ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 196, No. 2, September, pp. 465-474, 1979
Multiple
Forms of Cyclic Nucleotide Phosphodiesterase Bovine Carotid Artery Smooth Muscle1 TIMOTHY
Department
of Anatomy
J. MURTAUGH and Cardiovascular
AND RAMESH Center,
College
from
C. BHALLA
of Medicine,
University
of Iowa,
Iowa City, Iowa 52242 Received October 11, 1978; revised April 20, 1979 DEAE-cellulose chromatography, in the presence and absence of Ca*+, of the 16,000g supernatant from bovine carotid artery smooth muscle has been used to separate four different types of cyclic nucleotide phosphodiesterase (3’:5’-cyclic-nucleotide 5’-nucleotidohydrolase, EC 3.1.4.17) activity, designated types A, B, C, and D. Type A is a high affinity, cyclic AMP-specific form of phosphodiesterase (K, = 1.6 PM) and elutes at relatively high ionic strength. Type B is a high affinity (K, = 2 PM), cyclic GMP-specific form which elutes at low ionic strength. Type C is a mixed substrate form, displaying anomalous kinetics for the hydrolysis of both cyclic AMP and cyclic GMP. It elutes from DEAE-cellulose at an ionic strength intermediate to that of types A and B. Type D is also a mixed substrate form of phosphodiesterase. However, its elution pattern from DEAE-cellulose differs, depending on whether CaZ+ is present or not, suggesting a Ca2+-dependent interaction between this enzyme form and the acidic Caz+-dependent regulator protein (CDR). The hydrolytic activity of type D is stimulated by CDR, and activation requires the simultaneous presence of Ca2+ and CDR. Kinetic analysis of cyclic AMP hydrolysis by type D gives a linear double reciprocal plot; activation has no effect on the K, but increases the velocity approximately sixfold. Activation of cyclic GMP hydrolysis apparently affects both the K, and V. At all concentrations tested, the degree of activation is higher with cyclic AMP than with cyclic GMP. It is suggested that while the activable form of phosphodiesterase may play a relatively minor role in the overall hydrolysis of cyclic nucleotides, Ca2+-dependent activation may have a more important role in regulating the level of cyclic AMP than that of cyclic GMP in vascular smooth muscle.
Adenosine 3’:5’-monophosphate (cyclic AMP) and guanosine 3’:5’-monophosphate (cyclic GMP) have been implicated in the regulation of relaxation and contraction of vascular smooth muscle (l-4). It has been suggested that cyclic AMP promotes relaxation by regulating the intracellular levels of free Caz+ available to the contractile apparatus. This may be accomplished by a cyclic AMP-dependent phosphorylation of a microsomal membrane component and the subsequent stimulation of Ca2+ uptake by these membrane vesicles (5-7). While the physiological role of cyclic GMP in vascular smooth muscle is unclear, its levels in the 1 This work was supported by NIH Grant HL 19027 and predoctoral training grant PHS-GM07228 (to T. J. M.).
cell are increased by agents which promote contraction (8). Despite the evidence for cyclic nucleotide regulation of vascular smooth muscle tone, little is known regarding the control of cyclic nucleotide synthesis and degradation in this tissue. Because of the apparent involvement of cyclic AMP in regulating Ca2+ levels in vascular smooth muscle, it is of some interest to investigate the possible involvement of Ca2+ in a reciprocal regulation of cyclic nucleotide levels through the activation of phosphodiesterase activities. Recent studies in numerous tissues have demonstrated the presence of multiple forms of cyclic nucleotide phosphodiesterase (9-19). These enzymes show differences in substrate specificity, subcellular allosteric interactions, and distribution, 465
0003-9861/79/100465-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights
of reproduction
in any form reser~ red.
MURTAUGH
466
AND BHALLA
sensitivity to activation by a heat-stable, Ca2+-dependent regulator protein (CDR).2 There has, however, been little investigation into the possible forms of phosphodiesterase in vascular smooth muscle. Using pig coronary arteries, Wells et al. (20) isolated two peaks of phosphodiesterase activity by DEAE-cellulose chromatography. Peak I hydrolyzed both cyclic AMP and cyclic GMP, but with a much higher affmity for cyclic GMP, and peak II was specific for cyclic AMP hydrolysis. Only peak I was affected by CDR, giving increases in velocity for both substrates but no change in substrate affinity. Ho et al. (21) have shown that the elution pattern from DEAE-cellulose of the activatable form of phosphodiesterase from bovine heart depends on the state of its Ca2+-dependent interaction with CDR. We have taken advantage of this property by using DEAE-cellulose chromatography in the presence or absence of Ca2+ and have separated four peaks of phosphodiesterase activity, with differing kinetic properties, from bovine carotid artery smooth muscle. A portion of this work has been presented in preliminary form (22). EXPERIMENTAL
PROCEDURES
Materials. Dowex l-X8,200-400 mesh (Cl- form), cyclic AMP, cyclic GMP, snake venom (Crotalus atroz), Tris base, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid (EGTA), and O(diethylaminoethyl) (DEAE)-cellulose were purchased from Sigma Chemical Company, St. Louis, Missouri. Aquasol scintillation tluor, cyclic[3H]AMP (3.8 Cilmmol), cycliQH]GMP (7.8 Ci/mmol), [‘4C]adenosine (0.05 Cii mmol), and [Y&uanosine (0.5 Ci/mmol) were obtained from New England Nuclear, Boston, Massachusetts. Fresh bovine carotid arteries were obtained from a local slaughterhouse and transported on ice. The tissue was cleaned of loose fat and connective tissue, frozen in liquid nitrogen, and stored at -20°C until use. Preparation of tissue extracts. The buffer used for tissue homogenization, as well as for chromatographic procedures, consisted of 10 mM Tris-HCl, pH 7.2, 1 mM MgCl,, 2 mM 2-mercaptoethanol, and 5 mM sodium bisulfite. Approximately 80 g of frozen arteries was thawed in buffer, stripped of adventitial connective tissue, and minced, yielding approximately 50 g of 2 Abbreviations used: CDR, Ca2+-dependent regulator protein; EGTA, ethylene glycol bis(P-aminoethylether) N,N’-tetraacetic acid.
smooth muscle. The tissue was homogenized in 10 vol of buffer in a Waring Blendor (three pulses of 30 s each at high speed). The homogenate was filtered through two layers of cheesecloth, centrifuged for 15 min at l,OOOg, filtered through four layers of cheesecloth, recentrifuged for 15 min at lS,OOOg, and filtered through glass wool. A portion of this supernatant was stored in small aliquots at -20°C for use in tissue extract experiments. All steps were performed at 4°C. Assay procedures. The assay for phosphodiesterase was in principle that described by Butcher and Sutherland (23), using 3H-labeled cyclic nucleotides based on the procedure of Thompson and Appleman (24) with minor modifications. A two-step procedure was used. The reaction mixture consisted of 30 mM TrisHCl, pH 8.0,4 mM MgCl,, appropriate cyclic nucleotide concentration (including 3H-labeled substrate, giving approximately 100,000 cpm per assay), and an appropriate amount of enzyme in a total volume of 0.5 ml. The snake venom reaction was terminated by the addition of 1 ml of a Dowex slurry (3 H,O:l resin) containing 3 mM acetic acid (pH 3.0) for cyclic AMP assay or 115 mM formic acid (pH 2.1) for cyclic GMP assay, according to Boudreau and Drummond (25). Blank tubes were given the enzyme carrier buffer in place of enzyme. 14C-Labeled nucleoside recovery was monitored in each experiment and averaged 70-90%. The data reported are corrected for nucleoside recovery. All assays were carried out under conditions of linearity with respect to time and enzyme dilution. Specific activity of phosphodiesterase is expressed as nanomoles of cyclic nucleotide hydrolyzed per minute per milligram protein at 30°C. Protein was determined by the method of Lowry et al. (26) with bovine serum albumin as standard. Separation of phosphodiesteruses. The carotid artery supernatant fraction isolated above was dialyzed overnight against the homogenization buffer containing either 50 pM CaCl, or 100 pM EGTA. The dialyzed supernatant (approximately 400 ml) was applied to a DEAE-cellulose column (30 x 1.8 cm) previously equilibrated with the same buffer, and the column was washed with 3 column vol of buffer. The initial wash contained no detectable phosphodiesterase activity. The column was developed with 1000 ml of either the Ca2+-containing buffer or the EGTA-containing buffer, with a linear NaCl gradient (0 to 0.6 M). Fractions containing peak phosphodiesterase activity were pooled, concentrated by ultrafiltration in an Amicon apparatus with a UM-10 membrane, and dialyzed for rechromatography. Rechromatography on DEAE-cellulose (20 x 1 cm) with a 500-ml linear gradient (0 to 0.5 M NaCl) was performed using either the Ca2+-containing buffer or the EGTA-containing buffer, to separate peaks which eluted together under the ionic conditions of the fast chromatography. The appropriate peak fractions were again pooled, concentrated, dialyzed against 10 mM Tris-HCl, pH 7.4, 1 mM MgCl,, and 2 mM 2-
MULTIPLE
FORMS OF BOVINE
CYCLIC NUCLEOTIDE
mercaptoethanol, and stored in aliquots at -20°C. In subsequent discussions, “CaZ+-column” and “EGTAcolumn” will refer to DEAE-cellulose columns eluted in the presence of either 50 pM CaCl, or 100 pM EGTA, respectively. Preparation of CDR. A crude CDR preparation was made by boiling the carotid artery supernatant in a steam bath for 5 min followed by centrifugation to remove denatured material, based on the heat stability reported for the Ca*+-dependent regulator protein of phosphodiesterase (27,28). This preparation contained no detectable phosphodiesterase activity. Twenty microliters of this preparation gave maximal stimulation of an activator-deficient DEAE-cellulose peak. Partially purified CDR was isolated with DEAEcellulose chromatography in the presence of EGTA (Fig. 3). The peak activator fractions were pooled, boiled, dialyzed, and stored frozen in aliquots. This partially purified preparation was used in experiments involving the rechromatography of an activatordeficient phosphodiesterase peak. The CDR preparation was added to the enzyme peak prior to dialysis against Ca*+-containing buffer.
of Multiple
Separation
467
PHOSPHODIESTERASE
Phosphodiesterase
Activities The presence or absence of Ca2+ during DEAE-cellulose chromatography determines whether CDR will remain bound to phosphodiesterase during elution (21, 29). Due to the acidic nature of the CDR protein (29), an activatable form of phosphodiesterase will elute at different ionic strengths from DEAE-cellulose depending on whether it elutes bound to CDR or not. Two separate regimes of DEAE-cellulose chromatography were used in the following experiments. One involved chromatography of the smooth muscle 16,000g supernatant in the presence of Ca2+ (Fig. l), followed by rechromatography of the peak fractions in the presence of EGTA (Fig. 2). Alternately, the CAMP
RESULTS
Characteristics of Phosphodiesterase Activity in Tissue Extracts Cyclic AMP hydrolysis by the 16,000g supernatant exhibited nonlinear kinetics on a Lineweaver-Burk double reciprocal plot. The curve was concave downward, suggesting either negative cooperativity or the presence of multiple enzyme forms. Apparent K, values for cyclic AMP were 1.2 and 14 ,&I. Cyclic GMP hydrolysis gave a linear double reciprocal plot with an apparent K, of 2.5 ,uM. Hydrolysis of cyclic AMP at both high (50 PM) and low (2 PM) concentrations was inhibited approximately 30% by EGTA concentrations of 50 PM or above (data not shown). This inhibition was abolished by 100 PM Ca2+ but was reestablished by EGTA concentrations greater than 100 PM. Hydrolysis of 2 PM cyclic GMP was affected similarly. Increasing Ca2+ concentrations (from 10-6-10-3 M Ca2+) had no effect on either cyclic AMP or cyclic GMP hydrolysis (data not shown), presumably due to the presence of sufficient endogenous Ca2+ in the supernatant to allow full activation. Exogenous CDR also had no effect on the phosphodiesterase activities of the tissue supernatant.
04 03: 5 02
30
60 Fraction
90
G 8
120
Number
FIG. 1. DEAE-cellulose chromatography of a dialyzed 16,OOOgsupernatant. Chromatography was performed as described under Experimental Procedures. The column was eluted with a 0 to 0.6 M NaCl gradient in buffer containing 50 pM CaCl*. Fractions were assayed for cyclic AMP (top panel) and cyclic GMP (bottom panel) hydrolysis using a substrate concentration of 20 pM with either no additions (0) or in the presence of 100 pM EGTA (0). Assay with Ca2+ and CDR gave no change in activity from that obtained with no additions. Results are representative of five preparations. A, B, C, and D in this and subsequent figures refer to the respective phosphodiesterase types which are contained in the indicated pooled fractions.
MURTAUGHANDBHALLA
468 6c
..n.rn
LHIvIr
-No
5-
addition
-+5qN 4.
GP
+CDR
&
-05
cGMP
,,Y 54-
-04s
3-
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2-
-02
I-
-0 I
2 5
c-m IO
20
30
” 40
50
Fractton
8 60
‘1 70
130
single major peak eluting at 0.12 M NaCl, and two minor peaks representing contaminating components of the other peaks (Fig. 5B). The major peak hydrolyzed cyclic GMP preferrentially and was designated type B. Kinetic analysis of cyclic GMP hydrolysis by type B gave a linear double reciprocal plot with an apparent K, of 2.0 PM. Type B also had a low level of activity with cyclic AMP as substrate, with a K, of approximately 150 PM. Caz+ and CDR had no effect on the hydrolysis of either cyclic GMP or cyclic AMP by type B. Type C was isolated from an EGTA column (Fig. 3). It eluted from DEAEcellulose at an ionic strength intermediate -
01 90
No odd,t~on
Number
FIG. 2. Rechromatography of fractions 75-105 (Fig. 1) on DEAE-cellulose, eluted with a buffer containing 100 pM EGTA. Assays were as described for Fig. 1, with either no additions (0) or in the presence of 50 pM Ca2+ plus 20 ~1 of a boiled carotid artery supernatant (0). Results are representative of four preparations.
tissue extract was first chromatographed in the presence of EGTA (Fig. 3), followed by rechromatography of the peak fractions in the presence of Ca2+ and added CDR (Fig. 4). A combination of these procedures was used to separate four different types of phosphodiesterase activity, designated as types A, B, C, and D. Table I summarizes their kinetic characteristics and chromatographic behavior. Type A was isolated from an EGTA column (see Experimental Procedures) (Fig. 3). Rechromatography of this peak in the presence of either Ca2+ or EGTA gave a single major peak eluting at 0.3 M NaCl and a minor peak representing contaminating peaks (Fig. 5A). The major peak was highly specific for cyclic AMP hydrolysis and was designated type A. Kinetic analysis gave a linear double reciprocal plot with an apparent K, of 1.6 PM, and its activity was not affected by Ca*+ and CDR. Type B was isolated from a Ca2+ column (Fig. 1). Rechromatography of this peak in the presence of either Ca*+ or EGTA gave a
.P h c F u9 i
IO
05 04
s ? 03 2 9 02
5
01
$? 30° $3 200
Sk 83
100
cm -No addition -*50,,M Ca-
t
FIG. 3. DEAE-cellulose chromatography of a dialyzed 16,OOOg supernatant, in the presence of 100 pM EGTA. Fractions were assayed for cyclic AMP (top panel) and cyclic GMP (middle panel) hydrolysis with no additions (0) or in the presence of 50 pM Ca2+ plus 20 ~1 boiled carotid artery supernatant (0). Ca*+ or CDR alone had no effect on basal activity (not shown). Substrate concentration = 20 ELM. Lower panel: An aliquot of each fraction was boiled and assayed for its ability to stimulate cyclic AMP hydrolysis. Unboiled column fraction No. 36 was used as a source of activatable phosphodiesterase. Assays were performed in the presence (0) and absence (0) of 50 NM Ca*+. Results are representative of seven preparations.
MULTIPLE
FORMS OF BOVINE
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
469
strength (approximately 0.12 M NaCl) and is separated from types A and C. Con-No additm versely, if the tissue supernatant is first chromatographed in the presence of EGTA (Fig. 3), type D elutes at low ionic strength, in the same fractions as type B. When these fractions are rechromatographed in the presence of Ca*+ and exogenous CDR type D is separated from type B by eluting at high ionic strength (Fig. 4). Rechromatography in the presence of Ca*+ alone does not give this separation. This demonstrates that type D is capable of binding CDR in the presence of Ca2+. Type D was isolated by starting with an EGTA column (Fig. 3). The activity of the first peak from this column was stimulated 00 twoto threefold by assay in the presence 10 20 30 40 50 60 70 80 of Ca*+ and CDR. Ca*+ alone or CDR alone Fraction Number had no effect on the activity of this peak FIG. 4. Rechromatography of fractions 30-45 (Fig. (data not shown). Partially purified CDR 3) on DEAE-cellulose. Pooled fractions were mixed was also isolated from this column (Fig. 3). with heat-treated pooled CDR fraction (Fig. 3), dia- The pooled CDR fractions were heat treated lyzed, and chromatographed in the presence of 50 WM Ca*+. Fractions were assayed for cyclic AMP (top in a steam bath for 5 min and centrifuged. which had no panel) and cyclic GMP (bottom panel) hydrolysis in the This CDR preparation, phosphodiesterase activity, presence (0) or absence (0) of 100 pM EGTA. Assay endogenous in the presence of added Ca2+ and CDR gave no in- was mixed with the pooled activatable phoscrease in activity over that seen with no additions (not phodiesterase fractions. This mixture was shown). Substrate concentration = 20 pM. Results are concentrated and dialyzed against the Ca*+representative of four preparations. containing buffer. Upon rechromatography of this mixture in the presence of Ca*+, type D eluted at a higher ionic strength than type to that of types A and B (approximately B (Fig. 4). Neither peak was stimulated by 0.22 M NaCl). Rechromatography of this Ca*+ and CDR but the activity of type D was peak in the presence of either Ca*+ or EGTA reduced 60-70% by assay in the presence of gave a single major peak eluting at 0.22 M EGTA. In order to obtain a preparation of NaCl, which hydrolyzed both cyclic AMP type D free of endogenous CDR the fracand cyclic GMP (Fig. 5C). Type C displayed tions containing type D were pooled, connonlinear double reciprocal plots for the centrated, dialyzed against an EGTA-conhydrolysis of both cyclic AMP and cyclic taining buffer and rechromatographed in GMP (Table I). As with types A and B, Ca*+ the presence of EGTA. This procedure gave and CDR had no affect on the activity a single peak of activity eluting at 0.13 M kinetics of type C. NaCl which was stimulated three- to sixfold Type D is also a mixed-substrate form of by Ca*+ and activator (Fig. 6). The peak phosphodiesterase. However, its elution thus isolated was dialyzed to remove EGTA pattern from DEAE-cellulose differs de- and stored frozen for kinetic analysis. pending on whether Ca2+ is present or not. Cyclic AMP hydrolysis by type D was not In the presence of Ca*+, type D elutes at changed by increasing concentrations of high ionic strength, in essentially the same EGTA, Ca2+, or increasing amounts of CDR fractions as type A (approximately 0.3 M alone. Only in the presence of both Ca*+ and NaCl) (Fig. 1). Upon rechromatography of CDR was the activity increased approxithese fractions in the presence of EGTA mately sixfold (data not shown). Results (Fig. 2), type D elutes at a much lower ionic with cyclic GMP were similar. However,
470
MURTAUGH
AND BHALLA
TABLE
I
SUMMAR~OFKINETICSANDCHROMATOGRAPHICBEHAVIOROFMULTIPLEFORMS OFPHOSPHODIESTERASE FROMBOVINECAROTID ARTERY" DEAE elution pattern [NaCI] (M) V
(nmol/mg proteimmin)
Km (PM)
PDE
Substrate
Basal*
A
Cyclic AMP
1.6
B
Cyclic GMP
2.0
Cal+ + CDRc
In the presence of ca2+ (50pM) In the presence of + CDR EGTA (100 PM)
BasaP
Ca*+ + CDR”
1.7
4.4
4.8
0.30 (Figs. 1 and 5A)
1.8
1.2
1.2
0.13
0.12
(Figs. 1 and 4) C
D
Cyclic AMP
5.4;50
5.2;46
1.3;4.2
1.3;4.1
Cyclic GMP
0.6;27
0.825
1.5;3.1
1.4;3.3
Cyclic AMP
13.6
12.2
1.2
7.5
Cyclic GMP
2.4;16
2.3
1.9;4.3
4.9
0.30 (Figs. 2 and 3)
0.22 Figs. 1 and 5C) 0.30 (Figs. 1 and 4)
(Figs. 3 and 5B) 0.22 (Figs. 2 and 3) 0.12 (Figs. 2,3, and6)
’ Kinetic experiments were performed with a range of substrate concentrations from 0.1 pM to 2 mM.Chromatography was performed as described under Experimental Procedures. Results are means of duplicate determinations and are representative of experiments with at least three different preparations. Kinetic data were analyzed by a least-square regression analysis of a Lineweaver-Burk plot (r 2 0.95). b Activity was the samewith either no additions to the standard assay medium or in the presence of 0.1 mMEGTA. c Ca2+ + CDR, Activity in the presence of 50 pM Ca2+ and 25 Fg partially purified CDR.
maximal stimulation was only approximately threefold (data not shown). Kinetic analysis of cyclic AMP hydrolysis by type D is shown in Fig. ‘7A. Under basal conditions (the values being identical in the presence or absence of either EGTA or Ca2+), the double reciprocal plot was linear with an apparent K, of approximately 14 PM and a V of 1.2 nmol/mg proteimmin. In the presence of maximal Ca2+ and CDR the apparent K, was not affected (approximately 12 PM) while the V was increased approximately sixfold. The kinetics of cyclic GMP hydrolysis appear to be more complicated (Fig. ‘7B). Under basal conditions, the double reciprocal plot was nonlinear, exhibiting both high affinity (K, = 2.4 PM; V = 1.9 nmol/mg protein/min) and low affinity (K, = 16 pM; V = 4.3 nmol/mg protein/min) characteristics. Under conditions of maximal stimulation, however, a linear double reciprocal plot was observed. The apparent K, (2.3 PM) was the same as the high affinity K, seen under basal conditions,
while the V (4.9 nmol/mg proteimmin) was equal to the higher velocity of the low affinity activity. DISCUSSION
The results presented in this report demonstrate the presence of four different types of phosphodiesterase activity which can be isolated from vascular smooth muscle: a high affinity, cyclic AMP-specific form (type A); a high affinity, cyclic GMPspecific form (type B); a nonactivatable, mixed substrate form (type C); and an activatable, mixed substrate form (type D). We have not ruled out, however, the possibility that the chromatographic behavior or the hydrolytic activity of the native enzymes may have been altered by the action of endogenous proteases during the periods of purification used in our procedures. We have included sodium bisulfite, a protease inhibitor, in our buffers to minimize this possibility.
MULTIPLE
FORMS OF BOVINE
CYCLIC
Previous reports in smooth muscle have shown only two peaks of phosphodiesterase activity (20, 30, 31). Hidaka et al. (32) recently reported the separation of five phosphodiesterase peaks from human aorta. Two of the peaks which preferentially hydrolyzed cyclic GMP were activatable by Ca2+ and CDR. Wells et al. (20) have reported the presence of two peaks of phosphodiesterase activity from coronary artery, a nonactivatable, cyclic AMP-specific form and an activatable form which hydrolyzed both cyclic AMP and cyclic GMP. Since cyclic GMP was hydrolyzed only by the activatable form, they suggested that Ca2+-dependent activation is more im-
NUCLEOTIDE
PHOSPHODIESTERASE
471
8 CAMP 6 4
f
-
No addition
-
+5QM +CDR
Co”
n
0
0.0 0
20 Fraction
40 Number
60
FIG. 6. Rechromatography of type D (fractions 51-63 in Fig. 4) in the presence of 100 pM EGTA. Fractions were assayed for cyclic AMP (top panel) and cyclic GMP (bottom panel) hydrolysis with no additions (0) or in the presence of 50 pM Ca*+ and 25 pg partially purified CDR (0). Ca2+ or CDR alone had no effect on basal activity. Substrate concentration = 20 pM. Results are representative of three preparations.
FIG. 5. DEAE-cellulose rechromatography profiles for individual phosphodiesterase types. Fractions were assayed for cyclic AMP (0) and cyclic GMP (0) hydrolysis at a substrate concentration of 20 pM. Type A: Fractions 76-90 in Fig. 3 were rechromatographed in the presence of Ca*+ and CDR. Assay in the presence of 100 FM EGTA did not change the activity profile. Type B: Fractions 43-63 in Fig. 1 were rechromatographed in the presence of 100 pM EGTA. Assay in the presence of Ca*+ and CDR did not change the activity profile. Type C: Fractions 57-67 in Fig. 3 were rechromatographed in the presence of Caz+ and CDR. Assay in the presence of 100 pM EGTA did not change the activity profile.
portant in regulating cyclic GMP hydrolysis than cyclic AMP hydrolysis in vascular smooth muscle. However, we have shown the presence of a nonactivatable, cyclic GMP-specific enzyme, similar to that reported in liver and lung (11, 33-35). There are present, then, in vascular smooth muscle, high affinity forms for both cyclic AMP and cylic GMP which are not affected by CDR and Ca2+. Therefore, while Ca2+dependent activation may play a relatively minor role in regulating the levels of each cyclic nucleotide in the cell, it will have effects on both cyclic AMP and cyclic GMP. In the nonactivated state, type D has a higher affinity for cyclic GMP and has a higher level of activity with cyclic GMP as substrate at all concentrations. In the activated state, type D still has a higher level of activity with cyclic GMP at low substrate concentrations, whereas at substrate concentrations greater than 2 PM cyclic AMP hydrolysis is greater. However, at all substrate concentrations, the degree of activation of cyclic AMP hydrolysis is greater than that of cyclic GMP hydrolysis. Therefore, our data suggest that activation may play a more important role in regulating
472
MURTAUGHANDBHALLA
We have confirmed that DEAE-cellulose chromatography with a (NHJ2S04 gradient, as was used by Wells et al. (20), results in a profile similar to that which we obtain with chromatography in the presence of EGTA (Fig. 3) and that the activatable peak can be separated into two peaks (types B and D) by rechromatography in the presence of Ca2+ and activator, as in Fig. 4. Our data suggest that peak I of Wells et al. may consist of two enzyme forms (type B and type D) and that a change in the K, of the activatable form (type D) could have been masked by the presence of the high-affinity, nonactivatable cyclic GMP-specific form (type B). The major difference between our chromatography profile in the presence of EGTA and that of Wells et al. (20) is the presence in our profile of an enzyme form that elutes at an intermediate ionic strength, i.e., type C. While this discrepancy cannot be satisFIG. 7. Lineweaver-Burk plots for cyclic AMP(A) explained at this time, it may and cyclic GMP(B) hydrolysis by type D phospho- factorily diesterase. Substrate concentrations ranged from 0.1 represent species or tissue variation (bovine pM to 2 mM. 0, Basal activity (the same either in the carotid artery vs porcine coronary artery). presence or absence of 100 pM EGTA); 0, activity in A number of studies have shown that the the presence of 50 &bM Ca*+ and 25 pg partially purified concentration of CDR in many tissues is in CDR. Results are means of duplicate determinations excess of that necessary to fully activate the and are representative of experiments with three dif- Ca*+-sensitive phosphodiesterase activities ferent preparations. (38-42). This probably is the case for vascular smooth muscle as well. Tissue extracts by added Ca*+ and cyclic AMP levels. However, type D, in the cannot be stimulated CDR but can be inhibited by EGTA. In adactivated form, still demonstrates a higher dition, type D in the present study apaffinity for cyclic GMP than for cyclic AMP and in the presence of both substrates may parently elutes from DEAE-cellulose bound preferentially hydrolyze cyclic GMP. to CDR sufficient to keep it fully active Definition of the role of the activable en- when the column is eluted in the presence zyme in regulating cyclic AMP and cyclic of Ca*+. Therefore, regulation of the activatable phosphodiesterase in vascular GMP levels will, therefore, require futher study into its activity in the presence of smooth muscle will not be limited by the both substrates. The activity of phosphoavailability of CDR but will depend only on changes in Ca2+ concentration. Kroeger et diesterase from a number of sources against one nucleotide has been reported to be al. (31) have shown that agents which instimulated or inhibited by the presence of crease intracellular Ca*+ concentration in the other nucleotide (11, 33, 36, 37). uterine smooth muscle also lead to an acWells et al. (20) reported that activa- celerated loss of cyclic AMP levels following stimulation. Conversely, tion results in no change in affinity for cyclic isoproterenol GMP, only an increase in velocity, while our agents which stimulate an increase in cyclic data demonstrate an increase in both veloc- AMP levels in vascular and other smooth ity and affinity for cyclic GMP. This dis- muscles, as well as dibutyryl cyclic AMP crepancy could be due to the overriding itself, lead to relaxation of the smooth presence of the high affinity, cyclic GMP muscle, apparently as a result of changes enzyme (type B) in their activatable peak. in the level of intracellular free Ca2+ (2,3, 6,
MULTIPLE
FORMS OF BOVINE
CYCLIC NUCLEOTIDE
7). These observations as well as the results reported in this communication suggest that cyclic AMP and Ca2+ may have reciprocal roles in regulating the levels of each other in smooth muscle. ACKNOWLEDGMENTS Many thanks are extended to Dr. R. V. Sharma and Dr. D. Singh for their cooperation in some of the experiments and for helpful discussion. Miss Joanne Low participated in this work as an NSF Health Science Program trainee. The authors acknowledge with great appreciation her contributions and expert technical help. REFERENCES 1. SOMLYO, A. P., SOMLYO, A. V., AND SMIESKO, V. (1972) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 1, pp. 1’75-194, Raven Press, New York. 2. BAR, H. P. (1974) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 4, pp. 195-237, Raven Press, New York. 3. ANDERSSON, JOHANSSON,
R., NILSSON, K., WIKBERG, J., S., AND LUNDHOLM, L. (1975) in
Advances in Cyclic Nucleotide Research (Drummond, G. I., Greengard, P., and Robison, G. A., eds.), Vol. 5, pp. 491-518, Raven Press, New York. NAMM, D. H., AND LEADER, J. P. (1976) Blood Vessels 13, 24-47. BOUD~UIN-LEGROS, M., AND MEYER, P. (1973) Brit. J. Pharmacol. 47, 377-385. WEBB, R. C., AND BHALLA, R. C. (1976) J. Mol. Cell. Cardiol. 8, 145-157. BHALLA, R. C., WEBB, R. C., SINGH, D., AND BROCK, T. (1978) Amer. J. Physiol. 234,
H508-H514. 8. DUNHAM, E. W., HADDOX, 9. 10. 11.
M. K., AND GOLDBERG, N. D. (1974) Proc. Nat. Acad. Sci. USA 71, 815-819. THOMPSON, W. J., AND APPLEMAN, M. M. (1971) Biochemistry 10, 311-316. UZUNOV, P., AND WEISS, B. (1972) Biochim. Biophys. Acta 284,220-226. RUSSELL, T. R., TERASAKI, W. L., AND APPLEMAN, M. M. (1973) J. Bid. Chem. 248, 1334-
1340. 12. PLEDGER, 13.
W. J., STANCEL, G. M., THOMPSON, W. J., AND STRADA, S. J. (1974) Biochim. Biophys. Acta 370, 242-248. KAKIUCHI, S., YAMAZAKI, R., TESHIMA, Y., UENISHI, K., AND MIYAMOTO, E. (1975) Biochem. J. 146, 109-120.
14. BERGSTRAND, Biochemistry
PHOSPHODIESTERASE H.,
AND
LUNDQUIST,
473 B.
(1976)
15, 1727-1735.
15. Ho, H. C., TEO, T. S., DESAI, R., AND WANG, J. H. (1976) Biochim. Biophys. Acta 429,461473. 16. ADACHI, K., LEVINE, V., HALPERIN, K. M., LIZUKA, H., AND YOSHIKAWA, K. (1976) Biochim. Biophys. Acta 429, 498-507. 17. HIDAKA, H., YAMAKI, T., OCHIAI, Y., ASANO, T., AND YAMABE, H. (1977) Biochim. Biophys. Acta 484, 398-407. 18. VAN INWEGEN, R. G., SWAFFORD, R. L., STRADA, S. J., AND THOMPSON, W. J. (1977) Arch. Biochem. Biophys. 178, 58-68. 19. SAKAI, T., YAMANAKA, H., TANAKA, R., MAKINO, H., AND KASAI, H. (1977) Biochim. Biophys. Acta 483, 121-134. 20. WELLS, J. N., BAIRD, C. E., WV, Y. J., AND HARDMAN, J. G. (1975) Biochim. Biophys. Acta 384, 430-442. 21. Ho, H. C., WIRCH, E., STEVENS, F. C., AND WANG, J. H. (1977)J. Biol. Chem. 252,43-50. 22. MURTAUGH, T., SHARMA, R. V., AND BHALLA, R. C. (1978) in Advances in Cyclic Nueleotide
Research (Greengard, P., and Robison, G. A., eds.), Vol9, p. 758, Raven Press, New York. 23. BUTCHER, R. W., ANDSUTHERLAND, E. W. (1962) J. Biol. Chem. 237, 1244-1250. 24. THOMPSON, W. J., AND APPLEMAN, M. M. (1971) J. Biol. Chem. 246,3145-3150. 25. BOUDREAU, R. J., AND DRUMMOND, G. I. (1975) And. Biochem. 63, 388-399. 26. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 27. CHEUNG, W. Y. (1971) J. Biol. Chem. 246,28592869. 28. TEO, T. S., WANG, T. H., AND WANG, J. H. (1973) J. Biol. Chem. 248, 588-595. 29. TEO, T. S., AND WANG, J. H. (1973) J. Biol. Chem. 248, 5950-5955. 30. HIDAKA, H., ASANO, T., AND SHIMAMOTO, T. (1975) Biochim. Biophys. Acta 377, 103-116. 31. KROEGER, E. A., TEO, T. S., Ho, H., AND WANG, J. H. (1977) in Biochemistry of Smooth Muscle
(Stephens, N. L., ed.), pp. 641-652, University Park Press, Baltimore. 32. HIDAKA, H., YAMAKI, T., AND YAMABE, H. (1978) Arch. Biochem. Biophys. 187, 315-321. 33. Moss, J., MANGANIELLO, V. C., AND VAUGHAN, M. (1977) J. Biol. Chem. 252, 5211-5215. 34. DAVIS, C. W., AND Kuo, J. F. (1976) Biochim. Biophys. Acta 444, 554-562. 35. DAVIS, C. W., AND Kuo, J. F. (1977) J. Biol. Chem. 252,4078-4084. 36. TERASAKI, W. L., AND APPLEMAN, M. M. (1975) Metabolism 24, 311-319.
MURTAUGH
474
37. WELLS, J. N., Wu, W. J., BAIRD, C. E., HARDMAN, J. G. (1975) Mol. Phamacol.
775-783. 38. SMOAKE, J. A.,
AND BHALLA
AND
11,
SONG, S.-Y., AND CHEUNG, W. Y. (1974) Biochim. Biophys. Acta 341, 402-411. 39. WOLFF, D. J., AND BROSTROM, C. 0. (1974)Arch.
Biochem. Biophys. 163, 349-358.
40. WAISMAN, D., STEVENS, F. C., AND WANG, J. H. (1975) Biochem. Biophys. Res. Commun. 65, 975-982. 41. EGRIE, J. C., AND SIEGEL, F. L. (1975) Biochem. Biophys. Res. Commun. 67, 662-669. 42. UZUNOV, P., GNEGY, M. E., REVUELTA, A., AND COSTA, E. (1976) Biochem. Biophys. Res. Comma. 70, 132-138.
Multiple
Forms of Cyclic Nucleotide Phosphodiesterase Bovine Carotid Artery Smooth Muscle1 TIMOTHY
Department
of Anatomy
J. MURTAUGH and Cardiovascular
AND RAMESH Center,
College
from
C. BHALLA
of Medicine,
University
of Iowa,
Iowa City, Iowa 52242 Received October 11, 1978; revised April 20, 1979 DEAE-cellulose chromatography, in the presence and absence of Ca*+, of the 16,000g supernatant from bovine carotid artery smooth muscle has been used to separate four different types of cyclic nucleotide phosphodiesterase (3’:5’-cyclic-nucleotide 5’-nucleotidohydrolase, EC 3.1.4.17) activity, designated types A, B, C, and D. Type A is a high affinity, cyclic AMP-specific form of phosphodiesterase (K, = 1.6 PM) and elutes at relatively high ionic strength. Type B is a high affinity (K, = 2 PM), cyclic GMP-specific form which elutes at low ionic strength. Type C is a mixed substrate form, displaying anomalous kinetics for the hydrolysis of both cyclic AMP and cyclic GMP. It elutes from DEAE-cellulose at an ionic strength intermediate to that of types A and B. Type D is also a mixed substrate form of phosphodiesterase. However, its elution pattern from DEAE-cellulose differs, depending on whether CaZ+ is present or not, suggesting a Ca2+-dependent interaction between this enzyme form and the acidic Caz+-dependent regulator protein (CDR). The hydrolytic activity of type D is stimulated by CDR, and activation requires the simultaneous presence of Ca2+ and CDR. Kinetic analysis of cyclic AMP hydrolysis by type D gives a linear double reciprocal plot; activation has no effect on the K, but increases the velocity approximately sixfold. Activation of cyclic GMP hydrolysis apparently affects both the K, and V. At all concentrations tested, the degree of activation is higher with cyclic AMP than with cyclic GMP. It is suggested that while the activable form of phosphodiesterase may play a relatively minor role in the overall hydrolysis of cyclic nucleotides, Ca2+-dependent activation may have a more important role in regulating the level of cyclic AMP than that of cyclic GMP in vascular smooth muscle.
Adenosine 3’:5’-monophosphate (cyclic AMP) and guanosine 3’:5’-monophosphate (cyclic GMP) have been implicated in the regulation of relaxation and contraction of vascular smooth muscle (l-4). It has been suggested that cyclic AMP promotes relaxation by regulating the intracellular levels of free Caz+ available to the contractile apparatus. This may be accomplished by a cyclic AMP-dependent phosphorylation of a microsomal membrane component and the subsequent stimulation of Ca2+ uptake by these membrane vesicles (5-7). While the physiological role of cyclic GMP in vascular smooth muscle is unclear, its levels in the 1 This work was supported by NIH Grant HL 19027 and predoctoral training grant PHS-GM07228 (to T. J. M.).
cell are increased by agents which promote contraction (8). Despite the evidence for cyclic nucleotide regulation of vascular smooth muscle tone, little is known regarding the control of cyclic nucleotide synthesis and degradation in this tissue. Because of the apparent involvement of cyclic AMP in regulating Ca2+ levels in vascular smooth muscle, it is of some interest to investigate the possible involvement of Ca2+ in a reciprocal regulation of cyclic nucleotide levels through the activation of phosphodiesterase activities. Recent studies in numerous tissues have demonstrated the presence of multiple forms of cyclic nucleotide phosphodiesterase (9-19). These enzymes show differences in substrate specificity, subcellular allosteric interactions, and distribution, 465
0003-9861/79/100465-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights
of reproduction
in any form reser~ red.
MURTAUGH
466
AND BHALLA
sensitivity to activation by a heat-stable, Ca2+-dependent regulator protein (CDR).2 There has, however, been little investigation into the possible forms of phosphodiesterase in vascular smooth muscle. Using pig coronary arteries, Wells et al. (20) isolated two peaks of phosphodiesterase activity by DEAE-cellulose chromatography. Peak I hydrolyzed both cyclic AMP and cyclic GMP, but with a much higher affmity for cyclic GMP, and peak II was specific for cyclic AMP hydrolysis. Only peak I was affected by CDR, giving increases in velocity for both substrates but no change in substrate affinity. Ho et al. (21) have shown that the elution pattern from DEAE-cellulose of the activatable form of phosphodiesterase from bovine heart depends on the state of its Ca2+-dependent interaction with CDR. We have taken advantage of this property by using DEAE-cellulose chromatography in the presence or absence of Ca2+ and have separated four peaks of phosphodiesterase activity, with differing kinetic properties, from bovine carotid artery smooth muscle. A portion of this work has been presented in preliminary form (22). EXPERIMENTAL
PROCEDURES
Materials. Dowex l-X8,200-400 mesh (Cl- form), cyclic AMP, cyclic GMP, snake venom (Crotalus atroz), Tris base, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid (EGTA), and O(diethylaminoethyl) (DEAE)-cellulose were purchased from Sigma Chemical Company, St. Louis, Missouri. Aquasol scintillation tluor, cyclic[3H]AMP (3.8 Cilmmol), cycliQH]GMP (7.8 Ci/mmol), [‘4C]adenosine (0.05 Cii mmol), and [Y&uanosine (0.5 Ci/mmol) were obtained from New England Nuclear, Boston, Massachusetts. Fresh bovine carotid arteries were obtained from a local slaughterhouse and transported on ice. The tissue was cleaned of loose fat and connective tissue, frozen in liquid nitrogen, and stored at -20°C until use. Preparation of tissue extracts. The buffer used for tissue homogenization, as well as for chromatographic procedures, consisted of 10 mM Tris-HCl, pH 7.2, 1 mM MgCl,, 2 mM 2-mercaptoethanol, and 5 mM sodium bisulfite. Approximately 80 g of frozen arteries was thawed in buffer, stripped of adventitial connective tissue, and minced, yielding approximately 50 g of 2 Abbreviations used: CDR, Ca2+-dependent regulator protein; EGTA, ethylene glycol bis(P-aminoethylether) N,N’-tetraacetic acid.
smooth muscle. The tissue was homogenized in 10 vol of buffer in a Waring Blendor (three pulses of 30 s each at high speed). The homogenate was filtered through two layers of cheesecloth, centrifuged for 15 min at l,OOOg, filtered through four layers of cheesecloth, recentrifuged for 15 min at lS,OOOg, and filtered through glass wool. A portion of this supernatant was stored in small aliquots at -20°C for use in tissue extract experiments. All steps were performed at 4°C. Assay procedures. The assay for phosphodiesterase was in principle that described by Butcher and Sutherland (23), using 3H-labeled cyclic nucleotides based on the procedure of Thompson and Appleman (24) with minor modifications. A two-step procedure was used. The reaction mixture consisted of 30 mM TrisHCl, pH 8.0,4 mM MgCl,, appropriate cyclic nucleotide concentration (including 3H-labeled substrate, giving approximately 100,000 cpm per assay), and an appropriate amount of enzyme in a total volume of 0.5 ml. The snake venom reaction was terminated by the addition of 1 ml of a Dowex slurry (3 H,O:l resin) containing 3 mM acetic acid (pH 3.0) for cyclic AMP assay or 115 mM formic acid (pH 2.1) for cyclic GMP assay, according to Boudreau and Drummond (25). Blank tubes were given the enzyme carrier buffer in place of enzyme. 14C-Labeled nucleoside recovery was monitored in each experiment and averaged 70-90%. The data reported are corrected for nucleoside recovery. All assays were carried out under conditions of linearity with respect to time and enzyme dilution. Specific activity of phosphodiesterase is expressed as nanomoles of cyclic nucleotide hydrolyzed per minute per milligram protein at 30°C. Protein was determined by the method of Lowry et al. (26) with bovine serum albumin as standard. Separation of phosphodiesteruses. The carotid artery supernatant fraction isolated above was dialyzed overnight against the homogenization buffer containing either 50 pM CaCl, or 100 pM EGTA. The dialyzed supernatant (approximately 400 ml) was applied to a DEAE-cellulose column (30 x 1.8 cm) previously equilibrated with the same buffer, and the column was washed with 3 column vol of buffer. The initial wash contained no detectable phosphodiesterase activity. The column was developed with 1000 ml of either the Ca2+-containing buffer or the EGTA-containing buffer, with a linear NaCl gradient (0 to 0.6 M). Fractions containing peak phosphodiesterase activity were pooled, concentrated by ultrafiltration in an Amicon apparatus with a UM-10 membrane, and dialyzed for rechromatography. Rechromatography on DEAE-cellulose (20 x 1 cm) with a 500-ml linear gradient (0 to 0.5 M NaCl) was performed using either the Ca2+-containing buffer or the EGTA-containing buffer, to separate peaks which eluted together under the ionic conditions of the fast chromatography. The appropriate peak fractions were again pooled, concentrated, dialyzed against 10 mM Tris-HCl, pH 7.4, 1 mM MgCl,, and 2 mM 2-
MULTIPLE
FORMS OF BOVINE
CYCLIC NUCLEOTIDE
mercaptoethanol, and stored in aliquots at -20°C. In subsequent discussions, “CaZ+-column” and “EGTAcolumn” will refer to DEAE-cellulose columns eluted in the presence of either 50 pM CaCl, or 100 pM EGTA, respectively. Preparation of CDR. A crude CDR preparation was made by boiling the carotid artery supernatant in a steam bath for 5 min followed by centrifugation to remove denatured material, based on the heat stability reported for the Ca*+-dependent regulator protein of phosphodiesterase (27,28). This preparation contained no detectable phosphodiesterase activity. Twenty microliters of this preparation gave maximal stimulation of an activator-deficient DEAE-cellulose peak. Partially purified CDR was isolated with DEAEcellulose chromatography in the presence of EGTA (Fig. 3). The peak activator fractions were pooled, boiled, dialyzed, and stored frozen in aliquots. This partially purified preparation was used in experiments involving the rechromatography of an activatordeficient phosphodiesterase peak. The CDR preparation was added to the enzyme peak prior to dialysis against Ca*+-containing buffer.
of Multiple
Separation
467
PHOSPHODIESTERASE
Phosphodiesterase
Activities The presence or absence of Ca2+ during DEAE-cellulose chromatography determines whether CDR will remain bound to phosphodiesterase during elution (21, 29). Due to the acidic nature of the CDR protein (29), an activatable form of phosphodiesterase will elute at different ionic strengths from DEAE-cellulose depending on whether it elutes bound to CDR or not. Two separate regimes of DEAE-cellulose chromatography were used in the following experiments. One involved chromatography of the smooth muscle 16,000g supernatant in the presence of Ca2+ (Fig. l), followed by rechromatography of the peak fractions in the presence of EGTA (Fig. 2). Alternately, the CAMP
RESULTS
Characteristics of Phosphodiesterase Activity in Tissue Extracts Cyclic AMP hydrolysis by the 16,000g supernatant exhibited nonlinear kinetics on a Lineweaver-Burk double reciprocal plot. The curve was concave downward, suggesting either negative cooperativity or the presence of multiple enzyme forms. Apparent K, values for cyclic AMP were 1.2 and 14 ,&I. Cyclic GMP hydrolysis gave a linear double reciprocal plot with an apparent K, of 2.5 ,uM. Hydrolysis of cyclic AMP at both high (50 PM) and low (2 PM) concentrations was inhibited approximately 30% by EGTA concentrations of 50 PM or above (data not shown). This inhibition was abolished by 100 PM Ca2+ but was reestablished by EGTA concentrations greater than 100 PM. Hydrolysis of 2 PM cyclic GMP was affected similarly. Increasing Ca2+ concentrations (from 10-6-10-3 M Ca2+) had no effect on either cyclic AMP or cyclic GMP hydrolysis (data not shown), presumably due to the presence of sufficient endogenous Ca2+ in the supernatant to allow full activation. Exogenous CDR also had no effect on the phosphodiesterase activities of the tissue supernatant.
04 03: 5 02
30
60 Fraction
90
G 8
120
Number
FIG. 1. DEAE-cellulose chromatography of a dialyzed 16,OOOgsupernatant. Chromatography was performed as described under Experimental Procedures. The column was eluted with a 0 to 0.6 M NaCl gradient in buffer containing 50 pM CaCl*. Fractions were assayed for cyclic AMP (top panel) and cyclic GMP (bottom panel) hydrolysis using a substrate concentration of 20 pM with either no additions (0) or in the presence of 100 pM EGTA (0). Assay with Ca2+ and CDR gave no change in activity from that obtained with no additions. Results are representative of five preparations. A, B, C, and D in this and subsequent figures refer to the respective phosphodiesterase types which are contained in the indicated pooled fractions.
MURTAUGHANDBHALLA
468 6c
..n.rn
LHIvIr
-No
5-
addition
-+5qN 4.
GP
+CDR
&
-05
cGMP
,,Y 54-
-04s
3-
-03b
2-
-02
I-
-0 I
2 5
c-m IO
20
30
” 40
50
Fractton
8 60
‘1 70
130
single major peak eluting at 0.12 M NaCl, and two minor peaks representing contaminating components of the other peaks (Fig. 5B). The major peak hydrolyzed cyclic GMP preferrentially and was designated type B. Kinetic analysis of cyclic GMP hydrolysis by type B gave a linear double reciprocal plot with an apparent K, of 2.0 PM. Type B also had a low level of activity with cyclic AMP as substrate, with a K, of approximately 150 PM. Caz+ and CDR had no effect on the hydrolysis of either cyclic GMP or cyclic AMP by type B. Type C was isolated from an EGTA column (Fig. 3). It eluted from DEAEcellulose at an ionic strength intermediate -
01 90
No odd,t~on
Number
FIG. 2. Rechromatography of fractions 75-105 (Fig. 1) on DEAE-cellulose, eluted with a buffer containing 100 pM EGTA. Assays were as described for Fig. 1, with either no additions (0) or in the presence of 50 pM Ca2+ plus 20 ~1 of a boiled carotid artery supernatant (0). Results are representative of four preparations.
tissue extract was first chromatographed in the presence of EGTA (Fig. 3), followed by rechromatography of the peak fractions in the presence of Ca2+ and added CDR (Fig. 4). A combination of these procedures was used to separate four different types of phosphodiesterase activity, designated as types A, B, C, and D. Table I summarizes their kinetic characteristics and chromatographic behavior. Type A was isolated from an EGTA column (see Experimental Procedures) (Fig. 3). Rechromatography of this peak in the presence of either Ca2+ or EGTA gave a single major peak eluting at 0.3 M NaCl and a minor peak representing contaminating peaks (Fig. 5A). The major peak was highly specific for cyclic AMP hydrolysis and was designated type A. Kinetic analysis gave a linear double reciprocal plot with an apparent K, of 1.6 PM, and its activity was not affected by Ca*+ and CDR. Type B was isolated from a Ca2+ column (Fig. 1). Rechromatography of this peak in the presence of either Ca*+ or EGTA gave a
.P h c F u9 i
IO
05 04
s ? 03 2 9 02
5
01
$? 30° $3 200
Sk 83
100
cm -No addition -*50,,M Ca-
t
FIG. 3. DEAE-cellulose chromatography of a dialyzed 16,OOOg supernatant, in the presence of 100 pM EGTA. Fractions were assayed for cyclic AMP (top panel) and cyclic GMP (middle panel) hydrolysis with no additions (0) or in the presence of 50 pM Ca2+ plus 20 ~1 boiled carotid artery supernatant (0). Ca*+ or CDR alone had no effect on basal activity (not shown). Substrate concentration = 20 ELM. Lower panel: An aliquot of each fraction was boiled and assayed for its ability to stimulate cyclic AMP hydrolysis. Unboiled column fraction No. 36 was used as a source of activatable phosphodiesterase. Assays were performed in the presence (0) and absence (0) of 50 NM Ca*+. Results are representative of seven preparations.
MULTIPLE
FORMS OF BOVINE
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
469
strength (approximately 0.12 M NaCl) and is separated from types A and C. Con-No additm versely, if the tissue supernatant is first chromatographed in the presence of EGTA (Fig. 3), type D elutes at low ionic strength, in the same fractions as type B. When these fractions are rechromatographed in the presence of Ca*+ and exogenous CDR type D is separated from type B by eluting at high ionic strength (Fig. 4). Rechromatography in the presence of Ca*+ alone does not give this separation. This demonstrates that type D is capable of binding CDR in the presence of Ca2+. Type D was isolated by starting with an EGTA column (Fig. 3). The activity of the first peak from this column was stimulated 00 twoto threefold by assay in the presence 10 20 30 40 50 60 70 80 of Ca*+ and CDR. Ca*+ alone or CDR alone Fraction Number had no effect on the activity of this peak FIG. 4. Rechromatography of fractions 30-45 (Fig. (data not shown). Partially purified CDR 3) on DEAE-cellulose. Pooled fractions were mixed was also isolated from this column (Fig. 3). with heat-treated pooled CDR fraction (Fig. 3), dia- The pooled CDR fractions were heat treated lyzed, and chromatographed in the presence of 50 WM Ca*+. Fractions were assayed for cyclic AMP (top in a steam bath for 5 min and centrifuged. which had no panel) and cyclic GMP (bottom panel) hydrolysis in the This CDR preparation, phosphodiesterase activity, presence (0) or absence (0) of 100 pM EGTA. Assay endogenous in the presence of added Ca2+ and CDR gave no in- was mixed with the pooled activatable phoscrease in activity over that seen with no additions (not phodiesterase fractions. This mixture was shown). Substrate concentration = 20 pM. Results are concentrated and dialyzed against the Ca*+representative of four preparations. containing buffer. Upon rechromatography of this mixture in the presence of Ca*+, type D eluted at a higher ionic strength than type to that of types A and B (approximately B (Fig. 4). Neither peak was stimulated by 0.22 M NaCl). Rechromatography of this Ca*+ and CDR but the activity of type D was peak in the presence of either Ca*+ or EGTA reduced 60-70% by assay in the presence of gave a single major peak eluting at 0.22 M EGTA. In order to obtain a preparation of NaCl, which hydrolyzed both cyclic AMP type D free of endogenous CDR the fracand cyclic GMP (Fig. 5C). Type C displayed tions containing type D were pooled, connonlinear double reciprocal plots for the centrated, dialyzed against an EGTA-conhydrolysis of both cyclic AMP and cyclic taining buffer and rechromatographed in GMP (Table I). As with types A and B, Ca*+ the presence of EGTA. This procedure gave and CDR had no affect on the activity a single peak of activity eluting at 0.13 M kinetics of type C. NaCl which was stimulated three- to sixfold Type D is also a mixed-substrate form of by Ca*+ and activator (Fig. 6). The peak phosphodiesterase. However, its elution thus isolated was dialyzed to remove EGTA pattern from DEAE-cellulose differs de- and stored frozen for kinetic analysis. pending on whether Ca2+ is present or not. Cyclic AMP hydrolysis by type D was not In the presence of Ca*+, type D elutes at changed by increasing concentrations of high ionic strength, in essentially the same EGTA, Ca2+, or increasing amounts of CDR fractions as type A (approximately 0.3 M alone. Only in the presence of both Ca*+ and NaCl) (Fig. 1). Upon rechromatography of CDR was the activity increased approxithese fractions in the presence of EGTA mately sixfold (data not shown). Results (Fig. 2), type D elutes at a much lower ionic with cyclic GMP were similar. However,
470
MURTAUGH
AND BHALLA
TABLE
I
SUMMAR~OFKINETICSANDCHROMATOGRAPHICBEHAVIOROFMULTIPLEFORMS OFPHOSPHODIESTERASE FROMBOVINECAROTID ARTERY" DEAE elution pattern [NaCI] (M) V
(nmol/mg proteimmin)
Km (PM)
PDE
Substrate
Basal*
A
Cyclic AMP
1.6
B
Cyclic GMP
2.0
Cal+ + CDRc
In the presence of ca2+ (50pM) In the presence of + CDR EGTA (100 PM)
BasaP
Ca*+ + CDR”
1.7
4.4
4.8
0.30 (Figs. 1 and 5A)
1.8
1.2
1.2
0.13
0.12
(Figs. 1 and 4) C
D
Cyclic AMP
5.4;50
5.2;46
1.3;4.2
1.3;4.1
Cyclic GMP
0.6;27
0.825
1.5;3.1
1.4;3.3
Cyclic AMP
13.6
12.2
1.2
7.5
Cyclic GMP
2.4;16
2.3
1.9;4.3
4.9
0.30 (Figs. 2 and 3)
0.22 Figs. 1 and 5C) 0.30 (Figs. 1 and 4)
(Figs. 3 and 5B) 0.22 (Figs. 2 and 3) 0.12 (Figs. 2,3, and6)
’ Kinetic experiments were performed with a range of substrate concentrations from 0.1 pM to 2 mM.Chromatography was performed as described under Experimental Procedures. Results are means of duplicate determinations and are representative of experiments with at least three different preparations. Kinetic data were analyzed by a least-square regression analysis of a Lineweaver-Burk plot (r 2 0.95). b Activity was the samewith either no additions to the standard assay medium or in the presence of 0.1 mMEGTA. c Ca2+ + CDR, Activity in the presence of 50 pM Ca2+ and 25 Fg partially purified CDR.
maximal stimulation was only approximately threefold (data not shown). Kinetic analysis of cyclic AMP hydrolysis by type D is shown in Fig. ‘7A. Under basal conditions (the values being identical in the presence or absence of either EGTA or Ca2+), the double reciprocal plot was linear with an apparent K, of approximately 14 PM and a V of 1.2 nmol/mg proteimmin. In the presence of maximal Ca2+ and CDR the apparent K, was not affected (approximately 12 PM) while the V was increased approximately sixfold. The kinetics of cyclic GMP hydrolysis appear to be more complicated (Fig. ‘7B). Under basal conditions, the double reciprocal plot was nonlinear, exhibiting both high affinity (K, = 2.4 PM; V = 1.9 nmol/mg protein/min) and low affinity (K, = 16 pM; V = 4.3 nmol/mg protein/min) characteristics. Under conditions of maximal stimulation, however, a linear double reciprocal plot was observed. The apparent K, (2.3 PM) was the same as the high affinity K, seen under basal conditions,
while the V (4.9 nmol/mg proteimmin) was equal to the higher velocity of the low affinity activity. DISCUSSION
The results presented in this report demonstrate the presence of four different types of phosphodiesterase activity which can be isolated from vascular smooth muscle: a high affinity, cyclic AMP-specific form (type A); a high affinity, cyclic GMPspecific form (type B); a nonactivatable, mixed substrate form (type C); and an activatable, mixed substrate form (type D). We have not ruled out, however, the possibility that the chromatographic behavior or the hydrolytic activity of the native enzymes may have been altered by the action of endogenous proteases during the periods of purification used in our procedures. We have included sodium bisulfite, a protease inhibitor, in our buffers to minimize this possibility.
MULTIPLE
FORMS OF BOVINE
CYCLIC
Previous reports in smooth muscle have shown only two peaks of phosphodiesterase activity (20, 30, 31). Hidaka et al. (32) recently reported the separation of five phosphodiesterase peaks from human aorta. Two of the peaks which preferentially hydrolyzed cyclic GMP were activatable by Ca2+ and CDR. Wells et al. (20) have reported the presence of two peaks of phosphodiesterase activity from coronary artery, a nonactivatable, cyclic AMP-specific form and an activatable form which hydrolyzed both cyclic AMP and cyclic GMP. Since cyclic GMP was hydrolyzed only by the activatable form, they suggested that Ca2+-dependent activation is more im-
NUCLEOTIDE
PHOSPHODIESTERASE
471
8 CAMP 6 4
f
-
No addition
-
+5QM +CDR
Co”
n
0
0.0 0
20 Fraction
40 Number
60
FIG. 6. Rechromatography of type D (fractions 51-63 in Fig. 4) in the presence of 100 pM EGTA. Fractions were assayed for cyclic AMP (top panel) and cyclic GMP (bottom panel) hydrolysis with no additions (0) or in the presence of 50 pM Ca*+ and 25 pg partially purified CDR (0). Ca2+ or CDR alone had no effect on basal activity. Substrate concentration = 20 pM. Results are representative of three preparations.
FIG. 5. DEAE-cellulose rechromatography profiles for individual phosphodiesterase types. Fractions were assayed for cyclic AMP (0) and cyclic GMP (0) hydrolysis at a substrate concentration of 20 pM. Type A: Fractions 76-90 in Fig. 3 were rechromatographed in the presence of Ca*+ and CDR. Assay in the presence of 100 FM EGTA did not change the activity profile. Type B: Fractions 43-63 in Fig. 1 were rechromatographed in the presence of 100 pM EGTA. Assay in the presence of Ca*+ and CDR did not change the activity profile. Type C: Fractions 57-67 in Fig. 3 were rechromatographed in the presence of Caz+ and CDR. Assay in the presence of 100 pM EGTA did not change the activity profile.
portant in regulating cyclic GMP hydrolysis than cyclic AMP hydrolysis in vascular smooth muscle. However, we have shown the presence of a nonactivatable, cyclic GMP-specific enzyme, similar to that reported in liver and lung (11, 33-35). There are present, then, in vascular smooth muscle, high affinity forms for both cyclic AMP and cylic GMP which are not affected by CDR and Ca2+. Therefore, while Ca2+dependent activation may play a relatively minor role in regulating the levels of each cyclic nucleotide in the cell, it will have effects on both cyclic AMP and cyclic GMP. In the nonactivated state, type D has a higher affinity for cyclic GMP and has a higher level of activity with cyclic GMP as substrate at all concentrations. In the activated state, type D still has a higher level of activity with cyclic GMP at low substrate concentrations, whereas at substrate concentrations greater than 2 PM cyclic AMP hydrolysis is greater. However, at all substrate concentrations, the degree of activation of cyclic AMP hydrolysis is greater than that of cyclic GMP hydrolysis. Therefore, our data suggest that activation may play a more important role in regulating
472
MURTAUGHANDBHALLA
We have confirmed that DEAE-cellulose chromatography with a (NHJ2S04 gradient, as was used by Wells et al. (20), results in a profile similar to that which we obtain with chromatography in the presence of EGTA (Fig. 3) and that the activatable peak can be separated into two peaks (types B and D) by rechromatography in the presence of Ca2+ and activator, as in Fig. 4. Our data suggest that peak I of Wells et al. may consist of two enzyme forms (type B and type D) and that a change in the K, of the activatable form (type D) could have been masked by the presence of the high-affinity, nonactivatable cyclic GMP-specific form (type B). The major difference between our chromatography profile in the presence of EGTA and that of Wells et al. (20) is the presence in our profile of an enzyme form that elutes at an intermediate ionic strength, i.e., type C. While this discrepancy cannot be satisFIG. 7. Lineweaver-Burk plots for cyclic AMP(A) explained at this time, it may and cyclic GMP(B) hydrolysis by type D phospho- factorily diesterase. Substrate concentrations ranged from 0.1 represent species or tissue variation (bovine pM to 2 mM. 0, Basal activity (the same either in the carotid artery vs porcine coronary artery). presence or absence of 100 pM EGTA); 0, activity in A number of studies have shown that the the presence of 50 &bM Ca*+ and 25 pg partially purified concentration of CDR in many tissues is in CDR. Results are means of duplicate determinations excess of that necessary to fully activate the and are representative of experiments with three dif- Ca*+-sensitive phosphodiesterase activities ferent preparations. (38-42). This probably is the case for vascular smooth muscle as well. Tissue extracts by added Ca*+ and cyclic AMP levels. However, type D, in the cannot be stimulated CDR but can be inhibited by EGTA. In adactivated form, still demonstrates a higher dition, type D in the present study apaffinity for cyclic GMP than for cyclic AMP and in the presence of both substrates may parently elutes from DEAE-cellulose bound preferentially hydrolyze cyclic GMP. to CDR sufficient to keep it fully active Definition of the role of the activable en- when the column is eluted in the presence zyme in regulating cyclic AMP and cyclic of Ca*+. Therefore, regulation of the activatable phosphodiesterase in vascular GMP levels will, therefore, require futher study into its activity in the presence of smooth muscle will not be limited by the both substrates. The activity of phosphoavailability of CDR but will depend only on changes in Ca2+ concentration. Kroeger et diesterase from a number of sources against one nucleotide has been reported to be al. (31) have shown that agents which instimulated or inhibited by the presence of crease intracellular Ca*+ concentration in the other nucleotide (11, 33, 36, 37). uterine smooth muscle also lead to an acWells et al. (20) reported that activa- celerated loss of cyclic AMP levels following stimulation. Conversely, tion results in no change in affinity for cyclic isoproterenol GMP, only an increase in velocity, while our agents which stimulate an increase in cyclic data demonstrate an increase in both veloc- AMP levels in vascular and other smooth ity and affinity for cyclic GMP. This dis- muscles, as well as dibutyryl cyclic AMP crepancy could be due to the overriding itself, lead to relaxation of the smooth presence of the high affinity, cyclic GMP muscle, apparently as a result of changes enzyme (type B) in their activatable peak. in the level of intracellular free Ca2+ (2,3, 6,
MULTIPLE
FORMS OF BOVINE
CYCLIC NUCLEOTIDE
7). These observations as well as the results reported in this communication suggest that cyclic AMP and Ca2+ may have reciprocal roles in regulating the levels of each other in smooth muscle. ACKNOWLEDGMENTS Many thanks are extended to Dr. R. V. Sharma and Dr. D. Singh for their cooperation in some of the experiments and for helpful discussion. Miss Joanne Low participated in this work as an NSF Health Science Program trainee. The authors acknowledge with great appreciation her contributions and expert technical help. REFERENCES 1. SOMLYO, A. P., SOMLYO, A. V., AND SMIESKO, V. (1972) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 1, pp. 1’75-194, Raven Press, New York. 2. BAR, H. P. (1974) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 4, pp. 195-237, Raven Press, New York. 3. ANDERSSON, JOHANSSON,
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