Regulation of cardiac cyclic GMP-dependent protein kinase

230

Bioehimica et Biophysica Acta, 676 (1981) 230-244

Elsevier/North-HollandBiomedicalPress BBA 29681 REGULATION OF CARDIAC CYCLIC GMP-DEPENDENT PROTEIN KINASE THOMAS M. LINCOLN*,a and STANLEYL. KEELY ** a Department o f Pharmacology, School of Medicine, University o f South Carolina, Columbia, SC 29208 and Department of Physiology, School o f Medicine, Vanderbilt University, Nashville, TN 37232 (U.S.A.)

(Received January 27th, 1981)

Key words: Protein kinase; cyclic GMP; Regulation; Histone; (Rat]

An assay method based on the ability of high concentrations of Mg2+ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardiac cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0-4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0-4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMPdependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2-3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8-10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.

Work from several laboratories has shown that several agents or conditions which increase cyclic * To whom correspondence should be addressed. ** Present Address: McNeil Laboratories, Camp Hill Road, Fort Washington, PA 19034.

AMP levels produce a corresponding and immediate increase in the cardiac cyclic AMP-dependent protein kinase (cyclic AMP kinase) activity [1-6]. Much evidence has been accumulated which supports the hypothesis that the cyclic AMP kinase, in part at least, mediates the effects of many cardioactive

0304-4165/81/0000-0000/$02.50 © Elsevier/North-HollandBiomedicalPress

231 agents on both metabolism and function [1-9]. In addition to cyclic AMP, cyclic GMP has been found in relatively large amounts in the heart [10]. An increase in the levels of cyclic GMP is one of the earliest responses of the heart to treatment with acetylcholine [11-14]. The facts that acetylcholineinduced increases in cyclic GMP correlate, both in' time and degree, with the negative inotropic effect of the cholinergic agent, and that the 8-bromo analogue of cyclic GMP produces a decrease in contractile force in both atrial muscle and cardiac fibers [15,16] have led to suggestions that cyclic GMP may mediate at least some of the actions of acetylcholine in the heart. In addition, the ability of acetylcholine to attenuate the effects of /3-adrenergic agents on heart metabolism and function are associated with increased levels of cyclic GMP [8,13,14]. Several reports, however, have shown that agents such as sodium nitroprusside can produce large increases in cyclic GMP in heart, while not affecting contractile force [17-19]. Furthermore, Brooker [20] has found a clear dissociation between the levels of carbachol needed to raise cyclic GMP levels in guinea-pig atria and those needed to produce a negative inotropic effect. Thus, there is some controversy surrounding the role of cyclic GMP in mediating the actions of cholinergic agents in heart. Steiner et al. [21] have suggested that in many cases, changes in the tissue levels of cyclic nucleotides are not always associated with changes in physiological parameters caused by cyclic nucleotides, since changes in translocation and intracellular binding of cyclic AMP and cyclic GMP can occur in response to tissue stimulation. Besides cyclic AMP kinases, the heart also contains a relatively large amount of cyclic GMP-dependent protein klnase [22,23]. Since cyclic GMP probably mediates many of its effects through activation of the cyclic GMP kinase in heart, we felt it important to investigate the effects of agents which increase heart cyclic GMP on this enzyme in vitro, and to determine the relationship between cyclic GMP levels and the activity of the cyclic GMP kinase. We were also interested in the relationship between changes in cyclic GMP kinase activity and cardiac function. Techniques for the measurement of the cyclic GMP kinase activity ratio (activity assayed in the absence of cyclic GMP divided by activity assayed in the

presence of cyclic GMP), and a comparison of the effects of acetylcholine and nitroprusside on cyclic GMP levels, cyclic GMP kinase activity, and force of contraction are presented in this paper. The relationship between activation of the cyclic GMP kinase and force of contraction is also discussed.

Experimentalprocedure Characterization of cyclic GMP kinase in crude tissue extracts. White male Sprague-Dawley rats (200-300 g) were decapitated and the tissues were quickly excised and rinsed in cold 20 mM sodium phosphate/2 mM EDTA, pH 7.0 (buffer A). The tissues were weighed and homogenized in a Sorvall blender in 5 vol. (w/v) of buffer A, twice for 30 s each. The homogenates were centrifuged at 12000Xg for 20 min and the supematants were used as the source of enzyme. In most cases, the tissue extracts were diluted 1 : 1 0 in buffer A immediately before assay. Protein Mnase assay. Specific cyclic GMP kinase activity was assayed in a total volume of 0.1 ml containing 15 mM Tris-HC1 (pH 7.5), 75 mM magnesium acetate, 30 ~M [3,-S2P]ATP (500-2000 cpm/pmol in the assay), 0.1 mM 3-isobutyl-l-methyl xanthine, 50 btg F2 b histone, 100/,tg protein kinase inhibitor, 20 mM 2-mercaptoethanol, and 2 /,tM cyclic GMP where indicated. The reactions were initiated with the addition of 10 /~1 of the tissue extract supernatant (10-40 /~g protein) or 20 ~1 from fractions collected after DEAE-ceUulose chromatography. Assays were conducted at 30°C for the times indicated in the individual experiments. At the end of the incubation, 75 /.tl were spotted on 1 × 2 cm Whatman 3 MM falter papers, which were placed in a beaker of 10% trichloroacetic acid containing approx. 50 mM potassium phosphate. The papers were washed three times for 15 min each in 10% trichloroacetic acid according to the method of Corbin and Reimann [24], then once for 1 rain each with ethanol and diethyl ether. The papers were dried and counted in a xylene.based scintillant. 1 unit of activity is that amount of enzyme which catalyzes the transfer of 1 pmol of phosphate to the historic substrate in 1 min. Cyclic AMP kinase activity was measured by the method of Corbin and Reimann [24].

232

Cyclic GMP binding assay. Cyclic GMP binding was measured by a modification of the method described previously [22]. 10 /.tl of 5/,tM cyclic [aH]GMP (19 Ci/mmol), 10 /,tl of 10 /,tM cyclic AMP (nonradioactive) and 10/,tl of 2 mM 1-methyl3-isobutyl-xanthine were added to 12 × 75 mm test tubes. The assay was initiated by the addition of 70 /.tl of a 1 : 10 diluted tissue extract. The tubes were incubated for 45 min at 0-4°C. Free and bound cyclic [aH]GMP were separated by the Millipore f'dtration technique originally described by Gilman [251. DEAE-cellulose chromatography. 2ml of the various tissue extracts were chromatographed on 0.9 × 4 cm DEAE-cellulose columns equilibrated in buffer A. After applying the extracts, the columns were washed with 10 vol. of buffer A and kinase activity was eluted with a 100 ml linear gradient of 0-0.4 M NaCI in buffer A. Fractions of approx. 3 ml were collected and assayed for protein kinase activity with and without 2 ~M cyclic GMP as described above. Preparation of cellular fractions. To determine the subcellular distribution of cyclic GMP kinase activity, tissues were homogenized in 5 vol. of buffer A containing 0.3 M sucrose and 0.1 M KCI (buffer B) with 10 strokes using a Teflon pestle. The homogenate was centrifuged at 400 × g for 2 min, and the pellet was suspended in approx. 2 vol. of buffer B and homogenized for 10 s using a Polytron homogenizer (Brinkman Co.). This fraction is designated as the nuclear fraction. The supernatant from the first centrifugation step was next centrifuged at 10000 ×g for 20 min and the pellet was prepared in a similar fashion. This fraction is designated as the mitochondrial fraction. The supernatant was then centrifuged for 1 0 0 0 0 0 × g for 60 min in a Beckman T-21 fixedangle ultracentrifuge rotor. The pellet designated as 'microsomal' fraction was prepared as described above and the supernatant was saved. Each fraction was assayed for protein kinase activity with and without 2/,tM cyclic GMP. Cyclic GMP determination. Approx. 100 mg of frozen heart tissue were homogenized in 1 ml of 5% trichloroacetic acid and centrifuged at 10000 ×g for 10 min. Cyclic GMP was purified from the supernatant by a modification of the method described by Jakobs et al. [26] using neutral alumina and Dowex-

50 chromatography. A trace amount of cyclic [3H]GMP was added to the supernatant to determine the recovery which was usually 60-80%. Cyclic GMP was assayed by competitive binding using partially purified rat lung cyclic GMP kinase [22]. The assay was performed as follows: 20/xl of an assay mixture of 250 mM potassium phosphate, pH 8.0, 0.5 mg/ml type VIII-S histone (Sigma) and 25 pmol/ml cyclic [3H]GMP (0.5 pmol/tube) were added to 12 × 75 cm glass tubes. 10/sl of cyclic GMP standard (0.05-2.0 pmol) or unknown were added next. The assay was initiated by adding 20 /~I ( 5 - 1 0 /xg protein) of partially purified cyclic GMP kinase in 20 mM sodium phosphate, 2 mM EDTA, and 50 mM 2-mercaptoethanol, pH 7.0. The reaction mixture was incubated on ice for 60 min after which time the contents were diluted to 1 ml with cold 10 mM potassium phosphate/1 mM EDTA buffer (pH 6.8) and filtered through a 0.45/am cellulose ester filter. The tube was rinsed with another 1 ml of the buffer and the filter was washed with 10 ml of buffer. The filter was dried and counted in a xylene-based scintillant. The reproducible detection of less than 0.1 pmol of cyclic GMP was possible using this procedure. Heart perfusion. Fed male rats weighing 150-165 g were used. Sodium heparin (1 500 U/kg) was injected intraperitoneally 15 min before killing. The animals were anesthetized with sodium pentobarbital (100 mg/kg). Hearts were quickly excised, immersed in cold saline (4°C) until beating ceased, attached via the aorta to a perfusion cannula, and perfused at 37°C without recirculation at 60 mmHg with Krebs-Henseleit bicarbonate buffer containing 1.25 mM Ca 2÷ and 10 mM glucose. The buffer was equilibrated with O2/CO2 (95/5%). At the end of the perfusion period, hearts were quickly frozen between Wollenberger clamps which had been cooled in liquid nitrogen. The great vessels were trimmed away and the remaining tissue pulverized to a fine powder with a percussion mortar which had also been cooled in liquid nitrogen. The powders were stored at -70°C until assayed. Force of contraction was estimated by measuring peak tension development with a Statham strain-guage transducer attached to the apex of the heart by a stainless-steel suture. Diastolic tension was set at 2.5 g and tension development was recorded on a Sanborn 964 recorder. All hearts were paced at 240 beats/min by a Grass S-9 stimulator at a voltage

233 15% above threshold. In some cases, the working heart preparation of Neely et al. [27] was used. In these cases, hearts were paced at 300 beats/min at a voltage 15% above threshold. Preparation of heart tissues. For the determination of cyclic GMP kinase activity ratios, approx. 20-50 mg of frozen tissue were homogenized in 10 × 75 mm chilled plastic tubes with a motor-driven (Craftsman drill, Sears and Roebuck, Co.) Teflon pestle in 0.5 ml of ice-cold 10 mM potassium phosphate, 10 mM EDTA, 0.2 mM 3-isobutyl-l-methylxanthine, pH 7.0, for approx. 3-5 seconds. The tubes were placed in a chilled centrifuge rotor (such as a JA-20 Beckman rotor), and the rotor speed brought up to 10 000 rev./ min (approx. 30 seconds). The centrifugation was then quickly terminated, the tubes placed in ice, and the supernatants immediately assayed for cyclic GMP kinase activity. The processing procedure for six heart samples could be accomplished in under 5 min. Other methods. For some studies, bovine lung cyclic GMP kinase was purified to homogeneity as described previously [28]. Rat heart cyclic GMP kinase were partially purified through the Sepharose 6B chromatography step as described previously [22] except that 50 mM mercaptoethanol was used in all buffers. The enzymes were stored at 4°C. Protein kinase inhibitor was partially purified by the method of Schlender and Reimann [29]. The specific activity of the inhibitor preparation varied somewhat among preparations, but was usually capable of inhibiting 10000 units of catalytic subunit/mg protein. Protein was measured by the method of Lowry et al. [30]. [?-a2P]ATP was prepared by the method of Walseth and Johnson [31]. The eluted ATP was neutralized with 1 M Tris base, and made 1/,tM by the addition of nonradioactive ATP, yielding a final specific activity of approx. 1 • 106 cpm/pmol. Phosphodiesterase activity was determined by the method of Davis and Daly [321. Materials. [a2p] was from ICN or New England Nuclear, whereas cyclic [8-3H]GMP was purchased from Amersham-searle. DEAE-cellulose (DE-11) was from Whatman Co. Mixed historic (type IIA), F2b histone (type VII), arginine-rich histone (type VIII-S), acetylcholine chloride, sodium nitroprusside, and cyclic GMP were from Sigma Chemical Co., St. Louis, Me. Hf2 b histone was purchased from

Worthington Diagnostics, Freehold, NJ. 3-Isobutyl1-methylxanthine was a gift from Parke and Davis. All drug solutions were prepared fresh daffy. Results

Characterization of the cyclic GMP kinase assay. Because of the unusual kinetic properties of the cyclic GMP kinase, high concentrations of Mg~÷ and low concentrations of ATP were used in tissue extract assays. Takai et al. [33,34] originally observed that high concentrations of Mg2÷ stimulate cyclic GMP kinase from bovine cerebellum and silkworm when using histone as the substrate. This was due in part, at least, to a lowering of the Km of the enzyme for the ATP, and we confirmed these findings using the homogeneous cyclic GMP kinase from bovine lung [28]. Since a large amount of protein kinase activity in tissue extracts appeared to be due to the cyclic AMP kinase, it was necessary to inhibit this enzyme nearly completely. This was accomplished using a partially purified preparation of the cyclic AMP-dependent protein kinase inhibitor protein [35]. Cyclic AMP kinase activity was decreased about 99%, whereas cyclic GMP kinase activity was essentially unchanged when using partially purified inhibitor in the assay (Lincoln, T.M. and Keely, S.L., unpublished observations). If homogenous protein kinase inhibitor was also added to the cyclic GMP kinase assay to a final concentration of 10/.tM, no further effect was seen. Since this concentration of inhibitor inhibits the cyclic AMP kinase practically 100%, these data show that cyclic AMP kinase did not significantly contribute to histone phosphorylation in the cyclic GMP kinase assay. Under these conditions, the assay was linear with time up to 5 min and amount of protein up to 50/ag. Stimulation of the cyclic GMP kinase was specific for cyclic GMP (Ka = 20 rLM). Cyclic AMP was less effective (Ka = 800 nM). Essentially no stimulation of the cyclic GMP kinase in the extracts was observed at cyclic GMP concentrations below 5 nM. Tissue distribution of the cyclic GMP kinase and interference from other protein kinases. The protein kinase activity from several dissected tissues assayed under these conditions was found to be quite similar (60-80 U/mg protein in the presence of 2/,tM cyclic GMP). However, stimulation of protein kinase

234

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Fig. 1. DEAE-cellulose profiles of cyclic GMP kinase from tissue extracts. Approximately equal amounts of protein were applied to DEAE-ceUulose columns (0.9×4 cm) equilibrated with buffer A. The columns were washed with 10 vol. of buffer and eluted with a linear 0-0.4 M NaCI gradient as described in Experimental Procedure. 20 #1 of each fraction were assayed for cyclic GMP kinase activity in the absence (o e) or presence (× . . . . . . ×) of 2 ~tM cyclic GMP using F2 b histone (type VII). The arrows indicate the fraction at the beginning of the gradients.

activity was greatest in tissues containing large amounts o f cyclic GMP kinase (i.e., lung and heart). Activity ratios (-cyclic GMP/+cyclic GMP) varied from a low level o f 0.40 for lung to 0.94 for pancreas. This indicated the presence o f a cyclic GMPindependent protein kinase which was interfering in the assay for cyclic GMP-dependent protein kinase. In order to examine this possibility, the tissue extracts were chromatographed on DEAE-cellulose and the fractions were assayed in the presence and absence of 2/.tM cyclic GMP. The results are shown in Fig. 1. Two separate protein kinases were detected by the assay procedure: peak 1 was eluted at 0.12 M NaC1

and was stimulated by cyclic GMP; peak 2 was eluted at about 0.20 M NaC1 and was not affected by cyclic nucleotides. The highest cyclic GMP kinase activity was observed in lung and heart, while pancreas had the lowest. Peak2 from these tissues was not stimulated by cyclic AMP when using assay conditions designed to detect the cyclic AMP kinase, nor was it inhibited by the protein kinase inhibitor (Lincoln, T.M. and Keely, S.L., unpublished observations). These facts indicated that this enzyme was a cyclic nucleotide-independent protein kinase which had some characteristics similar to those o f the cyclic GMP kinase, and appeared to be identical to the protein kinase M initially described by Takai et al. [37]. It is interesting to note that the relative activity of peak 2 was similar for different tissues but that of peak 1 was quite variable. This supports previous work which indicates that the cyclic GMP kinase, like cyclic GMP itself, is tissue specific. Since two types of protein kinases were eluted from DEAE-cellulose, it was of interest to determine if the activity ratios o f the cyclic GMP kinase measured in the dissected tissue extracts could be accounted for by the presence o f these two kinases exclusively. The two peaks of enzyme activity were plotted on graph paper, and were separately cut out and weighed. The percentage o f cyclic GMP kinase activity was determined and the predicted activity ratio of the enzyme was calculated for each tissue, assuming that the cyclic GMP kinase expresses 10% of its activity in the absence o f cyclic GMP. Purified enzyme from which bound cyclic GMP has been removed is stimulated approx. 10-fold by cyclic GMP, indicating that the enzyme expresses 10% of its activity with histone as the substrate in the absence of cyclic GMP. When these values were compared to the actual activity ratios determined in the various tissue extracts, close agreement was seen using the two methods. Thus, the low activity ratios which were measured in the given tissue extracts were dependent on the relatively high concentration of cyclic GMP kinase which was present in the extract. The amount o f peak 2 present in many tissue extracts could have been due to the presence o f factors in the blood of the dissected tissues, since perfused tissues (heart for example) had lower activity ratios. It was not clear whether the independent protein kinase was derived from the formed elements in blood, or

235 TABLE I EFFECTS OF CHARCOALON THE CYCLICGMP KINASE ACTIVITY RATIOS IN THE PERFUSED RAT HEART Hearts were perfused by the Langandorff method in Expt. 1 and by the working method [27] in Expt. 2 with the indicated agents for 1 min. Cyclic GMP kinase was assayed using F2 b histone. Charcoal (Norite A) was added at the indicated concentrations to the buffer before homogenization. Experiment

Perfusateadditions

None None Acetylcholine (3/~M) Acetylcholine (3 t~M) Nitroprusside (25/~M) Nitroprusside (25 ~M) None None Acetylcholine (3 tiM) Acetylcholine (3 tzM) None None

Additions to • homogenization buffer

None Charcoal (2 mg/ml) None Charcoal(2 mg/ml) None Charcoal(2 mg/ml) None Charcoal (5 mg/ml) None Charcoal(5 mg/ml) cyclic GMP (0.2 t~M) cyclic GMP (0.2 ~M) + charcoal (5 mg/ml)

whether factors such as proteolytic enzymes were activating the independent kinase which was present in the dissected tissues. The possibility that bound cyclic GMP could also be contributing to the high cyclic GMP kinase activity ratios observed in some tissues such as liver, was tested by adding high amounts of charcoal (20 mg/ml) to the extract. It has previously been demonstrated that charcoal removes cyclic nucleotides from cyclic AMP kinases in tissue extracts [38], and from purified cyclic GMP kinase under some conditions [28]. This treatment had no effect on the high activity ratio of liver extracts (Lincoln, T.M. and Keely, S.L., unpublished observations). Furthermore, treatment of the liver extract with DEAE-cellulose in the presence of 0.13 M NaC1 in an attempt to remove selectively the peak 2 kinase resulted in a decrease in the activity ratio from 0.9 to 0.7. Thus, it was concluded that the high activity ratios in various tissues were due primarily to the presence of the peak 2 cyclic nucleotide-independent protein kinase. Finally, in an attempt to see if the bulk of cyclic GMP kinase was in subceUular fractions other than the soluble fractions of these tissues, a subceUular distribution study was performed in rat lung, liver and heart. In each case, over 95% of the

32p incorporation (epm)

Activity ratio (-cyclic GMP/+cyclic GMP)

-cyclic GMP

+cyclic GMP

3691 5943 4601 6706 6251 4580 3956 5845 6205 6908 7649

9 199 14 239 7 454 11 947 11 383 9 786 9 301 12 480 11 140 12 942 11 875

0.40 0.42 0.61 0.56 0.55 0.46 0.43 0.47 0.56 0.53 0.64

5608

12 260

0.46

enzyme activity was found in the 100 000 X g supernatant (Lincoln, T.M. and Keely, S.L., unpublished observations). Thus, the high background did not appear to be due to the absence of cyclic GMPstimulated activity in the soluble extracts.

Effects of tissue homogenization on the stability of the activity ratio. In order to study the effects of hormones and other agents on the cyclic GMP kinase in tissues, it was necessary to stabilize the equilibrium position of the active and inactive forms of cyclic GMP kinase in the tissue extract. Experiments were performed to determine: (i) if cyclic GMP dissociates from the enzyme during homogenization; (ii) whether sequestered cyclic GMP binds to the enzyme during homogenization; or (iii) if independent protein kinases are activated by test agents. Since separate regulatory and catalytic subunits of the cyclic GMP kinase do not normally exist [28,39], the inactivation of the enzyme would be dependent primarily on the rate of dissociation of cyclic GMP from the enzyme. As shown in Fig. 2A, when a 1 : 5 diluted lung extract was labelled by incubation with physiological concentrations of cyclic [3H]GMP and equilibrium was attained, the rate of dissociation of cyclic [aH]GMP from its binding site(s) after further

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Fig. 2. (A) Dissociation of cyclic GMP from the cyclic GMP

kinase. Rat lung was homogenized in 5 vol. (w/v) of 20 mM sodium phosphate/10 mM EDTA/0.2 mM 3-isobutyl-1methylxanthine, pH 7.0, and centrifuged at 10000 ×g for 20 min. The supernatant was made 5 • 10-s M with cyclic [3H]GMP ([3H]cGMP) and incubated for 30 min at 0-4°C. Aliquots (0.1 ml) were then removed and added to 0.9 ml of the homogenization buffer and incubated at 0-4°C for the various times indicated. At the end of the incubation time, the contents of the tubes were transferred to prewetted Millipore f'flters and free and bound cyclic [3H]GMP were separated. The percent of zero-time bound counts was determined and plotted against time of incubation at 0-4°C. Results shown are the average from two separate experiments. (B) Effect of dilution of heart extracts on the cyclic GMP kinase activity ratios. Rat hearts were perfused with or without 1 ,aM acetylcholine (ACh) for 1 min. At this time the hearts were processed as described in Experimental Procedure. To determine the activity ratio, approx. 50 mg of frozen tissue were homogenized in 0.5 ml of buffer A at 4°C and immediately centrifuged at 10 000 Xg for 1 min. The supernatants were diluted accordingly in buffer A and immediately assayed for protein kinase activity with and without 2 ~M cyclic GMP. Control hearts, (o. . . . . -e);acetylcholine-treated hearts, (o . . . . . . o).

dilution was relatively slow at 0 - 4 ° C . Hence, if the processing of tissue extracts was performed within 5 min, then only small amounts of cyclic GMP (less than 20%) would be liberated from the enzyme. These results were extended further in Fig. 2B. In this experiment, 1 gM acetylcholine was perfused to elevate tissue levels o f cyclic GMP approx. 3-fold over control values to 110 pmol/g wet wt. (data not shown). The activity ratio in this experiment was increased from a control value o f 0.40 to a stimulated level of 0.65. When the extracts were then diluted and quickly assayed, the activity ratios of the cyclic GMP kinase in hearts perfused in the presence or absence of 1 /.tM acetylcholine remained constant with dilution o f the extract. Thus, if cyclic GMP were

dissociating from the enzyme, it would have been expected that the activity ratio decreases, since the levels o f cyclic GMP would have been diluted below those necessary to observe activation. This indicates that there was little loss of bound cyclic GMP during the rapid tissue work-up, and suggests that theoretically, at least, binding of cyclic GMP was tight enough to the kinase to allow the estimation of the amount of cyclic GMP kinase present in the active form after tissue homogenization. The rapid tissue work-up approach has previously been used for the measurement of type II cyclic AMP-dependent protein kinase activity ratios in the perfused guineapig heart [3]. Another potential problem could be the binding of sequestered cyclic GMP to the enzyme during homogenization. Several different experiments were carried out to determine if the increases in the activity ratios during drug treatment were due to free cyclic GMP binding during homogenization. When hearts were perfused, each with acetylcholine and nitroprusside, two agents which increase cardiac cyclic GMP levels, the activity ratios were elevated above control values (Table I). When small amounts of charcoal were added to the homogenization medium to bind free cyclic GMP, the activity ratios of the acetylcholine-treated hearts dropped only slightly. With the nitroprusside-treated heart, however, the activity ratio was decreased from 0.53 to near-control values of 0.46. Control values were not significantly affected by the charcoal treatment. Thus, it appeared that free cyclic GMP could not account for activation of the cyclic GMP kinase with acetylcholine perfusion, although this could account for the increase in the activity ratio produced by nitroprusside. As discussed below, nitroprusside is able to increase cyclic GMP many-fold in heart, so that free cyclic GMP in the extract can reach levels capable o f activating the cyclic GMP kinase. Another experiment which was designed to remove endogenous cyclic GMP from extracts was performed. In this case, hearts were homogenized in the standard buffer containing the phosphodiesterase inhibitors 10 mM EDTA plus 0.2 mM 3-isobutyl-1methylxanthine, or in buffer lacking these ingredients but containing 10 mM Mg 2÷ and 2/.tM Ca 2÷ in order to enhance phosphodiesterase activity. Under the latter conditions, it was found that endogenous

237 TABLE II

TABLE III

EFFECT OF PHOSPHODIESTERASE INHIBITORS AND DIVALENT CATIONS ON CYCLIC GMP KINASE ACTIVITY RATIOS IN THE PERFUSED RAT HEART

EFFECTS OF EXOGENOUS cyclic GMP KINASE ADDITION ON cyclic GMP KINASE ACTIVITY RATIOS IN THE PERFUSED RAT HEART

Hearts were perfused for 1 min with saline or acetylcholine. Cyclic GMP kinase was assayed using mixed histone (type II-A) as the substrate. Endogenous cyclic GMP phosphodiesterase activity in the presence of 10 mM Mg2+ and 2 uM Ca2+ was 50 pmol GMP/min per extract at 0-4°C. In the presence of 10 mM EDTA and 0.2 mM 3-isobutyl-l-methylxanthine (IMX), cyclic GMP hydrolysis was undetectable. The results are the averages of two separate determinations.

Hearts were perfused using the working heart method [27] with the indicated agents for 1 min. Cyclic GMP kinase was assayed using mixed histone (type-IIA) as described in Experimental Procedure. Rat heart cyclic GMP kinase was partially purified through DEAE-cellulose chromatography, concentrated with 50% ammonium sulfate, and stored in buffer A. The f'mal specific activity was 440 U/ml with a basal activity ratio of 0.25. Hearts were homogenized without or with 44 exogenous cyclic GMP kinase added to extracts which contained approx. 40 U endogenous protein kinase using mixed histone (type II-A) as the substrate. Results are the averages of two separate determinations.

Perfusate additions

Homogenization buffer

Activity ratio (-cyclic GMP/ +cyclic GMP)

None Acetylcholine (1 t~M) None Acetylcholine (1 ~M)

EDTA + IMX EDTA + IMX Mg2÷ + Ca2+ Mg2÷+Ca2÷

0.42 0.57 0.47 0.66

cyclic GMP phosphodiesterase was capable of hydrolyzing 50 pmol/min per 0.5 ml extract at 0-4°C. With EDTA and 3-isobutyl-l-methylxanthine, however, cyclic GMP hydrolysis was undetectable. The rationale behind this approach was that if cyclic GMP were being released from its binding site on the kinase during homogenization, then the inclusion of phosphodiesterase inhibitors would prevent the hydrolysis o f cyclic GMP during tissue preparation and allow cyclic GMP to rebind to other binding sites. On the other hand, the inclusion o f Mg2+ and Ca 2÷ would favor cyclic GMP hydrolysis. Thus, if cyclic GMP binding was not preserved, the activity ratio would be expected to decrease in the presence of these cations during homogenization. As shown in Table II, however, the activity ratio o f cyclic GMP kinase was elevated by acetylcholine, and this activation was preserved even in the presence of the divalent cations. It can also be seen that the activity ratios for both control and acetylcholine-treated hearts were slightly increased with divalent cations. Perhaps this was due to an effect on the cyclic GMP kinase itself or on other cyclic GMPhndependent protein kinases. It would not appear to be an effect on cyclic GMP production, since the extracts were so dilute that GTP levels would have been insignificant.

Perfusate additions

Changes in homogenization buffer

Activity ratio (-cyclic GMP/ +cyclic GMP)

None Acetylcholine (1 I~M) Nitroprusside (25 I~M) Aeetylcholine (1 t~M) Nitroprusside (25 ~M)

None None None cGMPkinase eGMPkinase

0.43 0.57 0.58 0.42 0.52

A final experiment designed to test the possibility that cyclic GMP interacts with the cyclic GMP kinase during homogenization was performed. In this instance, partially purified cyclic GMP kinase from rat heart was added to the homogenization buffer. The rationale was that if sequestered cyclic GMP was released during homogenization, then both endogenous and exogenous cyclic GMP kinase would be activated. This approach was originally described by Palmer et al. [40] for the cyclic AMP kinase in perfused liver. As shown in Table III, the addition of the exogenous cyclic GMP kinase in an amount equal to that present in the extract lowered the activity ratio o f acetylcholine-treated hearts from 0.57 to 0.42. It would have been expected that if sequestered cyclic GMP were released during homogenization, then the added cyclic GMP kinase would also have been activated to an equal extent, thus partially preserving the elevated activity ratio. On the other hand, nitroprusside which can elevate cyclic GMP levels to a greater extent than acetylcholine, resulted in activation o f both the endogenous cyclic GMP kinase and the exogenous cyclic GMP kinase which

238 was added prior to homogenization. Although data obtained from this kind o f experiment are difficult to interpret due to the interference by the cyclic nucleotide-independent protein kinase and the unknown effects of other endogenous modulators of cyclic GMP kinase activity, they do support the conclusions drawn from the charcoal and phosphodiesterase experiments. Thus, it is important to dilute the extracts adequately upon homogenization when cyclic GMP levels are markedly elevated over basal levels, since artifactual activation of the enzyme can occur during homogenization.

Effects of acetylcholine on cyclic GMP-dependent and -independent protein kinases in the perfused rat heart. The cyclic GMP kinase activity ratio for the perfused heart was lower than that o f the dissected tissue, and this appeared to be due to the relatively small contribution of the cyclic nucleotideindependent protein kinase in the perfused heart, as indicated by DEAE-cellulose profiles o f the perfused tissue (approx. 5 0 - 7 5 % of the basal activity) (Lincoln, T.M. and Keely, S.L., unpublished observations). Furthermore, we could not demonstrate an increase in the peak 2 activity in hearts perfused with acetylcholine after separating the two kinases on DEAE-cellulose (Lincoln, T.M. and Keely, S.L., unpublished observations). This indicated that the increase in the activity ratio by acetylcholine was due primarily to activation of the cyclic GMP kinase, since proteolytic activation o f the peak 2 enzyme [37] was not apparent. This does not rule out the possibility, however, that peak 2 could have been activated by transient mechanisms which were not

detected after DEAE-cellulose chromatography. In order to examine further this possibility, hearts were perfused with saline or acetylcholine, and the extracts were treated with a specific cyclic GMP kinase antibody to inhibit the enzyme. The results of this experiment are shown in Table IV. The addition of the cyclic GMP kinase antibody effectively prevented 8 0 - 9 0 % of the cyclic GMP-stimulatable kinase activity in both control and drug-treated hearts. Total activity between the two preimmune serum controls was unchanged. In addition, protein kinase activity which remained after the addition o f the cyclic GMP kinase antibody was not increased in the acetylcholine-treated hearts (control = 38 -+6.6 U/mg protein; acetylcholine = 30 -+6.4 U/mg protein). These results suggest that the stimulation of protein kinase activity by acetylcholine was due to the stimulation o f the cyclic GMP-dependent enzyme. The correlation between cyclic GMP levels and cyclic GMP kinase activity in the perfused rat heart is shown in Fig. 3. Acetylcholine increased the level of cyclic GMP about 3-fold in rat heart, while the activity ratio was increased from 0.40 to 0.65. Since it was determined that about 5 0 - 7 5 % of the basal activity measured under these conditions appeared to be due to the peak 2 kinase in the perfused hearts, the true -cyclic GMP/+cyclic GMP activity ratio under basal conditions was probably closer to 0.2. This is indicated by the brackets in Fig. 3. Thus, in reality, there was an approx. 2-fold increase in the cyclic GMP kinase activity ratio in hearts perfused with acetylcholine. Although this correlated reasonably well with the increase in cyclic GMP

TABLE IV EFFECT OF cGMP KINASE ANTIBODIES ON PROTEIN KINASE ACTIVITIES IN THE PERFUSED RAT HEART Hearts were perfused using the working heart method [27] for 1 min with the agents above and processed as described in Experimental Procedure. Kinase activity was assayed using F2 b histone as the substrate. Antibody was added to a final dilution of 1 : 100 in the homogenization medium. Results are means ± S.E.M. of four determinations. Perfusate additions

Additions to homogenizing medium

Activity ratio (-cyclic GMP/ +cyclic GMP)

Total protein kinase activity (U/mg protein)

None None Acetylcholine (3/~M) Acetylcholine (3 ~M)

Preimmune serum Anti-cGMP kinase serum Preimmune serum Anti-cGMPkinase serum

0.41 0.86 0.56 0.89

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Fig. 3. Effects of acetylcholine on cyclic GMP (cGMP) and cyclic GMP ldnase activity ratios in the perfused rat heart. Hearts were perfused with or without 3/~M acetyleholine for 1 min, and processed as described in Experimental

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Fig. 4. Concentration dependence of acetylcholine and nit~oprusside. Hearts were perfused for 15 min with buffer alone and for an additional minute with buffer containing the individual concentration of acetyleholine (ACh) or sodium nitroprusside (SNP). Each point in panels A, C, and D represents the mean ± S.E. of eight to ten hearts; data in panel B were taken from three hearts; ACh, • =; SNP, o . . . . . . o.

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rat heart. Force measurements and freezing for subsequent assays were done after 60 s o f a steady rate o f infusion of either agent. As shown in panel A, nitroprusside at concentrations greater than 1 #M was capable o f elevating the levels o f cyclic GMP higher than could acetylcholine. At 0.1 ~M, acetylcholine produced a 2-fold increase in cyclic GMP and gave a significant increase in the cyclic GMP kinase activity ratio. As this agent produced further increases in cyclic GMP, a dose-dependent increase in the proteiia kinase activity ratio was observed. In panel C, nitroprusside, while increasing heart cyclic GMP to a much higher level than acetylcholine, was not capable of activating the cyclic GMP kinase except at very high concentrations. The data in Tables I and III suggested that the increased activity seen when hearts were treated with large doses of nitroprusside was probably due to activation o f the enzyme upon homogenization. On the other hand, it is possible that part of this small activation was due to compartmentalization of a small amount of protein kinase with the cyclic GMP produced by nitroprusside. Total protein kinase activity was not significantly increased with acetylcholine, demonstrating that only the cyclic GMP kinase was affected by the agent. Panel D in Fig. 4 shows the effects of acetylcholine and nitroprusside on contractile force in the perfused heart. AS we and others reported earlier [ 1 7 - 1 9 ] , nitroprusside had little or no effect

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Fig. 6. Additive effects of acetylcholine and nitroprusside. Hearts were perfused for 15 min with buffer alone and for an additional 1 min with buffer containing no additions (C), 10 /~M nitroprusside (NP), 1 ~M aeetyleholine (Ach), 10 ~M nitroprusside and 1 ~M acetylcholine (NP + Ach). Each point represents the mean ± S.E. of six hearts, cGMP, cyclic GMP. on force of contraction. Acetylcholine, however, produced a dose-dependent decrease in force, with significant decreases observed at 0 . 0 3 - 0 . 1 0 /.tM. As shown in Fig. 5, a good correlation between contractile force and cyclic GMP kinase activation was seen at doses between 0.03 and 3.0/.tM acetylcholine (n = 44, r = - 0 . 7 1 ) .

Additive effects of acetylcholine and nitroprusside. Fig. 6 shows the effects o f treating hearts with 1 /.tM acetylcholine alone or in combination with 10 ~M nitroprusside. When either agent was used alone, results similar to those described earlier were observed. Acetylcholine produced a 2-fold increase in cyclic GMP levels and protein kinase activity, while nitroprusside generated much higher cyclic GMP

241 levels, but had little or no effect on the cyclic GMP kinase or contractile force. When the two agents were perfused together, additive effects on cyclic GMP levels were seen. Even though very high cyclic GMP levels were generated using both agents, the increase in the activity ratio and the decrease in force of contraction were similar to those produced by acetylcholine when infused separately. These observations eliminated the possibility that nitroprusside was having two actions: one to increase cyclic GMP, and a second to prevent or reverse the activation of the cyclic GMP kinase by cyclic GMP. Effects of acetylcholine and nitroprusside on cyclic AMP and cyclic AMP-dependent protein kinase. As reported earlier [9,14,19], acetylcholine alone had no effect on cyclic AMP levels or on the cyclic AMP kinase activity ratio in the perfused heart. Similarly, nitroprusside was without effect on these parameters, thus confirming earlier work [ 17,18]. Discussion

The accurate detection of cyclic GMP kinase activity in crude tissue extracts in order to study activity ratios has been met with some difficulty by several investigators. On the other hand, measurement of the enzyme activity after preliminary purification (i.e., gel filtration or ion-exchange chromatography) has been successfully performed in many laboratories. The difficulty using crude extracts appears to stem from several causes: (i) the concentration of the cyclic GMP kinase is low compared to that of the cyclic AMP kinase concentrations; (ii) the specific activity of the cyclic GMP kinase using histone as the substrate is somewhat lower than that of the cyclic AMP kinase; (iii) cyclic nucleotide-independent kinases contribute to total enzyme activity measured in tissue extracts. Thus, the assay conditions used for the measurement of cyclic AMP kinase cannot be used for cyclic GMP kinase activity in crude extracts. We have used an adaptation of the assay initially described by Takai et al. [33] for assaying cyclic GMP kinase activity in crude extracts, since under the conditions of low ATP and high Mg2÷ concentrations, (i) most protein kinases including the cyclic AMP kinase are less active at high concentrations of Mg2÷, and (ii) the ATP concentration is below the Km for ATPases and several other kinases although it is well

above that for the cyclic GMP-dependent enzyme [28,39]. Even under these conditions, however, activity ratios of cyclic GMP kinase in some tissues are high. This appears to be due to the presence of a cyclic nucleotide-independent protein kinase which appears to be quite similar to the cyclic GMP kinase as far as substrate specificity and Mg2÷ stimulation are concerned. This enzyme is distributed rather evenly in dissected tissues (Fig. 1) although it is lower in perfused heart compared to dissected heart. Furthermore, several dissected tissues (liver and pancreas, for example) have only trace amounts of cyclic GMP kinase, and the large background of the independent kinase interferes with the assay of the cyclic GMP kinase. Improvement in the assay method could be realized if a specific inhibitor of the independent kinase were found. In contrast to the cyclic AMP kinase [38], different isozymic forms of cyclic GMP kinase have thus far not been found. It is still possible that the assay conditions for cyclic GMP kinase might have to be altered from one tissue to another because of the presence of varying independent kinases and other problems. Several of these problems may also be overcome using more homogeneous preparations of cells from tissues. For example, Kuo [42] has reported that the specific activity of cyclic GMP kinase in pancreatic islets is very high (comparable to lung). The bulk of tissue, i.e., pancreatic acinar tissue, would appear to be a poor source of cyclic GMP kinase. Therefore, it seems clear that the usefulness of studying cyclic GMP kinase activity ratios will be more limited than that of the cyclic AMP kinase because of the more limited distribution of the cyclic GMP kinase. Hormonal stimulation of cyclic AMP kinase activity correlates well with cyclic AMP production in several tissues, and likewise, hormonal stimulation of cyclic GMP kinase correlates reasonably well with cyclic GMP production in the perfused rat heart. However, a major problem encountered when studying the hormonal regulation of cyclic nucleotide-dependent protein kinases in tissue extracts is the preservation of the activation state of the kinase. For the cyclic AMP kinase, this has been accomplished by stabilizing the amount of dissociated catalytic subunit using salt or high dilution [38]. Thus, dissociation of cyclic AMP from the regulatory subunit is not an important factor in this assay. The cyclic GMP kinase

242 is a different case, however. Unlike the assay for cyclic AMP kinase activation (i.e., the assay for flee catalytic subunit), the cyclic GMP kinase activation assay is essentially an assay for enzyme-cyclic GMP complex. The relative amounts of activated and nonactivated cyclic GMP kinase are maintained primarily by the slow dissociation rate of cyclic GMP from the cyclic GMP kinase at 0-4°C. McCune and Gill [41] and Corbin and Rannels (personal communication) have found that the dissociation of cyclic GMP from the pure enzyme is slowed considerably at low temperatures, and the data in Fig. 2A with the crude extracts are in agreement with these findings. Thus, the speed with which homogenization and centrifugation can be performed is a limiting step in the assay. Normally, four to six hearts can be processed for assay within 5 min. During this time, small amounts of cyclic GMP dissociate from the enzyme so that a small amount of error is introduced to the assay. However, the reliability of the assay is evidenced by the stability of the cyclic GMP kinase activity ratio after dilution of extracts of hearts perfused with acetylcholine (Fig. 2B). If the assay were measuring unbound cyclic GMP, then dilution of the extract would result in a lowering of the activity ratio of the enzyme, since free cyclic GMP would be diluted below the concentration needed to activate the cyclic GMP kinase. The addition of charcoal to bind free cyclic GMP during homogenization does not prevent activation of the enzyme by acetylcholine (Table I). Furthermore, exogenous cyclic GMP kinase added to the extract does not pick up free cyclic GMP during homogenization (Table III). These experiments indicate that cyclic GMP does not appear to be activating the enzyme during homogenization when steps are taken to use appropriate tissue-buffer dilutions. With nitroprusside-treated hearts this is particularly important, since cyclic GMP levels can be raised several-fold over basal values. As with the measurement of any enzyme in broken cell preparations, one cannot be sure that the activation which one observes with acetylcholine is a true reflection of what has occurred in situ. Presumably, other factors and modulators besides cyclic GMP could be affecting cyclic GMP kinase activity, and these may not be preserved during extract preparations. Thus, data from experiments done in this fashion should be interpreted with the usual

amount of caution, since what is being measured may not be a true reflection of regulation in vivo. Even with this reservation, however, this is the first time that the amount of cyclic GMP-cyclic GMP kinase complex in a particular tissue has been estimated biochemically. Presumably, it is this pool of cyclic GMP which is the physiologically important one for cyclic GMP action in this tissue. Using this methodology for the heart, we have examined changes in the activity ratio of the cyclic GMP kinase with acetylcholine and nitroprusside. Acetylcholine, at concentrations which produce a decrease in contractile force, produces a significant increase in activity of the heart cyclic GMP kinase. While the basal activity ratio was 0.40, much of this background activity was due to a cyclic GMPindependent protein kinase (Fig. 3). If the estimated amount of the cyclic GMP-independent kinase activity is substracted, this would represent an approx. 2-fold increase in cyclic GMP kinase activity. In the same hearts, acetylcholine increased cyclic GMP levels 2-3-fold. There is, therefore, a qualitative correlation between the ability of acetylcholine to increase cyclic GMP and to activate the cyclic GMP kinase in heart tissues. Unlike the cyclic AMP kinase activity ratio, however, the cyclic GMP kinase activity ratio does not seem to be more sensitive, or even as sensitive a measure of cyclic GMP levels in heart tissue. Sodium nitroprusside, while capable of producing much larger increases in cyclic GMP than acetylcholine, does not activate the cyclic GMP kinase. A slight increase in kinase activity was detected in some hearts which had been treated with large doses of nitroprusside. This increase in kinase activity was most likely due to interaction of the cyclic nucleotide and enzyme after tissue homogenization. Thus, the activity ratio determination may be an important measurement of specific hormone-stimulated pools of cyclic GMP, such as those changing during intracellular translocation [21] or within specific cell types [43]. It was also observed that nitroprusside had no effect on the ability of acetylcholine to activate the cyclic GMP kinase, or to decrease contractile force. In hearts treated with both acetylcholine and nitroprusside, the values for the increases in the protein kinase activity ratio and the decrease in contractile force were similar to those seen in hearts

243 treated with acetylcholine alone (Fig. 6). Nitroprusside, therefore, does not appear to have an inhibitory action on the heart distal to the production of cyclic GMP. Several hypotheses may be formulated to explain why the nitroprusside-produced cyclic GMP is not coupled to cyclic GMP kinase activation. For example, the cyclic GMP produced by nitroprusside could be an altered form of cyclic GMP incapable of interacting with the kinase. This idea follows from the studies where it was shown that guanylate cyclase is somewhat nonspecific, and can produce 2-deoxycyclic GMP from 2-deoxy GTP [44] or cyclic AMP from ATP [45]. However, since we measured cyclic GMP levels using a cyclic GMP kinase competitivebinding method, then it is not clear why cyclic GMP would bind to the protein kinase in the binding assay but not in the protein kinase assay. Second, cellular compartmentalization of cyclic GMP and protein kinase could exist so that those pools of cyclic GMP produced by nitroprusside cannot be coupled to protein kinase activation. This could be due to a specific localization of the enzyme within the cell or perhaps to the inability of nitroprusside to affect a secondary process necessary for kinase activation. Finally, it is possible that the guanylate cyclases which are stimulated by nitroprusside and acetylcholine are located in different cell types in the heart, such that only acetylcholine stimulates the one compartmentalized with the cyclic GMP kinase. This idea seems particularly attractive, since the particulate guanylate cyclase is reported to be less sensitive to nitroprusside stimulation than the soluble form [46-48]. Thus, the particulate muscarinic receptor and the particulate guanylate cyclase, which is quite abundant in heart [49], could be localized primarily in myocytes, while soluble cyclase could be localized in other cell types. It is not unreasonable to suggest that the cyclic GMP kinase could be localized primarily in myocytes, since it is already known that cyclic GMP kinase has a restricted distribution in many mammalian organs and tissues [50]. The idea of intercellular compartmentalization is also compatible with the findings that nitroprusside and acetylcholine are additive with respect to cyclic GMP levels but not to kinase activation and contractile force. It is apparent, therefore, that the measurement of total cyclic GMP levels would be less meaningful

than the measurement of the physiologically relevant pool of cyclic GMP in myocytes. The role of cyclic GMP and the cyclic GMP kinase in cardiac function is obscure. Although good correlations between cyclic GMP kinase activation and the negative inotropic response to acetylcholine have been observed (Fig. 5), no direct data thus far implicate a role for cyclic GMP in mediating acetylcholine action in the heart. In fact, a significant body of evidence argues against this possibility. Brooker [20] has found that levels of cyclic GMP produced by carbachol do not correlate with the decrease in force of contraction in guinea-pig atrium with the drug; Diamond et al. [17] have obtained similar results with cat atrium. Acetylcholine and 8-bromo-cyclic GMP produce different ion fluxes in heart preparations [51]. Also, Ca2+-antagonist drugs such as verapamil inhibit contractile force without raising cyclic GMP levels [52]. Inhibitors of cyclic GMP hydrolysis are usually associated with positive inotropic effects [53,54]. However, it seems clear that agents such as nitroprusside which elevate cyclic GMP levels in heart but do not produce physiological and metabolic actions similar to acetylcholine cannot be used to dissociate cyclic GMP levels from these events since this agent, at least, is not capable of coupling cyclic GMP production to protein kinase activation. Acknowledgments We are grateful to Ms. Shelia Shay for skillful technical assistance. We are also indebted to Drs. C.G. and W.R. Ingebretsen and Ms. Charlyn HaweluJohnson from the University of South Carolina for the working heart samples used in some of these studies. We are also grateful to Dr. C.W. Davis from the University of South Carolina for performing the phosphodiesterase assays. We wish to thank Dr. Edmund Fischer, University of Washington, Seattle, WA, for his gift of purified protein kinase inhibitor and Drs. Penelope Miller and Paul Greengard, Yale University, New Haven, CT, for the cyclic GMP kinase antibody used in these studies. We are grateful to Dr. Jackie D. Corbin from Vanderbilt University for helpful discussions and guidance throughout much of this study. This research was supported by Program Project Grant AM7462, Research Grant

244 HL19181 to S.L.K. from the National Institutes of Health, and Research Grant PCM 8021205 to T.M.L. from the National Science Foundation. References 1 Keely, S.L., Corbin, J.D. and Park, C.R. (1975) J. Biol. Chem. 250, 4832-4840 2 Keely, S.L. and Corbin, J.D. (1977) Am. J. Physiol. 233, H269-H275 3 Keely, S.L. and Eiring, A. (1979) Am. J. Physiol. 236, H84-H91 4 Dobson, J.G. (1978) Am. J. Physiol. 234, H638-H645 5 Jesmok, G.J., Calbert, D.N. and Lech, J.J. (1976) J. Pharmacol. Exp. Ther. 200,187-194 6 Hayes, J.S., Brunton, L.L., Brown, J.H., Reese, J.P. and Mayer, S.E. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1570-1574 7 Kirchberger, M.A., Tada, M. and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173 8 Morkin, E. and LaRaia, P.J. (1974) N. Engl. J. Med. 290, 445-451 9 Keely, S.L., Lincoln, T.M. and Corbin, J.D. (1978) Am. J. Physiol. 234, H432-H438 10 Goldberg, N.D., Dietz, S.B. and O'Toole, A.G. (1969) J. Biol. Chem. 244, 4458-4466 11 George, W.J., Poison, J.B., O'Toole, A.G. and Goldberg, N.D. (1970) Proc. Natl. Acad. Sci. U.S.A. 66,398-403 12 George, W.J., Wilkerson, R.D. and Kadowitz, P.J. (1973) J. Pharmacol. Exp. Ther. 184,228-235 13 Watanabe, A.M. and Besch, H.R. (1975) Circ. Res. 37, 309-317 14 Gardner, R.M. and Allen, D.O. (1976) J. Cyclic Nucleotide Res. 2,171-178 15 Nawrath, H. (1976) Nature 262,509-511 16 Trautwein, W. and Trube, G. (1976) Pfliigers Arch. 366, 293 -295 17 Diamond, J., TenEich, R.E. and Trapani, A.M. (1977) Biochem. Biophys. Res. Commun. 79,912-918 18 Katsuki, S., Arnold, W. and Murad, F. (1977) J. Cyclic Nucleotide Res. 3,239-247 19 Keely, S.L. and Lincoln, T.M. (1978) Biochim. Biophys. Acta 543,251-257 20 Brooker, G. (1977) J. Cyclic Nucleotide Res. 3,407-413 21 Steiner, Koide, Y., Earp, H.S., Bechtel, P.J. and Beavo, J.A. (1978) Adv. Cyclic Nucleotide Res. 8,691-705 22 Lincoln, T.M., Hall, C.L., Park, C.R. and Corbin, J.D. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2559-2563 23 Flockerzi, V., Speichermann, N. and Hofmann, F. (1978) J. Biol. Chem. 253, 3395-3399 24 Corbin, J.D. and Reimann, E.M. (1975) Methods Enzymol. 38,287-290 25 Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312 26 Jakobs, K.H., Bohme, E. and Schultz, G. (1975) Acta Endocrinol. 80, Suppl. 199,431

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