- Email: [email protected]
[14] cGMP-dependent protein kinase activation in intact tissues
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filters are dried and counted in a fluid for nonaqueous samples, or dissolved in a fluid like "Filter Count" (Packard), there will be considerable quenching, and more so the higher the protein content of the sample. Final Remarks The ammonium sulfate precipitation method has been used to study cyclic nucleotide binding to mammalian cyclic nucleotide-dependent protein kinases 1,6-~3 and a related nonmammalian binding protein. TM It has also been used to study the binding of cAMP and adenosine to S-adenosylhomocysteinase from mammalian )°:5 and plant 16 sources, as well as cGMP binding to a cyclic nucleotide phosphodiesterase. ~7 In several of these cases the sulfate precipitation assay appeared superior to other commonly used assays. Thus, the inclusion of ammonium sulfate is essential to detect the binding of 8-azido-cAMP to the rapidly exchanging site of RI112:8 and of cGMP to the rapidly exchanging site of the cGMPdependent protein kinase. 7,~3 8 G. Illiano, G. P. E. Tell, M. I. Siegel, and P. Cuatrecasas, Proc. Natl. Acad. Sci. U.S.A. 70, 2443 (1973). 9 j. Van Sande, C. Decoster, and J. E. Dumont, Biochem. Biophys. Res. Commun. 62, 168 (1975). t0 S. O. DCskeland and P. M. Ueland, Biochem. Biophys. Res. Commun. 66, 606 (1975). H j. Tse, C. W. Mackenzie III, and T. E. Donnelly, Jr., Int. J. Biochem. 13, 1071 (1981). 12 D. 0greid and S. O. DCskeland, FEBS Lett. 150, 161 (1982). 13 C. W. Mackenzie III, J. Biol. Chem. 257, 5589 (1982). ~4 R. Rangel-Aldao, G. Tovar, and M. L. de Ruiz, J. Biol. Chem. 258, 6979 (1983). ~5 p. M. Ueland and S. O. DCskeland, J. Biol. Chem. 253, 1667 (1978). ~6 M. Giannattasio, G. Carratu, G. F. Tucci, and A. M. Carafa, Phytochemistry 18, 1613 (1979). ~7 F. Miot, P. J. M. Van Haastert, and C. Erneux, Eur. J. Biochem. 149, 59 (1985). ~8 A. R. Kerlavage and S. S. Taylor, J. Biol. Chem. 257, 1749 (1982).
[14] c G M P - D e p e n d e n t P r o t e i n K i n a s e A c t i v a t i o n in I n t a c t T i s s u e s B y R O N A L D R . FISCUS a n d FERID M U R A D
Cyclic GMP-dependent protein kinase (cGMP-kinase) has been detected in many tissues. However, in contrast to the distribution of cAMPdependent protein kinase (cAMP-kinase), levels of cGMP-kinase vary considerably from one tissue to another as determined enzymatically or METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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immunologically.l,2 Mammalian tissues that are especially rich in cGMPkinase include cerebellum and organs containing large amounts of smooth muscle, such as lung, 1,2 tracheal muscle, 3,4 and blood vessels. 1,2,5 cGMPkinase in these organs is believed to be a major target for the intracellular messenger cGMP. Since cGMP is thought to mediate the smooth muscle relaxant effects of three important classes of vasodilators, the nitrovasodilators, the endothelium-dependent vasodilators, and the atrial natriuretic factors, 5-~° the need for an assay that can measure cGMP-kinase activation in intact tissues has become increasingly more important. Unfortunately, the endogenous activation of cGMP-kinase has proved to be more difficult to demonstrate than the analogous activation of cAMPkinase. Equation (1) will help to explain one of the problems, cGMPkinase is represented as a dimer of the polypeptide RC that contains both regulatory and catalytic domains. ~ Unlike cAMP-kinase that dissociates into regulatory and catalytic subunits upon activation with cAMP, cGMPkinase remains as a holoenzyme upon binding of cGMP [see Eq. (1)]. 1 (RC)2 + 4cGMP ~ (RC)2' cGMP4 (less active) (fully active)
(1)
An increase in the activity of purified cGMP-kinase is observed after adding cGMP in sufficient amounts to occupy the cGMP-binding sites. 1In intact cells the enzyme is probably activated in a similar manner, i.e., the equilibrium of Eq. (1) is shifted to the right, whenever there is an elevation in cGMP concentrations in the vicinity of the kinase. Ideally, when measuring the activation of cyclic nucleotide-dependent protein kinases in intact tissues, this equilibrium should not be disturbed by the procedures of tissue extraction or assaying. In the case of cAMP-kinase, the fact that the regulatory and catalytic subunits dissociate upon activation helps to preserve the activation state of the enzyme when it is diluted into homogenizing buffer and further diluted into a kinase reaction solution for t T. M. Lincoln and J. D. Corbin, Adv. Cyclic Nucleotide Res. 15, 139 (1983). 2 S. M. Lohman and U. Walter, Adv. Cyclic Nucleotide Res. 18, 63 (1984). 3 T. J. Torphy, W. B. Freese, G. A. Rinard, L. L. Brunton, and S. E. Mayer, J. Biol. Chem. 257, 11609 (1982). 4 R. R. Fiscus, T. J. Torphy, and S. E. Mayer, Biochim. Biophys. Acta 805, 382 (1984). 5 R. R. Fiscus, R. M. Rapoport, and F. Murad, J. Cyclic Nucleotide Res. 9, 415 (1983). 6 S. Katsuki, W. P. Arnold, and F. Murad, J. Cyclic Nucleotide Res. 3, 239 (1977). 7 R. M. Rapoport and F. Murad, Circ. Res. 52, 352 (1983). 8 R. M. Rapoport and F. Murad, J. Cyclic Nucleotide Res. 9, 281 (1983). 9 R. J. Winquist, E. P. Faison, S. A. Waldman, K. Schwartz, F. Murad, and R. M. Rapoport. Proc. Natl. Acad. Sci. U.S.A. 81, 7661 (1984). to R. R. Fiscus, R. M. Rapoport, S. A. Waldman, and F. Murad, Biochim. Biophys. Acta 846, 179 (1985).
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assaying. In contrast, activated cGMP-kinase, under similar conditions, begins to lose activity,4 probably because of the rapid dissociation of cGMP from the enzyme as a result of dilution, 11 i.e., the equilibrium of Eq. (1) is shifted to the left. Therefore, special procedures are needed to minimize cGMP dissociation. We have found that by rapidly extracting the tissue and assaying at a reduced temperature (0°) with an abbreviated incubation time (2.5 min), endogenous cGMP-kinase activities can be assessed.<5, l0 Kinase activity in tissue homogenates is measured in the absence and presence of 2 ~ M cGMP and expressed as an activity ratio (activity without cGMP divided by activity with cGMP) in a manner similar to that first described by Lincoln and Keely 12 and analogous to the method described by Corbin 13for measuring cAMP-kinase activity ratios in tissues. With the assay for cGMP-kinase activity ratios, however, saturating amounts of the heat-stable inhibitor of cAMP-kinase (the protein kinase inhibitor of Walsh e t a/. 14) are added to avoid interference by cAMPkinase. Under the assay conditions described herein, 2 ~ M cGMP fully activates cGMP-kinase in smooth muscle homogenates. 3-5,m Therefore, the activity ratio obtained by this procedure gives an estimate of the proportion of cGMP-kinase in the active state in the tissue. Experimental Methods and Material
Tissue Preparation Two smooth muscle preparations, canine tracheal smooth muscle and rat thoracic aorta, have been used for studying endogenous activation of cGMP-kinaseY ,m In both cases, the smooth muscle tissues were cut into small strips and allowed to equilibrate for at least 1 hr with a physiological salt solution at 37° and aerated with 95% 02 : 5% CO 2. In the case of rat aortas, some strips were rubbed on the intimal surface to remove the endothelium. 5,7.8 The strips were exposed to agents that alter smooth muscle contractility, such as the vasodilators sodium nitroprusside, acetylcholine (endothelium dependent), and atrial natriuretic factor ( a t r i o p e p t i n II). 4-m At appropriate times the strips were removed from the physiological salt solution and rapidly frozen between metal clamps precooled in liquid nitrogen. Unless samples are immediately used for the H R. W. McCune and G. N. Gill, J. Biol. Chem. 254, 5083 (1979). r2 T. M. Lincoln and S. L. Keely, Biochim. Biophys. Acta 676, 230 (1981). 13 j. D. Corbin, this series, Vol. 99, p. 227. t4 D. A. Walsh, C. D. Ashby, C. Gonzalez, D. Calkins, E. H. Fischer, and E. G. Krebs, J. Biol. Chem. 246, 1977 (1971).
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cGMP-kinase assay, it is recommended that for maximum stability the samples are stored in liquid nitrogen until they can be assayed.
Procedure for Preparation of Tissue Extracts Frozen tissues are homogenized with a motor-driven ground glass pestle and homogenizing tubes in 16 vol of ice-cold 20 mM potassium phosphate, pH 7.0, l0 m M EDTA, 2 mM EGTA, 0.5 mM isobutylmethylxanthine, and 6 m M dithiothreitol.~° The EGTA is added to selectively inhibit calcium-dependent kinases that may interfere with the assay. The homogenates are rapidly transferred to cold microfuge tubes and centrifuged at 8000 g in a microfuge at 4° for 0.5 min and the supernatant fractions are used for the kinase assay. To minimize cGMP dissociation from the enzyme during the sample extract preparation, each sample is homogenized, centrifuged, and assayed individually with a total time of 2.0 rain from the beginning of homogenization to the beginning of the assay.
Procedure for Measuring cGMP-Kinase Activity Ratios Kinase activity is determined by measuring the amount of 32p transferred from [y-32p]ATP to histone H2b during a 2.5-min incubation at 0°. The reaction tubes are incubated in an ice-water bath with frequent agitation to assure constant temperature throughout the bath. Also, it is recommended that the assay be conducted in a walk-in cold room in order that all materials that come in contact with samples and reaction mixtures, such as pipet tips and filter paper squares, stay cold. The assays are initiated by adding 7 ~l of supernatant fraction of tissue homogenates to 35/~1 of a reaction mixture containing (in final concentration) the following: 15 m M potassium phosphate (pH 7.0), 7 mM magnesium acetate, 0.5 mg/ml histone H2b, 20 mM sodium fluoride, 0.25 mM isobutylmethylxanthine, 30 ~ M adenosine triphosphate (ATP) with [y-32p]ATP at 5000 cpm/pmol, and 50 t~g/assay tube of the partially purified heat-stable inhibitor of cAMP-kinase (protein kinase inhibitor). At this concentration protein kinase inhibitor inhibits rat aorta cAMP-kinase by >99% in a typical assay. 5 The heat-stable protein kinase modulator protein that has been reported to be necessary in some cGMP-kinase assay systems ~had no beneficial effect in this assay. The reaction is stopped by transferring 35 pA of the final reaction mixture to 3MM chromatography paper squares (1.7 × ! .7 cm) and immediately immersing them in icecold 10% trichloroacetic acid with 2.5% pyrophosphate. At least 15 ml of trichloroacetic acid solution per paper square should be used in order to keep background radioactivity low. The paper squares are washed in 5%
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trichloroacetic acid with 2.5% pyrophosphate by a procedure modified from that of Corbin and Reimann 15 and includes two hot (90°) washes for 15 min each and two washes at room temperature for 20 min each. The final wash is in 95% ethanol for 10 min and the paper squares are dried and counted in Betacount (Baker) by liquid scintillation spectrometry. Materials
The heat-stable inhibitor of cAMP-kinase (protein kinase inhibitor) ~4 was prepared by the procedure of McPherson et al. 16through the DEAEcellulose and dialysis steps. Synthetic atriopeptin II was purchased from Peninsula Laboratories (Belmont, CA), histone H2b from Worthington, [7-32p]ATP from Amersham, 3MM chromatography paper from Whatman, and trichloroacetic acid from Mallinckrodt. Other chemicals and drugs were purchased from Sigma. Application of the cGMP-Kinase Activity Ratio Method The assay technique described above was first applied to the study of cGMP-kinase regulation in canine tracheal smooth muscle? Sodium nitroprusside and the muscarinic agonist methacholine were selected as potential stimulatory agents, since both elevate cGMP concentrations in tracheal muscle. 4,17,1s However, these agents have quite different effects on contractility; methacholine contracts whereas sodium nitroprusside relaxes trachealis. 4,j7,Js In addition, cGMP elevations caused by muscarinic agonists are calcium dependent, whereas those to sodium nitroprusside are not. ~v Nevertheless, both agents were observed to elevate cGMPkinase activity ratios in canine trachealis, thus demonstrating a functional link between elevation of cGMP and activation of cGMP-kinase in intact airway smooth muscle? These data support the hypothesis that cGMP, via kinase activation and subsequent phosphorylation, mediates smooth muscle relaxation to sodium nitroprusside. Also, the data suggest that cGMP-kinase may be important in the proposed role of cGMP as a negative-feedback signal in regulating smooth muscle contraction to agents like methacholine. We have also used the activity ratio method to study cGMP-kinase regulation in rat aorta. 5,1° Representatives of three major classes of vasot5 j. ~6 J. 17 S. ~8 T.
D. Corbin and E. M. Reimann, this series, Vol. 38, p. 287. M. M c P h e r s o n , S. W h i t e h o u s e , and D. A. Walsh, Biochemistry 18, 4835 (1979). K a t s u k i and F. Murad, Mol. Pharmacol. 13, 330 (1977). J. Torphy, Z. Cong, S. M. Peterson, R. R. Fiscus, G. A. Rinard, and S. E. Mayer, J. Pharmacol. Exp. Ther. 233, 409 (1985).
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KINASE ACTIVATION
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dilators, the nitrovasodilator sodium nitroprusside, the endotheliumdependent vasodilator acetylcholine, and the atrial natriuretic factor atriopeptin II were tested and all were found to elevate cGMP-kinase activity ratios, responses that correlated with cGMP elevation and relaxation of the tissue. Table I shows the effects of sodium nitroprusside and acetylcholine on kinase activities in the absence and presence of exogenous cGMP as well as the calculated activity ratios. Sodium nitroprusside and acetylcholine (when the endothelium was intact) increased endogenous cGMP-kinase activity ( - c G M P activity in the kinase assay of Table I). Total cGMP-kinase activity (+cGMP activity in the kinase assay) was unaltered by drug treatment of the tissues. It is possible that the increase in the - c G M P activity represents activation of an undefined cGMP-independent protein kinase (see the following section). However, this is unlikely in this study since under these circumstances an equal increase in the total (+cGMP) activity would be expected and this was not observed with acetylcholine treatment. Also, since the +cGMP kinase activity, the denominator in the activity ratio calculation, is not significantly altered, a change in the activity ratio reflects solely the change in the - c G M P kinase activity and presumably also the change in cGMP-kinase activity in the tissue at the time of freezing. Therefore, it appears that the proportion of cGMP-kinase in the active state has increased in rat aorta treated with sodium nitroprusside or acetylcholine. In addition, the endothelium TABLE 1 EFFECT OF SODIUM NITROPRUSSIDE AND ACETYLCHOLINE ON cGMP-KINASE ACTIVITY IN RAT AORTAa Kinase activity (pmol P / m i n / m g protein) Treatment
Endothelium present
Group 1 Control S o d i u m n i t r o p r u s s i d e (50 n M ) Group 2 Control Acetylcholine (10/zM) Control Acetylcholine (10/zM)
+ +
cGMP-kinase activity ratios (-cGMP/+cGMP)
-cGMP
+cGMP
2.46 _+ 0.37 4.70 + 0.76 ~'
10.2 _+ 0.48 12.0 _+ 1.40
0.29 -+ 0.02 0.40 _+ 0.02 b
3.18 3.68 4.89 9.86
11.9 12.4 11.9 13.0
0.29 0.29 0.42 0.71
-+ 0.53 ÷ 0.58 _+ 0.32 h _+ 1.25 b.'
_+ 0.91 _+ 1.30 _+ 0.81 _+ 1.11
+-+ + +
0.02 0.01 0.03 b 0.04 ~'.'
" R a t a o r t a s w e r e e x p o s e d to s o d i u m n i t r o p r u s s i d e or a c e t y l c h o l i n e for 2 min. K i n a s e a c t i v i t y w a s m e a s u r e d w i t h a n d w i t h o u t c G M P ( 2 / ~ M ) in a 2.5-min a s s a y at 0 °. The d a t a r e p r e s e n t the m e a n + S E M . N - 5 for e a c h e x p e r i m e n t a l c o n d i t i o n . D a t a from F i s c u s e t a l . 5 h Significantly greater than control without endothelium. ' Significantly greater than control with endothelium.
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TABLE II EFFECT OF ATRIOPEPT1N II ON cGMP-KINASE ACTIVITY RATIOS IN RAT AORTAa
Treatment
cGMP-kinase activity ratios (-cGMP/+cGMP)
Control Atriopeptin ll (1 nM) Atriopeptin ll (10 nM) Atriopeptin lI (100 nM)
0.27 -+ 0.02 0.36 -+ 0.02;' 0.51 -+ 0.04b 0.66 -+ 0.04;'
Rat aortas lacking endothelium were exposed to atriopeptin II for 10 min. Kinase activity was measured as described in Table I. The data represent the mean -+ SEM. N = 5 for each treatment. Data from Fiscus e t a l 5 ° b Significantly greater than control.
by itself elevated endogenous cGMP-kinase activity in rat aorta, suggesting that there may be a tonic stimulatory effect of endothelial cells on vascular cGMP-kinase. There are several advantages to using the activity ratio over the - c G M P kinase activity for estimating endogenous enzyme activity. First of all, with the activity ratio method it is unnecessary to measure protein concentration. Second, activity ratio measurements are considerably more consistent from sample to sample than are kinase activity measurements based on tissue protein (see the data in Table I as an example). Table II shows the effects of atriopeptin II at three different concentrations on cGMP-kinase activity ratios in rat aorta. The c o n c e n t r a t i o n response relationship and the time course (not shown) of this response are closely correlated with those of cGMP elevation and relaxation, j° Because the actions of atriopeptin II are similar to those of acetylcholine and sodium nitroprusside, we have proposed that all three vasodilators share a c o m m o n molecular mechanism of action that includes cGMP elevation and cGMP-kinase activation. 5-1° Problems Encountered in Studying cGMP-Kinase Many of the potential pitfalls discussed previously concerning cAMPkinase activity ratio measurements also apply to cGMP-kinase.13 Of particular concern is the possibility that the activation of the enzyme reflects an alteration that occurs during the tissue extraction or assaying procedures, rather than in the intact tissue. Conceivably there could be a pool
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of cGMP of high concentration that is not in contact with cGMP-kinase within the cells, but upon homogenization the cellular compartments are disrupted and the released cGMP may activate the kinase. A further complication is the possibility that histone H2b may be increasing the affinity of cGMP binding to the enzyme during the assay.~9 If a sufficient amount of cellular cGMP is carried through the tissue extraction and assaying procedures, an artifactually high activity ratio may be obtained. To circumvent these problems, tissue samples must be adequately diluted during tissue homogenization and during the addition of homogenate to the assay reaction mixture. In addition, some samples should be homogenized in buffer to which charcoal (2 or 3 mg/ml) has been added. 12,~3 Theoretically, the charcoal will adsorb endogenous cGMP and prevent the spurious activation of the enzyme during homogenization. This is also a good way to prevent carryover of cGMP into the assay reaction mixture, since endogenous cGMP will be removed along with the charcoal during the brief centrifugation of the homogenates. Using this technique, we have found that responses to methacholine or sodium nitroprusside in canine trachealis or to acetylcholine or sodium nitroprusside in rat aorta are somewhat attenuated by the presence of 3 mg/ml charcoal in the homogenizing buffer. 4,5 Nevertheless, cGMP-kinase activity ratios are still significantly elevated by these agents when charcoal is present during homogenization. The interpretation of these results is complicated, however, since the attenuation of the responses by charcoal may be related to other effects not mentioned above, such as an increased rate of cGMP dissociation from the enzyme after homogenization or a selective removal of activated cGMP-kinase from the tissue extract. This latter explanation may be important, since charcoal at 3 mg/ml was observed to remove substantial amounts of protein from supernatant fractions of rat aorta (unpublished observations). Another potential pitfall is the possible presence of kinase activity other than that contributed by cGMP-kinase. Of course, the magnitude of this problem has been greatly reduced by adding protein kinase inhibitor and EGTA to selectively inhibit cAMP-dependent and calcium-dependent kinases, respectively. It is possible, however, that other kinases that can utilize histone H2b as a substrate may add to the observed enzyme activities measured both with and without cGMP. If this is the case, the activity ratio measurement may give a spurious value that is larger than the actual proportion of enzyme in the active state in the tissue. This problem would be greatest for the control values, where even a small contribution by an undefined kinase may be significant. For this reason, the actual propor19 j. Tse, C. W. Mackenzie III, and T. E. Donnelly, Jr., Int. J. Biochem. 13, 1071 (1981).
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tion of activated enzyme in control rat aortas may be less than the calculated activity ratio of 0.27 (Table II). Furthermore, the increase in the activity ratio in response to stimulatory agents may underestimate the actual changes in endogenous cGMP-kinase activity. There is another reason why the activity ratio method may underestimate the actual enzyme activation that occurs in the tissue. Two cGMPbinding sites, a higher affinity site 1 and a lower affinity site 2, have been found on cGMP-kinase. 2°,21 Activation of the enzyme at low cGMP concentrations is associated with binding to site 1, whereas activation at higher concentrations is associated with binding to both sites. 2°,21 Since the dissociation of cGMP from site 2 is very rapid (tl/2 < 10 sec even at 0°)2°,21 all of the site 2 binding may be lost during the tissue extraction and assaying procedures. How this alters enzyme activity under the assay conditions described herein is presently unknown, but it may mean that part of the endogenous cGMP-kinase activity, the part associated with site 2 binding, is lost almost immediately upon homogenization of the tissue. Therefore, the activity ratio technique may be measuring only the enzyme activation associated with site 1 binding. However, considering the low concentrations of endogenous cGMP and the small changes that are needed to produce a physiological response, 4-1° it is probably the site 1 binding that is most important for endogenous regulation of the enzyme. Other techniques may be utilized to assess endogenous site 2 binding. 2°,21 The assay procedure described herein has been developed for use with homogenates of vascular and airway smooth muscle. If other tissues are to be assayed by this procedure, appropriate controls and significant modifications may be necessary. The fact that most mammalian tissues, other than smooth muscle and cerebellum, have relatively low activities of cGMP-kinase presents a problem that may be difficult to overcome. One modification that may be beneficial, however, is the use of synthetic peptides rather than histones as specific substrates in the kinase assay. A heptapeptide corresponding to the amino acid sequence near the serine-32 phosphorylation site in histone H2b has been shown to be a relatively selective substrate for cGMP-kinase. 22 This peptide may be useful in validating and improving the cGMP-kinase activity ratio assay. However, we recommended that a change from histone to synthetic peptides be done cautiously, since the cGMP-binding characteristics of the enzyme during the assay procedure may be drastically altered by the absence of histone. 19,21 2o C. W. Mackenzie III, J. Biol. Chem. 257, 5589 (1982). 2~ j. D. Corbin and S. O. DCskeland, J. Biol. Chem. 258, 11391 (1983). 2z D. B. Glass and E. G. Krebs, J. Biol. Chem. 254, 9728 (1979).
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Acknowledgments This work was supported by research grants (AM30787 and HL 28474) from the National Institutes of Health, The Veterans Administration, the Council for Tobacco Research, U.S.A., Inc. and by a fellowship from the American Heart Association, California Affiliate (to R.R.F.).
[15] c A M P A n a l o g A n t a g o n i s t s o f c A M P A c t i o n D. ROTHERMEt,, and B E R N D JASTORFF
B y L Y N N E H . PARKER BOTELHO, JOHN
ROBERT V . COOMBS,
The isolation and purification of cAMP in 1957 by Sutherland fostered a period of intensive synthetic chemical involvement in the preparation of analogs of this molecule. The original goal was to synthesize a more potent tissue-specific agonist with a longer biological half-life. The result of these efforts in the decades subsequent to the discovery of cAMP was the synthesis of over 600 cyclic nucleotide analogs of varying potency. Apparently, however, no effort was made to design and synthesize a cAMP antagonist. The first cAMP antagonist was synthesized in 19741 as a mixture of the diastereomers of adenosine cyclic 3',5'-phosphorothioate. A stereospecific synthesis of these isomers was published in 1979, 2 but data demonstrating that the Rp diastereomer, (Rp)-cAMPS, could bind to the cAMP-dependent protein kinase holoenzyme without causing activation were not published until 1982. 3 The first direct evidence that (Rp)-cAMPS (Fig. 1) was a cAMP antagonist and could oppose the action of cAMP was a study done in hepatocytes in 1983. 4 This study demonstrated that (Rp)-cAMPS is an intracellular inhibitor of cAMP action. Subsequently, this analog has been used to study several cAMPdependent systems: glucagon-induced glucose production in hepatocytes4-7; isoproterenol-induced lipolysis in adipocytesS; cAMP activation F. Eckstein, L. P. Simonson, and H.-P. Baer, Biochemistry 13, 3806 (1974). 2 j. Baraniak, R. W. Kinas, K. Lesiak, and W. J. Stec, J. Chem. Soc. Chem. Commun. 20, 940 (1979). 3 R. J. DeWit, J. Hoppe, W. J. Stec, J. Baraniak, and B. Jastorff, Eur. J. Biochem. 122, 95 (1982). 4 j. D. Rothermel, W. J. Stec, J. Baraniak, B. Jastorff, and L. H. P. Botelho, J. Biol. Chem. 258, 12125 (1983). 5 j. D. Rothermel, B. Jastorff, and L. H. P. Botelho, J. Biol. Chem. 259, 8151 (1984). 6 j. D. Rothermel, N. L. Perillo, J. S. Marks, and L. H. P. Botelho, J. Biol. Chem. 259, 15294 (1984). 7 C. J. Dragland-Meserve, J. D. Rothermel, M. J. Houlihan, and L. H. P. Botelho, J. Cyclic Nucleotide Protein Phosphorylation Res. 10, 371 (1985).
METHODS IN ENZYMOLOGY. VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ACTION
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filters are dried and counted in a fluid for nonaqueous samples, or dissolved in a fluid like "Filter Count" (Packard), there will be considerable quenching, and more so the higher the protein content of the sample. Final Remarks The ammonium sulfate precipitation method has been used to study cyclic nucleotide binding to mammalian cyclic nucleotide-dependent protein kinases 1,6-~3 and a related nonmammalian binding protein. TM It has also been used to study the binding of cAMP and adenosine to S-adenosylhomocysteinase from mammalian )°:5 and plant 16 sources, as well as cGMP binding to a cyclic nucleotide phosphodiesterase. ~7 In several of these cases the sulfate precipitation assay appeared superior to other commonly used assays. Thus, the inclusion of ammonium sulfate is essential to detect the binding of 8-azido-cAMP to the rapidly exchanging site of RI112:8 and of cGMP to the rapidly exchanging site of the cGMPdependent protein kinase. 7,~3 8 G. Illiano, G. P. E. Tell, M. I. Siegel, and P. Cuatrecasas, Proc. Natl. Acad. Sci. U.S.A. 70, 2443 (1973). 9 j. Van Sande, C. Decoster, and J. E. Dumont, Biochem. Biophys. Res. Commun. 62, 168 (1975). t0 S. O. DCskeland and P. M. Ueland, Biochem. Biophys. Res. Commun. 66, 606 (1975). H j. Tse, C. W. Mackenzie III, and T. E. Donnelly, Jr., Int. J. Biochem. 13, 1071 (1981). 12 D. 0greid and S. O. DCskeland, FEBS Lett. 150, 161 (1982). 13 C. W. Mackenzie III, J. Biol. Chem. 257, 5589 (1982). ~4 R. Rangel-Aldao, G. Tovar, and M. L. de Ruiz, J. Biol. Chem. 258, 6979 (1983). ~5 p. M. Ueland and S. O. DCskeland, J. Biol. Chem. 253, 1667 (1978). ~6 M. Giannattasio, G. Carratu, G. F. Tucci, and A. M. Carafa, Phytochemistry 18, 1613 (1979). ~7 F. Miot, P. J. M. Van Haastert, and C. Erneux, Eur. J. Biochem. 149, 59 (1985). ~8 A. R. Kerlavage and S. S. Taylor, J. Biol. Chem. 257, 1749 (1982).
[14] c G M P - D e p e n d e n t P r o t e i n K i n a s e A c t i v a t i o n in I n t a c t T i s s u e s B y R O N A L D R . FISCUS a n d FERID M U R A D
Cyclic GMP-dependent protein kinase (cGMP-kinase) has been detected in many tissues. However, in contrast to the distribution of cAMPdependent protein kinase (cAMP-kinase), levels of cGMP-kinase vary considerably from one tissue to another as determined enzymatically or METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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immunologically.l,2 Mammalian tissues that are especially rich in cGMPkinase include cerebellum and organs containing large amounts of smooth muscle, such as lung, 1,2 tracheal muscle, 3,4 and blood vessels. 1,2,5 cGMPkinase in these organs is believed to be a major target for the intracellular messenger cGMP. Since cGMP is thought to mediate the smooth muscle relaxant effects of three important classes of vasodilators, the nitrovasodilators, the endothelium-dependent vasodilators, and the atrial natriuretic factors, 5-~° the need for an assay that can measure cGMP-kinase activation in intact tissues has become increasingly more important. Unfortunately, the endogenous activation of cGMP-kinase has proved to be more difficult to demonstrate than the analogous activation of cAMPkinase. Equation (1) will help to explain one of the problems, cGMPkinase is represented as a dimer of the polypeptide RC that contains both regulatory and catalytic domains. ~ Unlike cAMP-kinase that dissociates into regulatory and catalytic subunits upon activation with cAMP, cGMPkinase remains as a holoenzyme upon binding of cGMP [see Eq. (1)]. 1 (RC)2 + 4cGMP ~ (RC)2' cGMP4 (less active) (fully active)
(1)
An increase in the activity of purified cGMP-kinase is observed after adding cGMP in sufficient amounts to occupy the cGMP-binding sites. 1In intact cells the enzyme is probably activated in a similar manner, i.e., the equilibrium of Eq. (1) is shifted to the right, whenever there is an elevation in cGMP concentrations in the vicinity of the kinase. Ideally, when measuring the activation of cyclic nucleotide-dependent protein kinases in intact tissues, this equilibrium should not be disturbed by the procedures of tissue extraction or assaying. In the case of cAMP-kinase, the fact that the regulatory and catalytic subunits dissociate upon activation helps to preserve the activation state of the enzyme when it is diluted into homogenizing buffer and further diluted into a kinase reaction solution for t T. M. Lincoln and J. D. Corbin, Adv. Cyclic Nucleotide Res. 15, 139 (1983). 2 S. M. Lohman and U. Walter, Adv. Cyclic Nucleotide Res. 18, 63 (1984). 3 T. J. Torphy, W. B. Freese, G. A. Rinard, L. L. Brunton, and S. E. Mayer, J. Biol. Chem. 257, 11609 (1982). 4 R. R. Fiscus, T. J. Torphy, and S. E. Mayer, Biochim. Biophys. Acta 805, 382 (1984). 5 R. R. Fiscus, R. M. Rapoport, and F. Murad, J. Cyclic Nucleotide Res. 9, 415 (1983). 6 S. Katsuki, W. P. Arnold, and F. Murad, J. Cyclic Nucleotide Res. 3, 239 (1977). 7 R. M. Rapoport and F. Murad, Circ. Res. 52, 352 (1983). 8 R. M. Rapoport and F. Murad, J. Cyclic Nucleotide Res. 9, 281 (1983). 9 R. J. Winquist, E. P. Faison, S. A. Waldman, K. Schwartz, F. Murad, and R. M. Rapoport. Proc. Natl. Acad. Sci. U.S.A. 81, 7661 (1984). to R. R. Fiscus, R. M. Rapoport, S. A. Waldman, and F. Murad, Biochim. Biophys. Acta 846, 179 (1985).
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assaying. In contrast, activated cGMP-kinase, under similar conditions, begins to lose activity,4 probably because of the rapid dissociation of cGMP from the enzyme as a result of dilution, 11 i.e., the equilibrium of Eq. (1) is shifted to the left. Therefore, special procedures are needed to minimize cGMP dissociation. We have found that by rapidly extracting the tissue and assaying at a reduced temperature (0°) with an abbreviated incubation time (2.5 min), endogenous cGMP-kinase activities can be assessed.<5, l0 Kinase activity in tissue homogenates is measured in the absence and presence of 2 ~ M cGMP and expressed as an activity ratio (activity without cGMP divided by activity with cGMP) in a manner similar to that first described by Lincoln and Keely 12 and analogous to the method described by Corbin 13for measuring cAMP-kinase activity ratios in tissues. With the assay for cGMP-kinase activity ratios, however, saturating amounts of the heat-stable inhibitor of cAMP-kinase (the protein kinase inhibitor of Walsh e t a/. 14) are added to avoid interference by cAMPkinase. Under the assay conditions described herein, 2 ~ M cGMP fully activates cGMP-kinase in smooth muscle homogenates. 3-5,m Therefore, the activity ratio obtained by this procedure gives an estimate of the proportion of cGMP-kinase in the active state in the tissue. Experimental Methods and Material
Tissue Preparation Two smooth muscle preparations, canine tracheal smooth muscle and rat thoracic aorta, have been used for studying endogenous activation of cGMP-kinaseY ,m In both cases, the smooth muscle tissues were cut into small strips and allowed to equilibrate for at least 1 hr with a physiological salt solution at 37° and aerated with 95% 02 : 5% CO 2. In the case of rat aortas, some strips were rubbed on the intimal surface to remove the endothelium. 5,7.8 The strips were exposed to agents that alter smooth muscle contractility, such as the vasodilators sodium nitroprusside, acetylcholine (endothelium dependent), and atrial natriuretic factor ( a t r i o p e p t i n II). 4-m At appropriate times the strips were removed from the physiological salt solution and rapidly frozen between metal clamps precooled in liquid nitrogen. Unless samples are immediately used for the H R. W. McCune and G. N. Gill, J. Biol. Chem. 254, 5083 (1979). r2 T. M. Lincoln and S. L. Keely, Biochim. Biophys. Acta 676, 230 (1981). 13 j. D. Corbin, this series, Vol. 99, p. 227. t4 D. A. Walsh, C. D. Ashby, C. Gonzalez, D. Calkins, E. H. Fischer, and E. G. Krebs, J. Biol. Chem. 246, 1977 (1971).
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cGMP-kinase assay, it is recommended that for maximum stability the samples are stored in liquid nitrogen until they can be assayed.
Procedure for Preparation of Tissue Extracts Frozen tissues are homogenized with a motor-driven ground glass pestle and homogenizing tubes in 16 vol of ice-cold 20 mM potassium phosphate, pH 7.0, l0 m M EDTA, 2 mM EGTA, 0.5 mM isobutylmethylxanthine, and 6 m M dithiothreitol.~° The EGTA is added to selectively inhibit calcium-dependent kinases that may interfere with the assay. The homogenates are rapidly transferred to cold microfuge tubes and centrifuged at 8000 g in a microfuge at 4° for 0.5 min and the supernatant fractions are used for the kinase assay. To minimize cGMP dissociation from the enzyme during the sample extract preparation, each sample is homogenized, centrifuged, and assayed individually with a total time of 2.0 rain from the beginning of homogenization to the beginning of the assay.
Procedure for Measuring cGMP-Kinase Activity Ratios Kinase activity is determined by measuring the amount of 32p transferred from [y-32p]ATP to histone H2b during a 2.5-min incubation at 0°. The reaction tubes are incubated in an ice-water bath with frequent agitation to assure constant temperature throughout the bath. Also, it is recommended that the assay be conducted in a walk-in cold room in order that all materials that come in contact with samples and reaction mixtures, such as pipet tips and filter paper squares, stay cold. The assays are initiated by adding 7 ~l of supernatant fraction of tissue homogenates to 35/~1 of a reaction mixture containing (in final concentration) the following: 15 m M potassium phosphate (pH 7.0), 7 mM magnesium acetate, 0.5 mg/ml histone H2b, 20 mM sodium fluoride, 0.25 mM isobutylmethylxanthine, 30 ~ M adenosine triphosphate (ATP) with [y-32p]ATP at 5000 cpm/pmol, and 50 t~g/assay tube of the partially purified heat-stable inhibitor of cAMP-kinase (protein kinase inhibitor). At this concentration protein kinase inhibitor inhibits rat aorta cAMP-kinase by >99% in a typical assay. 5 The heat-stable protein kinase modulator protein that has been reported to be necessary in some cGMP-kinase assay systems ~had no beneficial effect in this assay. The reaction is stopped by transferring 35 pA of the final reaction mixture to 3MM chromatography paper squares (1.7 × ! .7 cm) and immediately immersing them in icecold 10% trichloroacetic acid with 2.5% pyrophosphate. At least 15 ml of trichloroacetic acid solution per paper square should be used in order to keep background radioactivity low. The paper squares are washed in 5%
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trichloroacetic acid with 2.5% pyrophosphate by a procedure modified from that of Corbin and Reimann 15 and includes two hot (90°) washes for 15 min each and two washes at room temperature for 20 min each. The final wash is in 95% ethanol for 10 min and the paper squares are dried and counted in Betacount (Baker) by liquid scintillation spectrometry. Materials
The heat-stable inhibitor of cAMP-kinase (protein kinase inhibitor) ~4 was prepared by the procedure of McPherson et al. 16through the DEAEcellulose and dialysis steps. Synthetic atriopeptin II was purchased from Peninsula Laboratories (Belmont, CA), histone H2b from Worthington, [7-32p]ATP from Amersham, 3MM chromatography paper from Whatman, and trichloroacetic acid from Mallinckrodt. Other chemicals and drugs were purchased from Sigma. Application of the cGMP-Kinase Activity Ratio Method The assay technique described above was first applied to the study of cGMP-kinase regulation in canine tracheal smooth muscle? Sodium nitroprusside and the muscarinic agonist methacholine were selected as potential stimulatory agents, since both elevate cGMP concentrations in tracheal muscle. 4,17,1s However, these agents have quite different effects on contractility; methacholine contracts whereas sodium nitroprusside relaxes trachealis. 4,j7,Js In addition, cGMP elevations caused by muscarinic agonists are calcium dependent, whereas those to sodium nitroprusside are not. ~v Nevertheless, both agents were observed to elevate cGMPkinase activity ratios in canine trachealis, thus demonstrating a functional link between elevation of cGMP and activation of cGMP-kinase in intact airway smooth muscle? These data support the hypothesis that cGMP, via kinase activation and subsequent phosphorylation, mediates smooth muscle relaxation to sodium nitroprusside. Also, the data suggest that cGMP-kinase may be important in the proposed role of cGMP as a negative-feedback signal in regulating smooth muscle contraction to agents like methacholine. We have also used the activity ratio method to study cGMP-kinase regulation in rat aorta. 5,1° Representatives of three major classes of vasot5 j. ~6 J. 17 S. ~8 T.
D. Corbin and E. M. Reimann, this series, Vol. 38, p. 287. M. M c P h e r s o n , S. W h i t e h o u s e , and D. A. Walsh, Biochemistry 18, 4835 (1979). K a t s u k i and F. Murad, Mol. Pharmacol. 13, 330 (1977). J. Torphy, Z. Cong, S. M. Peterson, R. R. Fiscus, G. A. Rinard, and S. E. Mayer, J. Pharmacol. Exp. Ther. 233, 409 (1985).
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KINASE ACTIVATION
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dilators, the nitrovasodilator sodium nitroprusside, the endotheliumdependent vasodilator acetylcholine, and the atrial natriuretic factor atriopeptin II were tested and all were found to elevate cGMP-kinase activity ratios, responses that correlated with cGMP elevation and relaxation of the tissue. Table I shows the effects of sodium nitroprusside and acetylcholine on kinase activities in the absence and presence of exogenous cGMP as well as the calculated activity ratios. Sodium nitroprusside and acetylcholine (when the endothelium was intact) increased endogenous cGMP-kinase activity ( - c G M P activity in the kinase assay of Table I). Total cGMP-kinase activity (+cGMP activity in the kinase assay) was unaltered by drug treatment of the tissues. It is possible that the increase in the - c G M P activity represents activation of an undefined cGMP-independent protein kinase (see the following section). However, this is unlikely in this study since under these circumstances an equal increase in the total (+cGMP) activity would be expected and this was not observed with acetylcholine treatment. Also, since the +cGMP kinase activity, the denominator in the activity ratio calculation, is not significantly altered, a change in the activity ratio reflects solely the change in the - c G M P kinase activity and presumably also the change in cGMP-kinase activity in the tissue at the time of freezing. Therefore, it appears that the proportion of cGMP-kinase in the active state has increased in rat aorta treated with sodium nitroprusside or acetylcholine. In addition, the endothelium TABLE 1 EFFECT OF SODIUM NITROPRUSSIDE AND ACETYLCHOLINE ON cGMP-KINASE ACTIVITY IN RAT AORTAa Kinase activity (pmol P / m i n / m g protein) Treatment
Endothelium present
Group 1 Control S o d i u m n i t r o p r u s s i d e (50 n M ) Group 2 Control Acetylcholine (10/zM) Control Acetylcholine (10/zM)
+ +
cGMP-kinase activity ratios (-cGMP/+cGMP)
-cGMP
+cGMP
2.46 _+ 0.37 4.70 + 0.76 ~'
10.2 _+ 0.48 12.0 _+ 1.40
0.29 -+ 0.02 0.40 _+ 0.02 b
3.18 3.68 4.89 9.86
11.9 12.4 11.9 13.0
0.29 0.29 0.42 0.71
-+ 0.53 ÷ 0.58 _+ 0.32 h _+ 1.25 b.'
_+ 0.91 _+ 1.30 _+ 0.81 _+ 1.11
+-+ + +
0.02 0.01 0.03 b 0.04 ~'.'
" R a t a o r t a s w e r e e x p o s e d to s o d i u m n i t r o p r u s s i d e or a c e t y l c h o l i n e for 2 min. K i n a s e a c t i v i t y w a s m e a s u r e d w i t h a n d w i t h o u t c G M P ( 2 / ~ M ) in a 2.5-min a s s a y at 0 °. The d a t a r e p r e s e n t the m e a n + S E M . N - 5 for e a c h e x p e r i m e n t a l c o n d i t i o n . D a t a from F i s c u s e t a l . 5 h Significantly greater than control without endothelium. ' Significantly greater than control with endothelium.
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TABLE II EFFECT OF ATRIOPEPT1N II ON cGMP-KINASE ACTIVITY RATIOS IN RAT AORTAa
Treatment
cGMP-kinase activity ratios (-cGMP/+cGMP)
Control Atriopeptin ll (1 nM) Atriopeptin ll (10 nM) Atriopeptin lI (100 nM)
0.27 -+ 0.02 0.36 -+ 0.02;' 0.51 -+ 0.04b 0.66 -+ 0.04;'
Rat aortas lacking endothelium were exposed to atriopeptin II for 10 min. Kinase activity was measured as described in Table I. The data represent the mean -+ SEM. N = 5 for each treatment. Data from Fiscus e t a l 5 ° b Significantly greater than control.
by itself elevated endogenous cGMP-kinase activity in rat aorta, suggesting that there may be a tonic stimulatory effect of endothelial cells on vascular cGMP-kinase. There are several advantages to using the activity ratio over the - c G M P kinase activity for estimating endogenous enzyme activity. First of all, with the activity ratio method it is unnecessary to measure protein concentration. Second, activity ratio measurements are considerably more consistent from sample to sample than are kinase activity measurements based on tissue protein (see the data in Table I as an example). Table II shows the effects of atriopeptin II at three different concentrations on cGMP-kinase activity ratios in rat aorta. The c o n c e n t r a t i o n response relationship and the time course (not shown) of this response are closely correlated with those of cGMP elevation and relaxation, j° Because the actions of atriopeptin II are similar to those of acetylcholine and sodium nitroprusside, we have proposed that all three vasodilators share a c o m m o n molecular mechanism of action that includes cGMP elevation and cGMP-kinase activation. 5-1° Problems Encountered in Studying cGMP-Kinase Many of the potential pitfalls discussed previously concerning cAMPkinase activity ratio measurements also apply to cGMP-kinase.13 Of particular concern is the possibility that the activation of the enzyme reflects an alteration that occurs during the tissue extraction or assaying procedures, rather than in the intact tissue. Conceivably there could be a pool
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INTACT TISSUE c G M P KINASE ACTIVATION
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of cGMP of high concentration that is not in contact with cGMP-kinase within the cells, but upon homogenization the cellular compartments are disrupted and the released cGMP may activate the kinase. A further complication is the possibility that histone H2b may be increasing the affinity of cGMP binding to the enzyme during the assay.~9 If a sufficient amount of cellular cGMP is carried through the tissue extraction and assaying procedures, an artifactually high activity ratio may be obtained. To circumvent these problems, tissue samples must be adequately diluted during tissue homogenization and during the addition of homogenate to the assay reaction mixture. In addition, some samples should be homogenized in buffer to which charcoal (2 or 3 mg/ml) has been added. 12,~3 Theoretically, the charcoal will adsorb endogenous cGMP and prevent the spurious activation of the enzyme during homogenization. This is also a good way to prevent carryover of cGMP into the assay reaction mixture, since endogenous cGMP will be removed along with the charcoal during the brief centrifugation of the homogenates. Using this technique, we have found that responses to methacholine or sodium nitroprusside in canine trachealis or to acetylcholine or sodium nitroprusside in rat aorta are somewhat attenuated by the presence of 3 mg/ml charcoal in the homogenizing buffer. 4,5 Nevertheless, cGMP-kinase activity ratios are still significantly elevated by these agents when charcoal is present during homogenization. The interpretation of these results is complicated, however, since the attenuation of the responses by charcoal may be related to other effects not mentioned above, such as an increased rate of cGMP dissociation from the enzyme after homogenization or a selective removal of activated cGMP-kinase from the tissue extract. This latter explanation may be important, since charcoal at 3 mg/ml was observed to remove substantial amounts of protein from supernatant fractions of rat aorta (unpublished observations). Another potential pitfall is the possible presence of kinase activity other than that contributed by cGMP-kinase. Of course, the magnitude of this problem has been greatly reduced by adding protein kinase inhibitor and EGTA to selectively inhibit cAMP-dependent and calcium-dependent kinases, respectively. It is possible, however, that other kinases that can utilize histone H2b as a substrate may add to the observed enzyme activities measured both with and without cGMP. If this is the case, the activity ratio measurement may give a spurious value that is larger than the actual proportion of enzyme in the active state in the tissue. This problem would be greatest for the control values, where even a small contribution by an undefined kinase may be significant. For this reason, the actual propor19 j. Tse, C. W. Mackenzie III, and T. E. Donnelly, Jr., Int. J. Biochem. 13, 1071 (1981).
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tion of activated enzyme in control rat aortas may be less than the calculated activity ratio of 0.27 (Table II). Furthermore, the increase in the activity ratio in response to stimulatory agents may underestimate the actual changes in endogenous cGMP-kinase activity. There is another reason why the activity ratio method may underestimate the actual enzyme activation that occurs in the tissue. Two cGMPbinding sites, a higher affinity site 1 and a lower affinity site 2, have been found on cGMP-kinase. 2°,21 Activation of the enzyme at low cGMP concentrations is associated with binding to site 1, whereas activation at higher concentrations is associated with binding to both sites. 2°,21 Since the dissociation of cGMP from site 2 is very rapid (tl/2 < 10 sec even at 0°)2°,21 all of the site 2 binding may be lost during the tissue extraction and assaying procedures. How this alters enzyme activity under the assay conditions described herein is presently unknown, but it may mean that part of the endogenous cGMP-kinase activity, the part associated with site 2 binding, is lost almost immediately upon homogenization of the tissue. Therefore, the activity ratio technique may be measuring only the enzyme activation associated with site 1 binding. However, considering the low concentrations of endogenous cGMP and the small changes that are needed to produce a physiological response, 4-1° it is probably the site 1 binding that is most important for endogenous regulation of the enzyme. Other techniques may be utilized to assess endogenous site 2 binding. 2°,21 The assay procedure described herein has been developed for use with homogenates of vascular and airway smooth muscle. If other tissues are to be assayed by this procedure, appropriate controls and significant modifications may be necessary. The fact that most mammalian tissues, other than smooth muscle and cerebellum, have relatively low activities of cGMP-kinase presents a problem that may be difficult to overcome. One modification that may be beneficial, however, is the use of synthetic peptides rather than histones as specific substrates in the kinase assay. A heptapeptide corresponding to the amino acid sequence near the serine-32 phosphorylation site in histone H2b has been shown to be a relatively selective substrate for cGMP-kinase. 22 This peptide may be useful in validating and improving the cGMP-kinase activity ratio assay. However, we recommended that a change from histone to synthetic peptides be done cautiously, since the cGMP-binding characteristics of the enzyme during the assay procedure may be drastically altered by the absence of histone. 19,21 2o C. W. Mackenzie III, J. Biol. Chem. 257, 5589 (1982). 2~ j. D. Corbin and S. O. DCskeland, J. Biol. Chem. 258, 11391 (1983). 2z D. B. Glass and E. G. Krebs, J. Biol. Chem. 254, 9728 (1979).
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Acknowledgments This work was supported by research grants (AM30787 and HL 28474) from the National Institutes of Health, The Veterans Administration, the Council for Tobacco Research, U.S.A., Inc. and by a fellowship from the American Heart Association, California Affiliate (to R.R.F.).
[15] c A M P A n a l o g A n t a g o n i s t s o f c A M P A c t i o n D. ROTHERMEt,, and B E R N D JASTORFF
B y L Y N N E H . PARKER BOTELHO, JOHN
ROBERT V . COOMBS,
The isolation and purification of cAMP in 1957 by Sutherland fostered a period of intensive synthetic chemical involvement in the preparation of analogs of this molecule. The original goal was to synthesize a more potent tissue-specific agonist with a longer biological half-life. The result of these efforts in the decades subsequent to the discovery of cAMP was the synthesis of over 600 cyclic nucleotide analogs of varying potency. Apparently, however, no effort was made to design and synthesize a cAMP antagonist. The first cAMP antagonist was synthesized in 19741 as a mixture of the diastereomers of adenosine cyclic 3',5'-phosphorothioate. A stereospecific synthesis of these isomers was published in 1979, 2 but data demonstrating that the Rp diastereomer, (Rp)-cAMPS, could bind to the cAMP-dependent protein kinase holoenzyme without causing activation were not published until 1982. 3 The first direct evidence that (Rp)-cAMPS (Fig. 1) was a cAMP antagonist and could oppose the action of cAMP was a study done in hepatocytes in 1983. 4 This study demonstrated that (Rp)-cAMPS is an intracellular inhibitor of cAMP action. Subsequently, this analog has been used to study several cAMPdependent systems: glucagon-induced glucose production in hepatocytes4-7; isoproterenol-induced lipolysis in adipocytesS; cAMP activation F. Eckstein, L. P. Simonson, and H.-P. Baer, Biochemistry 13, 3806 (1974). 2 j. Baraniak, R. W. Kinas, K. Lesiak, and W. J. Stec, J. Chem. Soc. Chem. Commun. 20, 940 (1979). 3 R. J. DeWit, J. Hoppe, W. J. Stec, J. Baraniak, and B. Jastorff, Eur. J. Biochem. 122, 95 (1982). 4 j. D. Rothermel, W. J. Stec, J. Baraniak, B. Jastorff, and L. H. P. Botelho, J. Biol. Chem. 258, 12125 (1983). 5 j. D. Rothermel, B. Jastorff, and L. H. P. Botelho, J. Biol. Chem. 259, 8151 (1984). 6 j. D. Rothermel, N. L. Perillo, J. S. Marks, and L. H. P. Botelho, J. Biol. Chem. 259, 15294 (1984). 7 C. J. Dragland-Meserve, J. D. Rothermel, M. J. Houlihan, and L. H. P. Botelho, J. Cyclic Nucleotide Protein Phosphorylation Res. 10, 371 (1985).
METHODS IN ENZYMOLOGY. VOL. 159
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