0013-7227/90/1264-2102$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 4 Printed in U.S.A.
Characterization of Human Placental Cytosolic Adenosine 3',5'-Monophosphate Phosphodiest erase by Inhibitors and Insulin Treatment* LIMIN XIONG (HSIUNG), THOMAS R. L E B O N , AND YOKO FUJITA-YAMAGUCHI Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010
cAMP PDE). The third form was a low Km cAMP PDE, but was only modestly sensitive to inhibition by cGMP or cilostamide. The fourth form was a cAMP PDE which showed high sensitivity to inhibition by cGMP or cilostamide. The IC50 values of the fourth form were comparable to those of rat adipose insulinsensitive PDE. However, its Km for cAMP was 2 nM, which is about 10 times higher than that of the rat enzyme. Insulin treatment on placenta tissues stimulated at least two PDEs, the third and fourth forms. To our knowledge, this is the first report to describe insulin-sensitive cAMP PDEs in the cytosolic fraction of human placenta. (Endocrinology 126: 2102-2109,1990)
ABSTRACT. To gain insight into the regulation of low Km cAMP phosphodiesterases (PDE) by insulin in human tissues, PDEs in human placenta were studied. Human placenta contained cAMP PDEs in particulate and cytosolic fractions. More than 99% of the total activity was localized in the cytosolic fraction. The cytosolic fraction exhibited at least four cyclic nucleotide PDEs when fractionated by DEAE-cellulose chromatography. The first form was a calmodulin-activated PDE which hydrolyzed both cGMP and cAMP. The second form was a high affinity cAMP PDE with a nonlinear kinetic characteristic, but was not inhibited by either cGMP or cilostamide (either compound is known to specifically inhibit rat insulin-sensitive
I
N 1966, Butcher et al. (1) reported that insulin decreases intracellular cAMP levels in fat cells when the cAMP levels are elevated by catecholamines. Later, other investigators were able to observe that insulin decreases both the cAMP concentration and lipolysis under physiological conditions (2, 3) and that insulin stimulates cAMP phosphodiesterases, the only enzymes that break down cAMP in mammalian cells. Insulin, when applied to rat adipocytes, increases low Km cAMP PDE activity 2- to 3-fold (4). The insulin-sensitive PDE is associated with a microsomal fraction, designated P2. The optimum concentration of insulin for the stimulation is 1-3 nM. The enzyme activity is also increased when adipocytes are exposed to lipolytic agents, such as catecholamines, ACTH, and TSH (5, 6). The studies performed with rat (5) and 3T3-L1 (7) adipocytes indicated that there are some differences between the effects of insulin and epinephrine; that is, the increase in the particulate PDE activity was maximal after incubation of the cells with insulin for about 5 min and was sustained Received September 18,1989. Address all correspondence and requests for reprints to: Yoko FujitaYamaguchi, Department of Molecular Genetics, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010. * This work was supported by NIH Grant DK-29970 and a research grant from American Diabetes Association, California Affiliate.
at least 30-60 min in the presence of insulin, whereas in the presence of epinephrine PDE activity was maximal between 5-10 min and declined thereafter. Activation of the hepatic enzyme by insulin and glucagon has also been extensively studied (8, 9). While activation of this enzyme in fat and liver by insulin as well as other hormones has been well documented at the physiological level, the molecular mechanism of this activation is not known. An involvement of cAMP-dependent protein kinase (A-kinase) in the activation of this enzyme in rat adipocyte P2 fraction has recently been suggested (10). However, it is not yet known whether low Km cAMP PDE is an immediate substrate of A-kinase. Purification of hormone-sensitive low Km cAMP PDEs should help in understanding the mechanisms of regulation of this enzyme. Putative insulin-sensitive enzymes have been purified from rat adipocytes and rat liver (11-13). To date, however, protein chemical studies to further analyze low Km cAMP PDEs have not been conducted due to a difficulty in purifying sufficient quantities of the enzyme. According to the order of elution from a DEAE-cellulose column and kinetics characteristics, PDEs from different tissues were originally classified into three major classes, D-I, D-II, and D-III, by Appleman and Terasaki (14). The third fraction, D-III, was classified as a
2102
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE cAMP-specific PDE and descriptively called low Km cAMP PDE. At least two subclasses of low Km cAMP PDEs can be distinguished on the basis of their physicochemical properties and sensitivity to specific PDE inhibitors (15, 16). One type is rather sensitive to inhibition by Ro 20-1724 and rolipram, and the other to cGMP and cilostamide. The particulate insulin-sensitive PDE has been shown to be inhibited potently by cGMP and cilostamide, but much less effectively by Ro 20-1724. Unlike studies on rat or mouse low Km cAMP PDE, studies on the regulatory mechanism of human PDE activity have been limited, except for the PDE from platelets (17). In our present study we have characterized low Km cAMP PDEs in human placenta. Placenta is not only a good source for purifying human enzymes but is also an important tissue for fetal growth. Effects of growth factors, such as insulin and insulin-like growth factors (IGFs), on placental cells are currently not well understood. We report that insulin stimulates cAMP PDEs in the cytosolic fraction of placenta.
Materials and Methods
2103
were resuspended in 2 vol of the same buffer and assayed immediately or stored at —75 C (particulate fraction). Insulin treatment and preparation of cytosolic fraction Placenta pieces, prepared as described above, were further cut into approximately 2-mm3 pieces, washed with saline by centrifuging at 2000 X g for 5 min, then washed in the same manner with Krebs-Ringer-HEPES buffer containing 2% BSA. Tissue pieces were preincubated in the buffer at 37 C for 20 min and then for another 10 min in the absence (for basal activity) or presence (for insulin-stimulated activity) of added insulin. The buffer was removed from placenta pieces by centrifugation at 2000 X g for 5 min. The placenta tissues were washed three times using the same centrifugation method with 10 mM TES buffer, pH 7.0, containing 0.25 M sucrose, 0.2 mM PMSF, and 1 fig/ml each of leupeptin, pepstatin-A, and aprotinin. The placenta pieces were homogenized with a Tekmar Tissumizer as described above. The homogenate was centrifuged at 2000 X g for 10 min, after which any placental debris was discarded. The supernatant obtained was centrifuged at 100,000 X g for 30 min. The 100,000 X g supernatant and pellet corresponded to the cytosolic fraction and the particulate fraction (P2 plus P3 fraction), respectively.
Materials
Low Km cAMP PDE assay
Porcine insulin was a gift from Eli Lilly (Indianapolis, IN). [2,8-3H]cAMP (30-50 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Crotalus atrox snake venom, fraction V BSA, phenylmethylsulfonylfluoride (PMSF), N"benzoyl-L-arginine ethyl ester (BAEE), leupeptin, pepstatin-A, aprotinin, and theophylline were obtained from Sigma (St. Louis, MO). Ca2+-free porcine brain calmodulin was purchased from Ocean Biologies (Edmonds, WA). Mono-Q and Superose12 columns were obtained from Pharmacia-LKB (Piscataway, NJ). Griseolic acid, cilostamide, and Ro 20-1724 were gifts from Dr. M. Yamazaki of Sankyo Co. Ltd. (Tokyo, Japan) (18), Dr. S. Hosozawa of Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan), and Dr. V. C. Manganiello of the NIH, respectively. All other chemicals used were reagent grade.
The enzyme activity was measured according to the two-step assay procedure of Thompson and Appleman (19) with some modifications. In the first step of the assay, the [2,8-3H]cAMP was converted by phosphodiesterase to 5'-[2,8-3H]cAMP, which was subsequently converted to [2,8-3H]adenosine by the added nucleotidase in the second step. In the first step, the cytosolic enzyme fraction was incubated for 10 min in 0.1 ml 50 mM Tris-HCl buffer, pH 7.4, containing 0.5 mM dithiothreitol, 5 mM MgCl2, and 0.5 nM cAMP ([2,8-3HlcAMP; 200,000 dpm/assay) at 30 C. The reaction was stopped by heating in a heating block at 100 C for 90 sec. After the reaction mixture was cooled at -20 C for 4 min, 10 ^l Crotalus atrox snake venom (5 mg/ml) were added. The second step reaction was allowed to proceed for 20 min at 37 C. Unreacted cAMP was then removed by mixing with 1 ml of a 33% slurry of Dowex AG-1X2 (Bio-Rad, Richmond, CA). The mixture was rocked at 4 C for 5 min and centrifuged for 5 min at 2,200 x g. A 0.5-ml aliquot of the supernatant was then counted by liquid scintillation. For the assay of cAMP PDE in particulate fractions, the procedure was similar to this, except that 40 mM TES buffer, pH 7.5, and 4 mM MgSO4 were used instead of Tris, MgCl2, and dithiothreitol according to the method of Kono et al. (4, 20). For kinetic analysis, assays for cAMP or cGMP hydrolysis were carried out using an identical procedure, with the substitution of 3H-labeled cAMP or cGMP (~6000 dpm/pmol) at concentrations of 0.14, 0.24, 0.36, 0.58,1.21, and 1.94 X 10"6 M. An assay blank, consisting of a corresponding amount of 3Hlabeled cAMP or cGMP with water in place of enzyme preparation, was carried through the assay procedure, and the background counts were subtracted from each sample.
Subcellular fractionation of human placenta Fresh normal human placentas were obtained within 1 h after delivery, kept on ice, trimmed of amnion and chorion, washed with 0.25 M sucrose, and cut into small pieces. The pieces were transferred to 1 vol 50 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM PMSF, 2 mM BAEE, and 1 /xg/ml each of leupeptin, pepstatin-A, and aprotinin, homogenized five times for 1 min each with a Tissumizer from Tekmar Co. (Cincinnati, OH) at a setting of 30, and centrifuged at 15,000 X g for 20 min at 4 C. The supernatant was then centrifuged at 100,000 X g for 1 h at 4 C. The supernatant (cytosolic fraction) was assayed immediately or stored at -75 C. The pellet was suspended in 10 vol 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM PMSF and 2 mM BAEE, homogenized with a Tekmer Tissumizer at a setting of 10, and centrifuged at 100,000 X g for 60 min at 4 C. The sedimented membranes
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE
2104 Data analysis
Endo • 1990 Voll26»No4
NH2
The values presented are the mean and SEM. The effect of insulin on PDE activity was analyzed using the one-sample t test. The effect of cilostamide was tested using linear regression, and the regression curves were compared at the different insulin levels using the analysis of covariance.
0=P-0 I OH
Results
NH2
HOOC
OH
cAMP
OH
Griseolic acid
Subcellular distribution of low Km cAMP PDE Participate and cytosolic fractions were prepared by centrifugation at 100,000 X g for 60 min from placental 15,000 X g supernatant. The participate fraction is equivalent to the fat P2 plus P3 fraction with which insulinsensitive PDE is associated. The specific activity of the particulate fraction was 28.2 ± 6.6 pmol/min/mg protein (Table 1). When the particulate fraction was prepared in the presence of PMSF and BAEE but in the absence of leupeptin, pepstatin-A, and aprotinin, only one tenth of the specific activity was found in the particulate fraction. This suggests that membrane-bound low Km cAMP PDE is very susceptible to proteinases. The specific activity of cytosolic enzymes (~50 pmol/min/mg protein) was significantly higher than that of the particulate fraction. Cytosolic PDE activity accounted for more than 99% of the total PDE activity in placenta. Effect of inhibitors on cAMP hydrolysis by particulate and cytosolic enzymes To determine whether so-called insulin-sensitive PDEs are present in placental tissue, we used five PDE inhibitors, as presented in Fig. 1, including cGMP and cilostamide, that are known to be specific inhibitors of hormone-regulated particulate PDE activity in rat adipocytes and mouse 3T3-L1 adipocytes (16). Cilostamide and cGMP significantly inhibited cytosolic PDE (40% at 10 /uM), but had no effect on particulate PDE. Of the three nonselective inhibitors, griseolic acid was the most potent, while theophylline and Ro 20-1724 were not as effective in inhibiting the PDE activity of either fraction (Table 2). Separation and characterization of cytosolic low Km PDEs
The cytosolic PDEs were fractionated by DEAE-cellulose chromatography at pH 6.5, and the fractions were TABLE 1. Subcellular distribution of low Km cAMP PDEs in human placenta SA Total activity (pmol/min/mg (nmol/min)a protein) Particulate PDE (n = 5) Cytosolic PDE (n = 5) 0
28.2 ± 6.6 51.7 ± 6.4
Total activity per placenta of about 400 g.
3.74 ± 0.47 433 ± 54
Ratio (%) 0.9 99.1
0
H
CH 3 0=P-0
Theophylline
OH
OH
cGMP /CH3 O(CH2)3CON OCH 2 (CH 2 ) 2 CH 3 0CH 3
Cilostamide (OPC 3689)
Ro 20-1724 FIG. 1. Structure of PDE inhibitors and cAMP.
TABLE 2. Effects of inhibitors on cAMP hydrolysis by low Km cAMP PDEs from human placenta IC60 (MM)' Griseolic acid Cilostamide cGMP Theophylline Ro 20-1724
Particulate
Cytosolic
0.08 >10 >10 300 >1000
0.08 -10* -10" 400 >1000
0
IC 50 is an average of two similar experiments. At a concentration of 10 /*M, the PDE activity was 60% of the control value. 6
assayed for both cAMP and cGMP hydrolysis. As shown in Fig. 2, peak 1 contained higher cGMP PDE activity than cAMP PDE activity, whereas peaks 2 and 3 exhibited higher cAMP PDE activity. Since peak 2 did not look like a typical single activity peak, fractions 2a and 2b from the earlier portion of the peak and the later portion of the peak, respectively, were separately analyzed further. cAMP PDE activity in each fraction, designated 1, 2a, 2b, or 3 in Fig. 2, was measured in the presence of different concentrations of the five inhibitors (Fig. 3). As we have previously observed with particulate and cytosolic PDEs from placental tissue (Table 2), griseolic acid, theophylline, and Ro 20-1724 nonspecifically inhibited the PDE activity of all fractions. Cilostamide and
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE
2105
DEI
8 ioo -/•fc
50 tif °
DE2a
"o
? 100 50 0 •o
DE2b
o
JZ
0
100
200
300
400
500
600
a. o CL GL
700
Volume (ml)
FIG. 2. DEAE-cellulose chromatography of human placental cytosolic fraction. Forty-six milliliters of the cytosolic fraction, 100,000 X g supernatant, were applied to a column (3 X 14 cm) of DE-52 (Whatman, Clifton, NJ) equilibrated with 70 mM sodium acetate buffer, pH 6.5, containing 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.2 mM PMSF. The column was washed with the sodium acetate buffer, pH 6.5, until the absorbance at 280 nm reached 0.12. The proteins were eluted by a linear gradient (70 mM to 0.5 M sodium acetate, pH 6.5) and then with 1.2 M sodium acetate, pH 6.5. The eluates were assayed for cAMP (•) and cGMP (•) PDE activities using a substrate concentration of 0.5 MM- The protein concentration was monitored by measuring absorbance at 280 nm (O). The salt concentration was determined by measuring conductivity ( ). Four PDE fractions we have chosen for further analysis are marked with arrows and numbered 1, 2a, 2b, and 3.
cGMP markedly inhibited the PDE activity in fraction 3, with IC50 values of 0.03 and 0.2 nM, respectively (Table 3). These two inhibitors inhibited the activity in fraction 2b with IC50 values of 0.4 and 2 JUM, respectively (Table 3). While cilostamide modestly inhibited the activity in fraction 2a (40% inhibition at 10 nM), it had no significant effect on the activity in fraction 1. These data indicate that fractions 2b and 3 are likely to be hormoneregulated PDEs. Kinetic analysis was carried out for the enzymes found in the four fractions. The results are included in Table 3. The Knl values for fraction 2b and 3 enzymes were a little higher than expected based on the previously reported values for rat adipose enzymes. The cAMP (or cGMP) PDE activity of each fraction was measured in the absence or presence of Ca2+ and calmodulin in order to test its calmodulin sensitivity. Fraction 1 PDE activity was activated by calmodulin (a 20-fold increase in cGMP PDE activity and an 1.6-fold increase in cAMP PDE activity). In contrast, calmodulin had no effect on PDEs in fractions 2a, 2b, and 3 (Table 3). Effect of insulin on low Km cAMP PDE in the placental cytosolic fraction Placenta pieces were incubated with or without 40 nM insulin, then the pieces were washed, homogenized, and
100
50
• Cilostamide (0PC3689)
0 DE3
10 -10
2 0.4
0.2 0.03
20-fold (cGMP) 1.6-fold (cAMP) IC5o for cAMP hydrolysis cGMP (MM) Cilostamide
>10 >10
0
cAMP (or cGMP) PDE activity was measured in the absence or presence of 1 pM CaCl2 and 50 /xM calmodulin.
fractionated as described. The cytosolic fractions were analyzed by DEAE-cellulose chromatography. One set of two similar experiments is shown in Fig. 4. cAMP PDE activity in peaks 2 and 3, which were separately eluted
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE
2106 10
< v> o
j
A
9
-
8
-
-INS"
B
1 ~ "-
7
1 »•
6
ill
5 4
e Q.
1
2.520
1
15"
/i '
3 2
I X l.o-
1
^ ^ 0 . 5 -
n
J
800
i
II
-
i
1000 1200
^y*
/
- 1 \, 1/ j S^L 1
800
»
( 1 ,o.
- l\
4-
.afi
5 \i%l ml) significantly increased the cytosolic cilostamide-sen-
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE C +INS, 50/xg/ml
FIG. 5. Effect of insulin dose on the PDEs in the placental cytosolic fraction. Fresh placenta (60 g each) was processed and treated with buffer only (—INS) or insulin (+INS) at different concentrations (5, 50, and 200 /ug/ml), as described in Materials and Methods. One milliliter of each of the cytosolic fractions prepared from the four samples was analyzed by Mono-Q (Pharmacia) chromatography. The elution conditions were described in Fig. 4. The eluates were assayed for cAMP PDE activity using 0.5 tiM cAMP (O). The protein concentration was monitored by absorbance at 280 nm (—). The salt gradient was determined by measuring conductivity (•). A, B, C, and D show Mono-Q chromatographs of basal and insulin-stimulated (in the presence of 5, 50, and 200 ng/m\) PDEs, respectively.
12-
6
\ s
\
-0.5
Vol. 126, No. 4 Printed in U.S.A.
Characterization of Human Placental Cytosolic Adenosine 3',5'-Monophosphate Phosphodiest erase by Inhibitors and Insulin Treatment* LIMIN XIONG (HSIUNG), THOMAS R. L E B O N , AND YOKO FUJITA-YAMAGUCHI Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010
cAMP PDE). The third form was a low Km cAMP PDE, but was only modestly sensitive to inhibition by cGMP or cilostamide. The fourth form was a cAMP PDE which showed high sensitivity to inhibition by cGMP or cilostamide. The IC50 values of the fourth form were comparable to those of rat adipose insulinsensitive PDE. However, its Km for cAMP was 2 nM, which is about 10 times higher than that of the rat enzyme. Insulin treatment on placenta tissues stimulated at least two PDEs, the third and fourth forms. To our knowledge, this is the first report to describe insulin-sensitive cAMP PDEs in the cytosolic fraction of human placenta. (Endocrinology 126: 2102-2109,1990)
ABSTRACT. To gain insight into the regulation of low Km cAMP phosphodiesterases (PDE) by insulin in human tissues, PDEs in human placenta were studied. Human placenta contained cAMP PDEs in particulate and cytosolic fractions. More than 99% of the total activity was localized in the cytosolic fraction. The cytosolic fraction exhibited at least four cyclic nucleotide PDEs when fractionated by DEAE-cellulose chromatography. The first form was a calmodulin-activated PDE which hydrolyzed both cGMP and cAMP. The second form was a high affinity cAMP PDE with a nonlinear kinetic characteristic, but was not inhibited by either cGMP or cilostamide (either compound is known to specifically inhibit rat insulin-sensitive
I
N 1966, Butcher et al. (1) reported that insulin decreases intracellular cAMP levels in fat cells when the cAMP levels are elevated by catecholamines. Later, other investigators were able to observe that insulin decreases both the cAMP concentration and lipolysis under physiological conditions (2, 3) and that insulin stimulates cAMP phosphodiesterases, the only enzymes that break down cAMP in mammalian cells. Insulin, when applied to rat adipocytes, increases low Km cAMP PDE activity 2- to 3-fold (4). The insulin-sensitive PDE is associated with a microsomal fraction, designated P2. The optimum concentration of insulin for the stimulation is 1-3 nM. The enzyme activity is also increased when adipocytes are exposed to lipolytic agents, such as catecholamines, ACTH, and TSH (5, 6). The studies performed with rat (5) and 3T3-L1 (7) adipocytes indicated that there are some differences between the effects of insulin and epinephrine; that is, the increase in the particulate PDE activity was maximal after incubation of the cells with insulin for about 5 min and was sustained Received September 18,1989. Address all correspondence and requests for reprints to: Yoko FujitaYamaguchi, Department of Molecular Genetics, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010. * This work was supported by NIH Grant DK-29970 and a research grant from American Diabetes Association, California Affiliate.
at least 30-60 min in the presence of insulin, whereas in the presence of epinephrine PDE activity was maximal between 5-10 min and declined thereafter. Activation of the hepatic enzyme by insulin and glucagon has also been extensively studied (8, 9). While activation of this enzyme in fat and liver by insulin as well as other hormones has been well documented at the physiological level, the molecular mechanism of this activation is not known. An involvement of cAMP-dependent protein kinase (A-kinase) in the activation of this enzyme in rat adipocyte P2 fraction has recently been suggested (10). However, it is not yet known whether low Km cAMP PDE is an immediate substrate of A-kinase. Purification of hormone-sensitive low Km cAMP PDEs should help in understanding the mechanisms of regulation of this enzyme. Putative insulin-sensitive enzymes have been purified from rat adipocytes and rat liver (11-13). To date, however, protein chemical studies to further analyze low Km cAMP PDEs have not been conducted due to a difficulty in purifying sufficient quantities of the enzyme. According to the order of elution from a DEAE-cellulose column and kinetics characteristics, PDEs from different tissues were originally classified into three major classes, D-I, D-II, and D-III, by Appleman and Terasaki (14). The third fraction, D-III, was classified as a
2102
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE cAMP-specific PDE and descriptively called low Km cAMP PDE. At least two subclasses of low Km cAMP PDEs can be distinguished on the basis of their physicochemical properties and sensitivity to specific PDE inhibitors (15, 16). One type is rather sensitive to inhibition by Ro 20-1724 and rolipram, and the other to cGMP and cilostamide. The particulate insulin-sensitive PDE has been shown to be inhibited potently by cGMP and cilostamide, but much less effectively by Ro 20-1724. Unlike studies on rat or mouse low Km cAMP PDE, studies on the regulatory mechanism of human PDE activity have been limited, except for the PDE from platelets (17). In our present study we have characterized low Km cAMP PDEs in human placenta. Placenta is not only a good source for purifying human enzymes but is also an important tissue for fetal growth. Effects of growth factors, such as insulin and insulin-like growth factors (IGFs), on placental cells are currently not well understood. We report that insulin stimulates cAMP PDEs in the cytosolic fraction of placenta.
Materials and Methods
2103
were resuspended in 2 vol of the same buffer and assayed immediately or stored at —75 C (particulate fraction). Insulin treatment and preparation of cytosolic fraction Placenta pieces, prepared as described above, were further cut into approximately 2-mm3 pieces, washed with saline by centrifuging at 2000 X g for 5 min, then washed in the same manner with Krebs-Ringer-HEPES buffer containing 2% BSA. Tissue pieces were preincubated in the buffer at 37 C for 20 min and then for another 10 min in the absence (for basal activity) or presence (for insulin-stimulated activity) of added insulin. The buffer was removed from placenta pieces by centrifugation at 2000 X g for 5 min. The placenta tissues were washed three times using the same centrifugation method with 10 mM TES buffer, pH 7.0, containing 0.25 M sucrose, 0.2 mM PMSF, and 1 fig/ml each of leupeptin, pepstatin-A, and aprotinin. The placenta pieces were homogenized with a Tekmar Tissumizer as described above. The homogenate was centrifuged at 2000 X g for 10 min, after which any placental debris was discarded. The supernatant obtained was centrifuged at 100,000 X g for 30 min. The 100,000 X g supernatant and pellet corresponded to the cytosolic fraction and the particulate fraction (P2 plus P3 fraction), respectively.
Materials
Low Km cAMP PDE assay
Porcine insulin was a gift from Eli Lilly (Indianapolis, IN). [2,8-3H]cAMP (30-50 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Crotalus atrox snake venom, fraction V BSA, phenylmethylsulfonylfluoride (PMSF), N"benzoyl-L-arginine ethyl ester (BAEE), leupeptin, pepstatin-A, aprotinin, and theophylline were obtained from Sigma (St. Louis, MO). Ca2+-free porcine brain calmodulin was purchased from Ocean Biologies (Edmonds, WA). Mono-Q and Superose12 columns were obtained from Pharmacia-LKB (Piscataway, NJ). Griseolic acid, cilostamide, and Ro 20-1724 were gifts from Dr. M. Yamazaki of Sankyo Co. Ltd. (Tokyo, Japan) (18), Dr. S. Hosozawa of Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan), and Dr. V. C. Manganiello of the NIH, respectively. All other chemicals used were reagent grade.
The enzyme activity was measured according to the two-step assay procedure of Thompson and Appleman (19) with some modifications. In the first step of the assay, the [2,8-3H]cAMP was converted by phosphodiesterase to 5'-[2,8-3H]cAMP, which was subsequently converted to [2,8-3H]adenosine by the added nucleotidase in the second step. In the first step, the cytosolic enzyme fraction was incubated for 10 min in 0.1 ml 50 mM Tris-HCl buffer, pH 7.4, containing 0.5 mM dithiothreitol, 5 mM MgCl2, and 0.5 nM cAMP ([2,8-3HlcAMP; 200,000 dpm/assay) at 30 C. The reaction was stopped by heating in a heating block at 100 C for 90 sec. After the reaction mixture was cooled at -20 C for 4 min, 10 ^l Crotalus atrox snake venom (5 mg/ml) were added. The second step reaction was allowed to proceed for 20 min at 37 C. Unreacted cAMP was then removed by mixing with 1 ml of a 33% slurry of Dowex AG-1X2 (Bio-Rad, Richmond, CA). The mixture was rocked at 4 C for 5 min and centrifuged for 5 min at 2,200 x g. A 0.5-ml aliquot of the supernatant was then counted by liquid scintillation. For the assay of cAMP PDE in particulate fractions, the procedure was similar to this, except that 40 mM TES buffer, pH 7.5, and 4 mM MgSO4 were used instead of Tris, MgCl2, and dithiothreitol according to the method of Kono et al. (4, 20). For kinetic analysis, assays for cAMP or cGMP hydrolysis were carried out using an identical procedure, with the substitution of 3H-labeled cAMP or cGMP (~6000 dpm/pmol) at concentrations of 0.14, 0.24, 0.36, 0.58,1.21, and 1.94 X 10"6 M. An assay blank, consisting of a corresponding amount of 3Hlabeled cAMP or cGMP with water in place of enzyme preparation, was carried through the assay procedure, and the background counts were subtracted from each sample.
Subcellular fractionation of human placenta Fresh normal human placentas were obtained within 1 h after delivery, kept on ice, trimmed of amnion and chorion, washed with 0.25 M sucrose, and cut into small pieces. The pieces were transferred to 1 vol 50 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM PMSF, 2 mM BAEE, and 1 /xg/ml each of leupeptin, pepstatin-A, and aprotinin, homogenized five times for 1 min each with a Tissumizer from Tekmar Co. (Cincinnati, OH) at a setting of 30, and centrifuged at 15,000 X g for 20 min at 4 C. The supernatant was then centrifuged at 100,000 X g for 1 h at 4 C. The supernatant (cytosolic fraction) was assayed immediately or stored at -75 C. The pellet was suspended in 10 vol 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM PMSF and 2 mM BAEE, homogenized with a Tekmer Tissumizer at a setting of 10, and centrifuged at 100,000 X g for 60 min at 4 C. The sedimented membranes
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE
2104 Data analysis
Endo • 1990 Voll26»No4
NH2
The values presented are the mean and SEM. The effect of insulin on PDE activity was analyzed using the one-sample t test. The effect of cilostamide was tested using linear regression, and the regression curves were compared at the different insulin levels using the analysis of covariance.
0=P-0 I OH
Results
NH2
HOOC
OH
cAMP
OH
Griseolic acid
Subcellular distribution of low Km cAMP PDE Participate and cytosolic fractions were prepared by centrifugation at 100,000 X g for 60 min from placental 15,000 X g supernatant. The participate fraction is equivalent to the fat P2 plus P3 fraction with which insulinsensitive PDE is associated. The specific activity of the particulate fraction was 28.2 ± 6.6 pmol/min/mg protein (Table 1). When the particulate fraction was prepared in the presence of PMSF and BAEE but in the absence of leupeptin, pepstatin-A, and aprotinin, only one tenth of the specific activity was found in the particulate fraction. This suggests that membrane-bound low Km cAMP PDE is very susceptible to proteinases. The specific activity of cytosolic enzymes (~50 pmol/min/mg protein) was significantly higher than that of the particulate fraction. Cytosolic PDE activity accounted for more than 99% of the total PDE activity in placenta. Effect of inhibitors on cAMP hydrolysis by particulate and cytosolic enzymes To determine whether so-called insulin-sensitive PDEs are present in placental tissue, we used five PDE inhibitors, as presented in Fig. 1, including cGMP and cilostamide, that are known to be specific inhibitors of hormone-regulated particulate PDE activity in rat adipocytes and mouse 3T3-L1 adipocytes (16). Cilostamide and cGMP significantly inhibited cytosolic PDE (40% at 10 /uM), but had no effect on particulate PDE. Of the three nonselective inhibitors, griseolic acid was the most potent, while theophylline and Ro 20-1724 were not as effective in inhibiting the PDE activity of either fraction (Table 2). Separation and characterization of cytosolic low Km PDEs
The cytosolic PDEs were fractionated by DEAE-cellulose chromatography at pH 6.5, and the fractions were TABLE 1. Subcellular distribution of low Km cAMP PDEs in human placenta SA Total activity (pmol/min/mg (nmol/min)a protein) Particulate PDE (n = 5) Cytosolic PDE (n = 5) 0
28.2 ± 6.6 51.7 ± 6.4
Total activity per placenta of about 400 g.
3.74 ± 0.47 433 ± 54
Ratio (%) 0.9 99.1
0
H
CH 3 0=P-0
Theophylline
OH
OH
cGMP /CH3 O(CH2)3CON OCH 2 (CH 2 ) 2 CH 3 0CH 3
Cilostamide (OPC 3689)
Ro 20-1724 FIG. 1. Structure of PDE inhibitors and cAMP.
TABLE 2. Effects of inhibitors on cAMP hydrolysis by low Km cAMP PDEs from human placenta IC60 (MM)' Griseolic acid Cilostamide cGMP Theophylline Ro 20-1724
Particulate
Cytosolic
0.08 >10 >10 300 >1000
0.08 -10* -10" 400 >1000
0
IC 50 is an average of two similar experiments. At a concentration of 10 /*M, the PDE activity was 60% of the control value. 6
assayed for both cAMP and cGMP hydrolysis. As shown in Fig. 2, peak 1 contained higher cGMP PDE activity than cAMP PDE activity, whereas peaks 2 and 3 exhibited higher cAMP PDE activity. Since peak 2 did not look like a typical single activity peak, fractions 2a and 2b from the earlier portion of the peak and the later portion of the peak, respectively, were separately analyzed further. cAMP PDE activity in each fraction, designated 1, 2a, 2b, or 3 in Fig. 2, was measured in the presence of different concentrations of the five inhibitors (Fig. 3). As we have previously observed with particulate and cytosolic PDEs from placental tissue (Table 2), griseolic acid, theophylline, and Ro 20-1724 nonspecifically inhibited the PDE activity of all fractions. Cilostamide and
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE
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DE2b
o
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100
200
300
400
500
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Volume (ml)
FIG. 2. DEAE-cellulose chromatography of human placental cytosolic fraction. Forty-six milliliters of the cytosolic fraction, 100,000 X g supernatant, were applied to a column (3 X 14 cm) of DE-52 (Whatman, Clifton, NJ) equilibrated with 70 mM sodium acetate buffer, pH 6.5, containing 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.2 mM PMSF. The column was washed with the sodium acetate buffer, pH 6.5, until the absorbance at 280 nm reached 0.12. The proteins were eluted by a linear gradient (70 mM to 0.5 M sodium acetate, pH 6.5) and then with 1.2 M sodium acetate, pH 6.5. The eluates were assayed for cAMP (•) and cGMP (•) PDE activities using a substrate concentration of 0.5 MM- The protein concentration was monitored by measuring absorbance at 280 nm (O). The salt concentration was determined by measuring conductivity ( ). Four PDE fractions we have chosen for further analysis are marked with arrows and numbered 1, 2a, 2b, and 3.
cGMP markedly inhibited the PDE activity in fraction 3, with IC50 values of 0.03 and 0.2 nM, respectively (Table 3). These two inhibitors inhibited the activity in fraction 2b with IC50 values of 0.4 and 2 JUM, respectively (Table 3). While cilostamide modestly inhibited the activity in fraction 2a (40% inhibition at 10 nM), it had no significant effect on the activity in fraction 1. These data indicate that fractions 2b and 3 are likely to be hormoneregulated PDEs. Kinetic analysis was carried out for the enzymes found in the four fractions. The results are included in Table 3. The Knl values for fraction 2b and 3 enzymes were a little higher than expected based on the previously reported values for rat adipose enzymes. The cAMP (or cGMP) PDE activity of each fraction was measured in the absence or presence of Ca2+ and calmodulin in order to test its calmodulin sensitivity. Fraction 1 PDE activity was activated by calmodulin (a 20-fold increase in cGMP PDE activity and an 1.6-fold increase in cAMP PDE activity). In contrast, calmodulin had no effect on PDEs in fractions 2a, 2b, and 3 (Table 3). Effect of insulin on low Km cAMP PDE in the placental cytosolic fraction Placenta pieces were incubated with or without 40 nM insulin, then the pieces were washed, homogenized, and
100
50
• Cilostamide (0PC3689)
0 DE3
10 -10
2 0.4
0.2 0.03
20-fold (cGMP) 1.6-fold (cAMP) IC5o for cAMP hydrolysis cGMP (MM) Cilostamide
>10 >10
0
cAMP (or cGMP) PDE activity was measured in the absence or presence of 1 pM CaCl2 and 50 /xM calmodulin.
fractionated as described. The cytosolic fractions were analyzed by DEAE-cellulose chromatography. One set of two similar experiments is shown in Fig. 4. cAMP PDE activity in peaks 2 and 3, which were separately eluted
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE
2106 10
< v> o
j
A
9
-
8
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B
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ill
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1
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1
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n
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i
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-
i
1000 1200
^y*
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- 1 \, 1/ j S^L 1
800
»
( 1 ,o.
- l\
4-
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5 \i%l ml) significantly increased the cytosolic cilostamide-sen-
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INSULIN-SENSITIVE HUMAN PLACENTAL cAMP PDE C +INS, 50/xg/ml
FIG. 5. Effect of insulin dose on the PDEs in the placental cytosolic fraction. Fresh placenta (60 g each) was processed and treated with buffer only (—INS) or insulin (+INS) at different concentrations (5, 50, and 200 /ug/ml), as described in Materials and Methods. One milliliter of each of the cytosolic fractions prepared from the four samples was analyzed by Mono-Q (Pharmacia) chromatography. The elution conditions were described in Fig. 4. The eluates were assayed for cAMP PDE activity using 0.5 tiM cAMP (O). The protein concentration was monitored by absorbance at 280 nm (—). The salt gradient was determined by measuring conductivity (•). A, B, C, and D show Mono-Q chromatographs of basal and insulin-stimulated (in the presence of 5, 50, and 200 ng/m\) PDEs, respectively.
12-
6
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