± 0.23b 0.10 ± 0.0
(73%)
6.BO±0.15
(75%)
12.10±0.05 11.70±0.22
0.29±0.01 3.65±0.12
(22%)
boiled native
5.03±0.05 3.37 ± 0.74
0.17 ± 0.05 2.16±0.06
(37%)
Pig brain
boiled native
3.2B±0.12 1.01 ± O.OB
±0.06 1.50 ±0.03
(60%)
Bovine aortic endothelial cells e
boiled native
E5l
76
5.3 9.5
(5%)
Rat aortic smooth muscle cells e
boiled native
75
6.1
2.20±0.22
38
o
71
(63%)
Each value represents an average of 2 determinations or the mean ± SEM of 3 to 4 determinations. Percent of total cGMP-hydrolyzing activity which was immuno-aclaorbed is in parenthesis. Confluent endothelial cells (passage 5) and smooth muscle cells (passage 4) were used as enzyme sources. aNot inhibited by 1 mM EGTA but inhibited by dipyridamole (IC50. 8 J,iM); blnhibited by 1 mM EGTA; cpmol cGMP hydrolyzed/min/lOO mm plate of cultured cells.
193
TABLE 2
PURIFICATION OF PIG AORTIC CaM-PDE
Homogenate 100 K Supernatant DEAE-sephacel CaM-affigel
Vol (ml)
Protein (mg)
1,500 1,352 90 40
36,900 26,499 251 5.1
Total Activity CaiCaM EGTA (nmol/min) 2,945 1,494 193 72
Specific Activity CaiCaM (nmol/mg/min)
1,749 1,455 80 22
0.08 0.06 0.77 14
The starting material was 300 g of pig aortic media. Each fraction was assayed for cGMP-hydrolyzing activity in the presence of Ca (1 mM)/CaM (0.15 11M) or EGTA (1 mM) using 1 J.1M cGMP.
TABLE 3
PROPERTIES OF VASCULAR AND BRAIN CaM-PDE ISOZ¥MES Enzyme Source
Km (11M) cG cA
cG
cA
pH optimum
Stimulation by CaM
Porcine aorta
2.8
12
177
41
8.0
2-3 fold
100%
yes d
Bovine brain
1.6
4.5
1531
710
8.0
3-5 fold
95%
yes d
Vmaxa
Binding to ACC-1b C1 c
Except for kinetic assays, assays were performed with 1 11M of cGMP or cAMP in the presence of CaM (0.5 IlM)/CaCI2 (0.1 mM) at 30°C for 5-15 min. The immunoadsorption experiments were carried out by incubating each enzyme with 0.1 ml of Pansorbin cells liked to ACC-1 (1llg) for 2 hr or 0.1 ml of packed sepharose 4B linked to C1 monoclonal antibody for 30 min at 5°C. anmol cyclic nucleotide hydrolyzed/mg protein/min; bmonoclonal antibody to CaM bound to brain CaM-PDE (10); cmonoclonal antibody to bovine brain 60 KDa CaM-PDE (14); d100 III of packed sepharose linked to C1 specifically immunoadsorbed aortic CaM-PDE (0.41 nmollmin) and brain CaM-PDE (0.13 nmol/min).
were eluted using a step gradient as previously reported (12). The first peak containing CaM-stimulated activity was eluted with PDE isolation buffer containing 350 mM sodium acetate. The peak fractions were pooled, concentrated, dialyzed and was adjusted to 1 mM CaCl2 and 100 mM NaCl and immediately applied to a CaM affigel column (1 ml bed vo1.l2.5 mg protein). The column was washed with the same buffer. CaM-PDE was eluted with 2 column volumes of PDE buffer containing 4 mM EGTA and 100 mM NaCl, concentrated and stored in 0.2% BSA at -70°C. RESULTS Table 1 summarizes results of imunoadsorption studies with various tissue and cell extracts. The percent of cGMP-hydrolyzing activity immunoadsorbed onto solid phase ACC-1 varied widely from one tissue or cell to another. The immunoadsorbed activity was stimulated by CalCaM 194
and represented CaM-PDE. The unadsorbed activity appeared to be mainly cG-PDE based on its selective inhibition by dipyridamole and M&B 22948, selective inhibitors of cG-PDE (data not shown). Thus, activities in the pellet and supernatant indicate CaM-PDE and cG-PDE activities respectively. For porcine tissues the highest immunoadsorbed CaM-PDE activity was found in coronary artery and aorta, with an intermediate activity in heart and the least activity in lung. In contrast, the immunoadsorbed CaM-PDE contributed less than 20% of total cAMP hydrolysis in all tissues examined (aorta, heart, lung and brain). These data indicate that cAMP is mainly hydrolyzed by other enzymes, probably low Krn cAMP-PDE. Sixty-three percent and 5% of the total cGMP-hydrolyzing activity were immunoadsorbed onto the monoclonal antibody in smooth muscle and endothelial cell extracts, respectively. The remaining activity in the supernatant was not stimulated by Ca/CaM and was inhibited by dipyridamole with respective IC50'S of 4 to 8 ~M. These data indicate that endothelial cells contain little CaM-PDE while smooth muscle cells contain both CaM-PDE and cG-PDE. Table 2 shows purification of CaM-PDE from porcine aorta media. About 200-fold purification could be achieved by the successive ion exchange and CaM affinity chromatography. CaM-PDE was similarly purified about 300-fold also from bovine brain (data not shown). Table 3 summarizes results of studies comparing properties of porcine aortic and bovine brain CaM-PDEs. Krn values of aortic and brain CaM-PDEs for cGMP and cAMP were comparable, but Vrnax values of aortic CaM-PDE was 1/9 to 1117 that of brain enzyme. CaM-stimulated activity of aortic homogenate was also 1/5 that of brain. Thus, these data suggest a lower concentration of CaM-PDE in aorta than in brain. Both CaM-PDEs showed similar properties in pH optimum, CaM-stimulability and cross-reactivity with monoclonal antibodies C-1 and ACC1 to brain CaM-PDE or CaM bound to brain CaMPDE. DISCUSSION AND CONCLUSION In this study, CaM-PDE was separated from the rest of PDEs using a monoclonal antibody (ACC-1) selective for CaM-PDE, and cG-PDE was identified by selective inhibitors of cG-PDE after the removal ofCaM-PDE by immunoadsorption in the tissue or cell extracts (10,13). The relative importance of CaM-PDE or cG-PDE was assessed by determining respective contribution of these two PDEs to the total cGMP hydrolysis in the extracts. Results indicate that, in addition to aorta, coronary artery and nonvascular tissues contained both CaM-PDE and cG-PDE, which accounted for most of cGMP hydrolysis. An exception was endothelial cells, which contained cG-PDE but little CaM-PDE. The relative contribution of CaMPDE and cG-PDE to cGMP hydrolysis varied markedly among different tissues. For example, in the coronary artery and aorta, CaM-PDE hydrolyzed a large portion of cGMP. In lung, cG-PDE appeared to hydrolyze most cGMP. This finding suggests a possibility that a selective CaM-PDE inhibitor may exhibit some degree of tissue selectivity in raising cGMP. CaM-PDE contributed minimally to cAMP hydrolysis in tissues examined. Since both cGMP-inhibited cAMP-PDE and rolipram-sensitive cAMP-PDE are known to occur in the aorta and other tissues (2,11), cAMP may be mainly hydrolyzed by these cAMP-PDEs in these tissues. Our data indicate the presence of cG-PDE activity in porcine coronary artery, where only CaM-PDE and low Krn cAMP-PDE were previously detected (3). 195
The present study also compared properties of CaM-PDEs isolated from a vascular tissue (aorta) and a non-vascular tissue (brain). Although CaM-PDE was purified to homogeneity from several non-vascular tissues (2), vascular CaM-PDE has not been purified beyond an ion exchange chromatographic separation step (5-7). Our earlier immunoadsorption study indicated that a DEAE-sephacel column-purified rabbit aortic CaMPDE preparation contained 28% of non-CaM-PDE activity (11). In contrast, the CaM-affinity column-purified porcine aortic CaM-PDE appears to contain no contaminating PDEs since its entire activity could be immunoadsorbed by ACC-1 monoclonal antibody. These findings point out the desirability of using CaM-affinity purified CaM-PDE. Comparison of similarly purified aortic and brain CaM-PDEs shows that aortic and brain CaM-PDEs have similar kinetic properties, pH optimum and induce a similar conformation of CaM bound to them. These results extend our earlier studies showing a minimal difference in drug sensitivity of brain and aortic CaM-PDEs (6). Brain contained 60k Da and 63k Da subunits of CaM-PDE (14). C1 monoclonal antibody selectively interacted with the 60k Da subunit (14). Effective immunoadsorption of brain and aortic CaM-PDEs to C1 antibody in this study indicates that aortic CaM-PDE is related to the 60k Da subunit. Lung and heart CaM-PDE isozymes were reported to be also related to 60k Da but not 63k Da subunit (14). In conclusion, aortic smooth muscle, vascular and non-vascular tissues contain CaM-PDE and cG-PDE in varying ratios. Endothelial cells lack CaM-PDE. In addition, this study combined with an earlier finding (6) indicates a similar property of vascular and non-vascular CaM-PDE isozymes in terms of kinetic behavior, antigenic structure and drug sensitivity. ACKNOWLEDGMENTS The authors wish to thank Drs. J. Beavo and J. Wang for kindly providing ACC-1 and C1 monoclonal antibodies. REFERENCES 1.
2. 3. 4 5. 6. 7.
196
Murad F. Cyclic guanosine monophosphate as a mediator of vasodilation. J Clin Invest 78: 1-5, 1986. Beavo JA. Multiple isozymes of cyclic nucleotide phosphodiesterase. Adv Second Messenger Phosphoprotein Res 22: 1-38, 1988. Lorenz KL, Wells IN. Potentiation of the effects of sodium nitroprusside and of isoproterenol by selective phosphodiesterase inhibitors. Mol Pharmacol 23: 424-430, 1983. Hidaka H, Endo T. Selective inhibitors of three forms of cyclic nucleotide phosphodiesterases-basic and potential clinical applications. Adv Cyclic Nucleotide Res 16: 245-259, 1984. Lugnier C, Schoeffter P, LeBec A, Strouthou E, Stoclet JC. Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta. Biochem Pharmacol35:1743-1751, 1986. Ahn HS, Crim W, Romano M, Moroney S, Pitts B. Effects of selective inhibitors on cyclic nucleotide phosphodiesterases (PDEs) of rabbit and pig aorta. Pharmacologist 29: 522,1987. Hagiware M, Endo T, Hidaka H. Effects of vinpocetine on cyclic nucleotide metabolism in vascular smooth muscle. Biochem Pharmacol33: 453-457,1984.
8.
9.
10. 11. 12. 13.
14.
Bergstrand H, Kristoffersson J, Lundquist B, Schurmann A. Effects of antiallergic agents, compounds 48/80, and some reference inhibitors on the activity of partially purified human lung tissue adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3'5'monophosphate phosphodiesterases. Mol Pharmacol13: 38-43, 1977. Ahn HS, Crim W, Romano M, Moroney SJ, Sybertz EJ, Pitts B. Calmodulin dependent phosphodiesterase (CaM-PDE) and cyclic GMP specific PDE (cG-PDE) distribution and response to inhibitors in aorta and non-vascular tissues. Pharmacologist 30: A213, 1988. Hansen RS, Beavo AJ. Differential recognition of calmodulinenzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody. J Bioi Chem 261: 14636-14645, 1986. Ahn HS, Crim W, Romano M, Sybertz EJ, Pitts B. Effects of selective inhibitors on cyclic nucleotide phosphodiesterases of rabbit aorta. Biochem Pharmacol 38: 3331-3339, 1989. Ahn HS, Eardley D, Watkins R, Prioli N. Effects of several newer cardiotonic drugs on cardiac cyclic AMP metabolism. Biochem Pharmacol34: 1113-1121, 1986. Ahn HS, Crim W, Pitts B, Sybertz EJ. Ca/CaM-stimulated and cGMP specific phosphodiesterases: tissue distribution, drug sensitivity and regulation of cGMP levels. Adv Second Messenger Phosphoprotein Res, Vol. 24, In Press, 1990. Sharma RK, Wang JH. Isolation of bovine brain calmodulindependent cyclic nucleotide phosphodiesterase isozyme. Methods in Enzymology 159: 582-594, 1988.
197
Cellular and Molecular Mechanisms in Hypertension Edited by R.H. Cox. Plenum Press. New York. 1991
191
METHODS
PDEAssays PDE activity was measured by a radio enzymatic assay as previously described (11). Enzyme or tissue extract was incubated with 1-2 JlM of unlabelled substrate and tritiated substrate (about 60,000 cpm of 3H-cGMP or 3H-cAMP) in 50 mM Tris-HCI buffer, pH 7.5, containing 2 mM MgCI2, 0.1 J.!.M CaM and 0.2 mM CaCl2 at 30°C for 15 min. Culture ofBovi.ne Aortic Endothelial Cells and Rat Aortic Smooth Muscle Cells
Both cell cultures were propagated from frozen cells at early (2 to 4) passages. Bovine aortic endothelial cells were cultivated in RPM! 1640 medium (Gibco, NY) supplemented with 20% fetal calf serum (Hyclone, UT), penicillin (100 units), streptomycin (100 Jlg/ml) and 2 mM of Lglutamine at 37°C in a humidified atmosphere of 95% air-5% C02. Rat aortic smooth muscle cells were cultured in Dulbecco's modified essential medium supplemented with 10% fetal calf serum and penicillinstreptomycin as above. Preparation of Cell and Tissue Extracts Confluent bovine aortic endothelial or rat aortic smooth muscle cells were washed once with Dulbecco's phosphate buffered saline and scraped in 2 ml of a homogenization buffer from 3-100 mm plates. The harvested cells were homogenized using an all glass Kontes dual homogenizer. The homogenization buffer was 50 mM Tris-HCI, pH 7.5, containing 5 mM MgCI2, 0.1 mM of phenylmethylsulfonyl fluoride (PMSF), 10 J.!.M leupeptin and 0.2 mg/ml BSA. Various porcine tissues were homogenized in 3 to 10 volume (v/w) of the homogenization buffer. The cell and tissue homogenates were centrifuged at 2000 g for 20 min at 5°C. These supernatants were used in immunoadsorption assays. Immunoadsorption of CaM-PDE in Isolated CaM·PDE Preparation, Tissue and Cultured Cell Extracts A solid phase CaM-PDE monoclonal antibody (ACC-1 linked to Pansorbin cell) was prepared and immunoadsorption assays carried out as previously described (11). Aliquots (200-300 Jll) of an isolated CaM-PDE or the 2000 g supernatant of tissue and cell extracts were incubated with the native or boiled solid phase ACC-1 (1 Jlg) or C1 in the presence of CaM (0.5 Jlg/0.2 mL) and CaCl2 (0.1 mM) at 5°C for 2 to 3 hr, and were centrifuged at 10,000 g. The supernatants and resuspended pellets were assayed for PDE activity using 2 JlM cGMP as substrate. Purification of CaM·PDE CaM-PDE was purified from porcine aorta media layer by successive chromatography on DEAE-sephacel column and CaM affigel 15 column. The minced porcine aortic media was homogenized in 3 vol (v/w) of 50 mM Tris-acetate buffer (pH 6.0) containing 3.75 mM 2-mercaptoethanol, 0.1 mM PMSF and 10 J.!.M leupeptin (PDE buffer) using a Brinkman PT-10 polytron (2 x 15 sec bursts at the maximal speed). The homogenate was centrifuged at 100,000 g for 60 min. The resulting supernatant was applied to a DEAEsephacel column equilibrated with the PDE isolation buffer. PDE peaks 192
TABLEl IMMUNOADSORPTION OF cGMP-HYDROLYZING ACTIVITY BY SOLID PHASE CaM MONOCLONAL ANTIBODY IN EXTRACTS OF VARIOUS TISSUES AND CELIB Tissue or Cell
Conditions of Antibody
SUEematant Pellet cGMP-hydrolyzing activity (pmol/tube/min)
Pig aorta
boiled native
13.60±0.03 3.92 ± 0.17a
Pig coronary artery
boiled native
7.BO ± 0.22
Pig lung
boiled native
Pig heart
0.46±0.07 11.20 ± 0.23b 0.10 ± 0.0
(73%)
6.BO±0.15
(75%)
12.10±0.05 11.70±0.22
0.29±0.01 3.65±0.12
(22%)
boiled native
5.03±0.05 3.37 ± 0.74
0.17 ± 0.05 2.16±0.06
(37%)
Pig brain
boiled native
3.2B±0.12 1.01 ± O.OB
±0.06 1.50 ±0.03
(60%)
Bovine aortic endothelial cells e
boiled native
E5l
76
5.3 9.5
(5%)
Rat aortic smooth muscle cells e
boiled native
75
6.1
2.20±0.22
38
o
71
(63%)
Each value represents an average of 2 determinations or the mean ± SEM of 3 to 4 determinations. Percent of total cGMP-hydrolyzing activity which was immuno-aclaorbed is in parenthesis. Confluent endothelial cells (passage 5) and smooth muscle cells (passage 4) were used as enzyme sources. aNot inhibited by 1 mM EGTA but inhibited by dipyridamole (IC50. 8 J,iM); blnhibited by 1 mM EGTA; cpmol cGMP hydrolyzed/min/lOO mm plate of cultured cells.
193
TABLE 2
PURIFICATION OF PIG AORTIC CaM-PDE
Homogenate 100 K Supernatant DEAE-sephacel CaM-affigel
Vol (ml)
Protein (mg)
1,500 1,352 90 40
36,900 26,499 251 5.1
Total Activity CaiCaM EGTA (nmol/min) 2,945 1,494 193 72
Specific Activity CaiCaM (nmol/mg/min)
1,749 1,455 80 22
0.08 0.06 0.77 14
The starting material was 300 g of pig aortic media. Each fraction was assayed for cGMP-hydrolyzing activity in the presence of Ca (1 mM)/CaM (0.15 11M) or EGTA (1 mM) using 1 J.1M cGMP.
TABLE 3
PROPERTIES OF VASCULAR AND BRAIN CaM-PDE ISOZ¥MES Enzyme Source
Km (11M) cG cA
cG
cA
pH optimum
Stimulation by CaM
Porcine aorta
2.8
12
177
41
8.0
2-3 fold
100%
yes d
Bovine brain
1.6
4.5
1531
710
8.0
3-5 fold
95%
yes d
Vmaxa
Binding to ACC-1b C1 c
Except for kinetic assays, assays were performed with 1 11M of cGMP or cAMP in the presence of CaM (0.5 IlM)/CaCI2 (0.1 mM) at 30°C for 5-15 min. The immunoadsorption experiments were carried out by incubating each enzyme with 0.1 ml of Pansorbin cells liked to ACC-1 (1llg) for 2 hr or 0.1 ml of packed sepharose 4B linked to C1 monoclonal antibody for 30 min at 5°C. anmol cyclic nucleotide hydrolyzed/mg protein/min; bmonoclonal antibody to CaM bound to brain CaM-PDE (10); cmonoclonal antibody to bovine brain 60 KDa CaM-PDE (14); d100 III of packed sepharose linked to C1 specifically immunoadsorbed aortic CaM-PDE (0.41 nmollmin) and brain CaM-PDE (0.13 nmol/min).
were eluted using a step gradient as previously reported (12). The first peak containing CaM-stimulated activity was eluted with PDE isolation buffer containing 350 mM sodium acetate. The peak fractions were pooled, concentrated, dialyzed and was adjusted to 1 mM CaCl2 and 100 mM NaCl and immediately applied to a CaM affigel column (1 ml bed vo1.l2.5 mg protein). The column was washed with the same buffer. CaM-PDE was eluted with 2 column volumes of PDE buffer containing 4 mM EGTA and 100 mM NaCl, concentrated and stored in 0.2% BSA at -70°C. RESULTS Table 1 summarizes results of imunoadsorption studies with various tissue and cell extracts. The percent of cGMP-hydrolyzing activity immunoadsorbed onto solid phase ACC-1 varied widely from one tissue or cell to another. The immunoadsorbed activity was stimulated by CalCaM 194
and represented CaM-PDE. The unadsorbed activity appeared to be mainly cG-PDE based on its selective inhibition by dipyridamole and M&B 22948, selective inhibitors of cG-PDE (data not shown). Thus, activities in the pellet and supernatant indicate CaM-PDE and cG-PDE activities respectively. For porcine tissues the highest immunoadsorbed CaM-PDE activity was found in coronary artery and aorta, with an intermediate activity in heart and the least activity in lung. In contrast, the immunoadsorbed CaM-PDE contributed less than 20% of total cAMP hydrolysis in all tissues examined (aorta, heart, lung and brain). These data indicate that cAMP is mainly hydrolyzed by other enzymes, probably low Krn cAMP-PDE. Sixty-three percent and 5% of the total cGMP-hydrolyzing activity were immunoadsorbed onto the monoclonal antibody in smooth muscle and endothelial cell extracts, respectively. The remaining activity in the supernatant was not stimulated by Ca/CaM and was inhibited by dipyridamole with respective IC50'S of 4 to 8 ~M. These data indicate that endothelial cells contain little CaM-PDE while smooth muscle cells contain both CaM-PDE and cG-PDE. Table 2 shows purification of CaM-PDE from porcine aorta media. About 200-fold purification could be achieved by the successive ion exchange and CaM affinity chromatography. CaM-PDE was similarly purified about 300-fold also from bovine brain (data not shown). Table 3 summarizes results of studies comparing properties of porcine aortic and bovine brain CaM-PDEs. Krn values of aortic and brain CaM-PDEs for cGMP and cAMP were comparable, but Vrnax values of aortic CaM-PDE was 1/9 to 1117 that of brain enzyme. CaM-stimulated activity of aortic homogenate was also 1/5 that of brain. Thus, these data suggest a lower concentration of CaM-PDE in aorta than in brain. Both CaM-PDEs showed similar properties in pH optimum, CaM-stimulability and cross-reactivity with monoclonal antibodies C-1 and ACC1 to brain CaM-PDE or CaM bound to brain CaMPDE. DISCUSSION AND CONCLUSION In this study, CaM-PDE was separated from the rest of PDEs using a monoclonal antibody (ACC-1) selective for CaM-PDE, and cG-PDE was identified by selective inhibitors of cG-PDE after the removal ofCaM-PDE by immunoadsorption in the tissue or cell extracts (10,13). The relative importance of CaM-PDE or cG-PDE was assessed by determining respective contribution of these two PDEs to the total cGMP hydrolysis in the extracts. Results indicate that, in addition to aorta, coronary artery and nonvascular tissues contained both CaM-PDE and cG-PDE, which accounted for most of cGMP hydrolysis. An exception was endothelial cells, which contained cG-PDE but little CaM-PDE. The relative contribution of CaMPDE and cG-PDE to cGMP hydrolysis varied markedly among different tissues. For example, in the coronary artery and aorta, CaM-PDE hydrolyzed a large portion of cGMP. In lung, cG-PDE appeared to hydrolyze most cGMP. This finding suggests a possibility that a selective CaM-PDE inhibitor may exhibit some degree of tissue selectivity in raising cGMP. CaM-PDE contributed minimally to cAMP hydrolysis in tissues examined. Since both cGMP-inhibited cAMP-PDE and rolipram-sensitive cAMP-PDE are known to occur in the aorta and other tissues (2,11), cAMP may be mainly hydrolyzed by these cAMP-PDEs in these tissues. Our data indicate the presence of cG-PDE activity in porcine coronary artery, where only CaM-PDE and low Krn cAMP-PDE were previously detected (3). 195
The present study also compared properties of CaM-PDEs isolated from a vascular tissue (aorta) and a non-vascular tissue (brain). Although CaM-PDE was purified to homogeneity from several non-vascular tissues (2), vascular CaM-PDE has not been purified beyond an ion exchange chromatographic separation step (5-7). Our earlier immunoadsorption study indicated that a DEAE-sephacel column-purified rabbit aortic CaMPDE preparation contained 28% of non-CaM-PDE activity (11). In contrast, the CaM-affinity column-purified porcine aortic CaM-PDE appears to contain no contaminating PDEs since its entire activity could be immunoadsorbed by ACC-1 monoclonal antibody. These findings point out the desirability of using CaM-affinity purified CaM-PDE. Comparison of similarly purified aortic and brain CaM-PDEs shows that aortic and brain CaM-PDEs have similar kinetic properties, pH optimum and induce a similar conformation of CaM bound to them. These results extend our earlier studies showing a minimal difference in drug sensitivity of brain and aortic CaM-PDEs (6). Brain contained 60k Da and 63k Da subunits of CaM-PDE (14). C1 monoclonal antibody selectively interacted with the 60k Da subunit (14). Effective immunoadsorption of brain and aortic CaM-PDEs to C1 antibody in this study indicates that aortic CaM-PDE is related to the 60k Da subunit. Lung and heart CaM-PDE isozymes were reported to be also related to 60k Da but not 63k Da subunit (14). In conclusion, aortic smooth muscle, vascular and non-vascular tissues contain CaM-PDE and cG-PDE in varying ratios. Endothelial cells lack CaM-PDE. In addition, this study combined with an earlier finding (6) indicates a similar property of vascular and non-vascular CaM-PDE isozymes in terms of kinetic behavior, antigenic structure and drug sensitivity. ACKNOWLEDGMENTS The authors wish to thank Drs. J. Beavo and J. Wang for kindly providing ACC-1 and C1 monoclonal antibodies. REFERENCES 1.
2. 3. 4 5. 6. 7.
196
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