2111.full

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 257. No. 4, Issue of February 25. pp. 2111-2120,1982

Effects of Cholinergic and Adrenergic Agonists on Phosphorylation of a 165,000-dalton Myofibrillar Protein in Intact Cardiac Muscle*

(Received for publication, January 15, 1981, and in revised form, September 22, 1981)

H. Criss Hartzell and Louisa Titus With the technical assistance of Jan Zwemer-Collins From the Department of Anatomy, Emory University School of Medicine, Atlanta, Georgia 30322

“he purpose of this investigation was to examine the effects of &adrenergic and muscarinic cholinergic agonists on protein phosphorylation in intact frog atrium. 8-Adrenergic agonists increase and muscarinic agonists decrease “P incorporation into a 165,000-dalton (165K) protein within less than 1 min. The concentrations of isoproterenol that produce increases in 32P incorporation into the 165K protein and in systolic tension are similar. Further, the changes in 32P incorporation and tension produced by isoproterenol occur with similar time courses. Carbamylcholine decreases tension somewhat more quickly and at lower concentrations than it decreases 32P incorporation, however. Isoproterenol-stimulated 32P incorporation is thought to be mediated by CAMP-dependent protein kinase because bath application of dibutyryl CAMP, cholera toxin, or phosphodiesterase inhibitors increase 32P incorporation into the 165K protein in intact atria. When heart homogenates are incubated in the presence of [y3*P]ATP, cAMP stimulates the incorporation of 32P into the 165K protein. cGMP is much less effective. We suggest that carbamylcholine decreases 32P incorporation into the 165K protein by a mechanism independent of cAMP levels because carbamylcholine inhibits the stimulation of 32P incorporation into the 165K band produced by 8-bromo cAMP in intact cells. Phospho- rylation of the 165K protein occurs in cardiac muscle but not in other tissues. We hypothesize that the 165K protein is C-protein, because the 165K- and C-proteins have similar solubilities and are associated with the myofibril. Further, antibodies produced against the 165K protein bind to C-protein purified from rabbit heart and also bind to the same region of the myofibril where C-protein is found.

The autonomic nervous system neurotransmitters, noradrenaline and acetylcholine, have opposite effects on the heart. Noradrenaline increases heart rate and contractile force, while ACh’ reduces rate and force and also inhibits the

* Supported by Grants R01-HL21195 and R01-HL25090 from the National Institutes of Health, an award from the Emory University McCandless Fund, and National Institutes of Health Research Career Development Award K04-HL00435. A preliminary account of this work was presented at the Eleventh Annual Meeting of the American Society of Neurochemistry, March 4, 1980 (Trans. Am. SOC. Neuro- chem. (1980) 11, 119). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‘ The abbreviations used are: ACh, acetylcholine; CCh, carbamylcholine; SDS, sodium dodecyl sulfate; Hepes, 4-(2-hydroxyethyl)-I- piperazineethanesulfonic acid, Pipes, 1,4-piperazinediethanesulfonic acid; EGTA, ethylene glycol bis(P-aminoethyl ether)-N,N,N‘,N’- tetracetic acid; 165K, 165,000-dalton; db, dibutyryl.

stimulatory effects of noradrenaline. The response of the myocardium to adrenergic agents is thought to be mediated by the adenylate cyclase-CAMP-protein kinase system (see reviews by Entmann, 1974; Tsien, 1977). Although it is clear that /&adrenergic agonists increase cAMP levels and protein kinase activity in heart tissue, little is known about the target proteins whose phosphorylation may effectuate the action of

agonists on heart beat. It has been suggested that p agonists may regulate heart beat by phosphorylation of (i) sarcolemmal proteins, including the ionic channels that mediate electrical activity (Walsh et al., 1979; Jones et al., 1979), (ii) a sarco- plasmic reticulum protein, phospholamban, involved in calcium sequestration and release (Tada et al., 1979,1974; LaRaia and Morkin, 1974), and (iii) proteins of the contractile apparatus such as troponin and myosin light chain (see reviews by Stull, 1980; Adelstein and Eisenberg, 1980).

The mechanism of action of ACh is less well understood than the response to adrenergic agents. Because ACh increases cGMP and decreases cAMP in the heart, it has been suggested that some of the effects of ACh are mediated by activation of the guanylate cyclase-cGMP-protein kinase system (George et al., 1970; Lee et al., 1972; Watanabe and Besch, 1975; Gardner and Allen, 1976a, 1976b; but compare: Brooker, 1977; Nawrath, 1977; Diamond et al., 1977; Taniguchi et al., 1979; M h o et al., 1979; Linden and Brooker, 1979) or by inactivation of the adenylate cyclase system (Murad et al., 1962; LaRaia and Sonnenblick, 1971; Gardner and Allen, 1977; Keeley et al., 1978; Jakobs et al., 1979). It is not known whether the opposite physiological effects of ACh and noradrenaline are due to the reversible phosphorylation-dephosphorylation of the same target phosphoproteins.

Our approach for investigating the mechanisms of action of ACh and noradrenaline was to ask whether we could identify a phosphoprotein whose incorporation of 32P was affected in opposite directions by cholinergic and adrenergic agonists in intact atria. We found that /3-adrenergic agonists stimulate the CAMP-dependent phosphorylation of a 165,000-dalton phosphoprotein localized in the contractile apparatus. Cholin- ergic agonists cause a decrease in the phosphorylation of this protein. The effect of cholinergic agonists is most pronounced when phosphorylation of the 165K protein is previously en- hanced by adrenergic agonists. We present evidence that this protein is C-protein (Offer et al., 1973), a protein found in the region of overlap between thick and thin filaments in the contractile apparatus (Offer, 1972; Rome, 1972; Moos, 1972; Pepe and Drucker, 1975). This location suggests C-protein may play a role in regulation of cardiac muscle contraction.

EXPERIMENTAL PROCEDURES

Materials and Solutions-Catalytic subunit of CAMP-dependent protein kinase (prepared by the method of Bechtel et al., 1977; specific activity, 15 pmol of 32P transferred from 32P-ATP to histone H2B/

2111

2112 Phosphorylation in Intact Cardiac Muscle

min/mg) and purified cGMP-dependent protein kinase (prepared by the method of Glass and Krebs, 1979; specific activity, 5-6 pmol of 32P/min/mg; activity ratio, 2.5) were the generous gifts of Dr. David Glass, Department of Pharmacology, Emory University. Quinuclidi- nyl benzilate and R07-2956 were gifts from Hoffman-LaRoche (Nut- ley, NJ). Purified rabbit heart C-protein was kindly provided by Dr. Carl Moos, State University of New York, Stony Brook. Normal frog Ringer solution contained 115 m~ NaC1, 4 mM KCl, 2 II~M CaClz, 1 mM MgC12, and 10 mM Na-Hepes, pH 7.4. Mn-Ringer solution contained 105 mM NaC1,2 m KC1, 1 mM CaC12,4 m MnC12, and 1 mM Na-Hepes, pH 6.8. HCOs-Ringer solution contained 98 m NaCl, 25 m NaHC03, 4.3 mM KCl, 0.7 mM MgClz, and 1.4 mM CaC12. HC03- Ringer was gassed with 5% c o ~ / 9 5 % air.

Incubation of Frog Atria-The relative incorporation of 32P into proteins was studied in isolated frog atria incubated with 32P in vitro. Atria were dissected in normal frog Ringer solution, pinned onto dishes coated with cured Sylgard resin (Dow-Corning, Midland, MI), and allowed to recover from the dissection for 30 min. Atria were then incubated in Ringer containing 100 pCi/ml of 32P (orthophosphate) for 3 h a t 20-22 "C. Experiments were done in two different Ringer solutions: HCOs-Ringer, in which atria were beating spontaneously, and Mn-Ringer, which was used to stop spontaneous beating. At the end of the '*P incubation, the tissue was rinsed thoroughly and placed into Ringer containing drugs (ACh, isoproterenol, etc). The incubation was terminated by two methods. Method A: the atrium was homogenized in 2 ml of 1% SDS, 1 n w EDTA, 10 mM NaF, and 5 mM Tris-HC1, pH 9.3 (SDS-homogenization buffer). Method B: the atrium was frozen with clamps cooled with liquid nitrogen. The frozen atrium was homogenized in 2 ml of 10 mM EDTA, 15.3 mM NaHaP04, 135 mM Na2HPO4 (EDTA-PO4) at 4 "C. An aliquot was made to 1.3% SDS, 3% 2-mercaptoethanol. In both methods, homogenization was carried out for 3 s in a Polytron homogenizer (Brinkmann Instru- ments, Westbury, NY) with a PT-10 probe at a speed setting of 3-4. The homogenates were often concentrated by lyophilization or ultra- fdtration prior to SDS-polyacrylamide gel electrophoresis.

' To determine whether protein phosphorylation occurred during homogenization, an atrium which had not been exposed to 3zP was homogenized in buffer that contained 0.1 mCi/ml of [y-"PIATP (specific activity, 3846 cpm/pmol; final concentration, 26 p ~ ) . No 32P was incorporated into proteins under these conditions. Phosphatase activity during homogenization was evaluated by measuring changes in "P content of 32P-labeled proteins added to the homogenization medium. Washed myofibrils were prepared and labeled with '*P by incubation in 20 mM Na-Hepes buffer, 2 mM MgCL, 2 pg/ml of catalytic subunit of CAMP-dependent protein kinase, and 20 p~ [y- "PIATP. The reaction was stopped by addition of 10 mM EDTA. The major protein incorporating "P was the 165K band (Fig. 11). The 32P- labeled proteins were dialyzed to remove ATP and added to the homogenization buffer. No change in 32P content of the 165K protein or other proteins was observed during homogenization. Further, the content of "P in the 165K protein did not change with time (up to 12 h at 4 "C) of storage in EDTA-PO4 buffer or in SDS.

Phosphorylation of Homogenates-Atria or whole hearts were homogenized in 25 mM Tris-HC1, 0.3 M sucrose, 5 mM MgC12, 1 mM EGTA, and M isobutylmethyl xanthine, (Tris-IMBX buffer) pH 7.5, in the Polytron. The final reaction (50-100 pl) contained -1 mg/ ml of protein, 2 mM MgC12, 5 X M isobutylmethyl xanthine, 0.5 mM EGTA, 100 p~ [Y-~~PIATP (specific activity, 600-5000 cpm/ pmol), 25 mM Tris-HC1, pH 7.5, and other additions as noted. The reaction was initiated by addition of homogenate, carried out at 4 "C for 15 s, and terminated by addition of 1/5 volume of 10% SDS, 10% 2-mercaptoethanol.

Preparation of (y-3ZPjATP-[y-32P]ATP was prepared by the method of Glynn and Chappell (1964). The specific activities of the [3zP]ATP, determined by optical density a t 259 nm and Cherenkov radiation in water, were between 2500-5500 cpm/pmol. Purity of the ["PIATP was greater than 98%, as determined by thin layer chromatography on polyethyleneimine cellulose in 2 M sodium formate, pH 3.6, or in 0.85 M KH2P04, pH 3.4.

Electrophoresis-SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (1970) and Maize1 (1971). The slab gel was 0.75 mm thick and composed of a 3% acrylamide stacking gel (20 mm) and a 5 or 8% resolving gel (100 mm). Acry1amide:bisacryl- amide ratio was 37.5:l. Electrophoresis was carried out overnight a t 2 mA/gel. Stained and dried gels were placed in contact with Kodak X-Omat R x-ray film for autoradiography. Chemography of the film was eliminated by placing a layer of 0.5-ml polyethylene sheet between the fim and gel. Autoradiograms were exposed at room tem-

perature for 1-7 days and developed in a Kodak R P X-Omat proces- sor.

Characterization of the Phosphorylated Bands-The chemical nature of the phosphorylated bands was determined by several extraction and enzymatic treatments. Hearts were homogenized in 2 ml of 25 mM morpholinopropanesulfonic acid, 5 m MgC12, pH 7. Ali- quots of 200 pl were pipetted into tubes containing 1 CAMP and 200 pM [y-3*P]ATP. Phosphorylation was carried out for 15 s a t room temperature and the reaction was terminated by addition of 200 p1 of 20% trichloroacetic acid. The trichloroacetic acid-precipitated proteins were collected by centrifugation (1100 X g, 10 min) and treated with 10 pg of pronase or 20 pg of ribonuclease A, extracted with chloroform:methanol (2:1), or exposed to acid, base, or acidic hydroxylamine, as described by Krueger et al. (1977).

Cell ATP-ATP was purified from KOH-neutralized perchloric acid extracts of cells by thin layer chromatography on polyethyleneimine cellulose in 0.85 M KHzP04, pH 3.4 (Cashel et al., 1969). ATP spots, identified by autoradiography and the position of standards, were scraped and washed with 0.02 M ammonium bicarbonate. ATP was eluted with 2 M ammonium bicarbonate, concentrated by lyophilization, and quantified by the luciferin-luciferase assay (Stanley and Williams, 1969). 32P was measured by Cherenkov radiation in water. The specific activity of the [y-3ZP]ATP was assumed to be one-half of the total specific activity of [32P]ATP. In both erythrocytes and liver, extracellular "P equilibrates with the p- and y-phosphates of ATP (Niehaus and Hammerstedt, 1976; Mayer and Krebs, 1970). Also, in control experiments, the specific activity of the [32P]ATP was found to be twice that of creatine phosphate determined by the method of Barany et al. (1974). ATP specific activities averaged 122 * 46 cpm/ pmol (mean * SD, n = 91). No significant difference was observed between groups treated with different drugs.

Quantification of Results-The relative incorporation of 32P into the 165K band on SDS-polyacrylamide gels was determined by den- sitometry with a Transidyne 2500/2510 scanning densitometer. 3zP incorporation was expressed as the optical density (at 520 nm) of the

595 nm) of the 165K band in the Coomassie-stained gel. The 32P 165K band in the autoradiogram divided by the optical density (at

incorporation was corrected for the specific activity of the ATP, determined in an aliquot from the same atrial homogenate. To ensure quantitative comparison between samples in an individual experiment, samples were prepared on the same day and run on the same slab gel.

Subcellular Fractionation-Washed myofibrils were prepared by the method of Stull and Buss (1977). Hearts were homogenized in 10 volumes of 50 mM KCl, 20 m Tris, 1 mM EGTA, 2 m EDTA, and 15 mM 2-mercaptoethanol, pH 7.9 (homogenization buffer), in a VirTis Micro 23 homogenizer at maximum speed for three periods of 6-10 s. The suspension was filtered through cheesecloth, and the myofibrils were collected by centrifugation at 5000 X g for 10 min. The crude myofibrillar pellet was rehomogenized in 3 volumes of the same buffer containing 1% (v/v) Triton X-100 and centrifuged at 6000 X g for 10 min. The Triton extraction was repeated once. The pellet was washed 3 additional times by rehomogenization in 3 volumes of homogenization buffer. Subcellular fractions were phosphorylated in a standard reaction mixture containing 1 pg/ml of catalytic subunit of CAMP- dependent protein kinase.

Tension Measurements-Atria were isolated with the sinus venosus attached. The atrium was pinned at the atrioventricular junction to a Sylgard resin-coated chamber. A fine thread was tied around connective tissue near the sinus venosus and attached to a Grass FTO3C force transducer. Transducer output was amplified with a

AD522AD) and recorded on a Gould/Brush 220 chart recorder. The bridge amplifier (Analog Devices instrumentation amplifier

preparation chamber (volume, -0.5 m l ) was perfused at 2 d / m i n with HCOs-Ringer solution. The perfusion tubing was directed into the atrial cavity. The muscle was stretched to the peak of the length- tension relationship in Ringer without drugs.

Antibody Production-The 165K protein was extracted from Tri- ton-washed myofibrils with 100 m~ EDTA buffered to pH 7.5 with Tiis base and run on an SDS-polyacrylamide gel. The gel was s t a i e d and destained, and the 165K band was excised. The excised band (-200 pg of protein) was homogenized with Freund's complete adju- vant and injected subdermally into 8-10 sites in New Zealand rabbits. The animals were boosted by a second injection 10 days later. Bleed- ing began 30 days after the second immunization. Preimmune sera were collected before immunization. Immune sera were pooled. IgG was purified by ammonium sulfate precipitation and chromatography on DEAE-Sephacel (Levy and Sober, 1960). Immunofluorescent la-

Phosphorylation in Intact Cardiac Muscle 2113

beling of myofibrils was performed as described by Granger and Lazarides (1979).

To determine the purity of the antibody, proteins were transferred electrophoretically from SDS gels onto nitrocellulose paper (Towbin et al., 1979). The proteins which were recognized by the antiserum were detected by incubating the paper 1 h in antiserum diluted 1:100, rinsing and incubating in horseradish peroxidase-conjugated goat- anti-rabbit IgC. The location of the reacting protein bands was detected by a standard peroxidase color reaction (Towbin et al., 1979). Antiserum reacted with one band at 165K. No detectable reaction was seen with preimmune sera.

Immunoprecipitation-Various protein samples were dissolved in 10 mM EDTA, 15.3 mM NaH2P04, 135 mM Na2HP04, IO-$ M pepstatin A, and lo-’ M phenylmethylsulfonyl fluoride. The solutions were incubated 1 h at room temperature or 18 h at 4 “C with immune or preimmune IgG, followed by 1-h incubation with Staphylococcus aureus (IgG Sorb, The Enzyme Center, Boston, MA). The mixtures were centrifuged at 15,000 X g (5 min), and the supernates made to 1.3% SDS, 3% 2-mercaptoethanol, and run on SDS-polyacrylamide gel electrophoresis.

Statistical Analysis-Statistical analysis of the difference between means was determined by one-way analysis of variance and the Student-Newman-Keuls’ test. (Steel and Tome, 1980).

RESULTS

The main finding of this study is that P-adrenergic agonists (isoproterenol) and cholinergic agonists (ACh or CCh) have opposite effects on incorporation of 3‘P into a 165,000-dalton protein in intact frog atrium. Autoradiograms and densitometer tracings of SDS gels from a typical experiment are shown in Figs. 1 and 2. Intact atria not exposed to drugs incorporate

(a) (b) (c) ( d l I s 0 +

P r o t e i n I s 0 CCh Ringer

-.. M y o s i n ”* 9,

FIG. 1. Effect of isoproterenol and ACh on ‘*P incorporation into intact atria. a, Coomassie-stained gel; 6 to d, autoradiograms of SDS gel. Intact atria were incubated in HCOJ-Ringer solution containing 100 pCi/ml of 32P for 3 h, rinsed extensively, and exposed to IO-’ M isoproterenol for 5 min ( b ) , IO-’ M isoproterenol for 5 min, followed by IO-’ M isoproterenol and IO-’ M CCh for 15 min (c), and HCO1-Ringer for 5 min (d). Processed by Method B (see “Experi- mental Procedures”).

A I

I

- I s 0

- I s 0 -“control

I I I

Migration - FIG. 2. Densitometric tracing of autoradiograms illustrating

difference in 32P incorporation. Atria were treated as described in Fig. 1. Autoradiograms of the gels were scanned with a Transidyne scanning densitometer. A, comparison of %’P incorporation in an atrium incubated in HC03-Ringer alone (dotted line) and one incubated 5 min in Ringer containing lo-’ M isoproterenol (solid line). B, comparison of ‘”P incorporation in an atrium incubated in lo-’ M isoproterenol for 5 min (solid line) and in lo-’ M isoproterenol for 5 min, followed by lo-’ M CCh and lo-’ M isoproterenol for 15 min (dotted line).

32P into many protein bands (Figs. Id, 2 A ) . Isoproterenol M, 4 min) causes a dramatic increase in 32P incorporation into the 165K band (Figs. lb, 2A). ACh or CCh M, 20 min) produce a decrease in ”P incorporation into the 165K band compared to basal incorporation (Fig. 7, d and e) . The decrease is small but reproducible. ACh has a more pronounced effect in decreasing the 32P incorporation stimulated by isoproterenol (Figs. IC, 2B). Control experiments (“Methods”) show that no detectable changes in “P incorporation occur during homogenization and processing of homogenates.

Chemical Nature of the 165K Band The results in Table 1 show that the 32P in the 165K band

is bound to protein. The radioactivity and protein-staining in the 165K band are removed by protease treatment, but are not affected by treatments that extract phospholipids (chloroform-methanol) or degrade RNA (ribonuclease). The finding that the 165K radioactivity is stable in warm dilute alkali or cold acid and is resistant to acidic hydroxylamine suggest that the 32P is linked via a phosphoester bond. The lability of


the 165K radioactivity in hot acid and hot alkali is due to degradation of the protein and not to hydrolysis of the phosphate bond.

Dose-Response Relationships

Isoproterenol-Fig. 3 shows the results of an experiment in which intact atria were incubated in different concentrations of isoproterenol for 5 min. The amount of 32P incorporated into the 165K-protein increases with increasing isoproterenol concentration. A statistically significant (p < O.Ol), 2-fold increase in 32P incorporation is observed with IO-@ M isoproterenol. The isoproterenol dose-response relationships for 32P

TABLE I Stability of 165K-protein

Treatment Relative

band B

‘*P in 165K Comments

Control 100 Protease (5 pg/ml) 2 Eliminates protein

Ribonuclease A (100 pg/ml) 96 Chloroform:methanol (2:l) 88 0.5 N NaOH, 30’, 37 “C 100 0.5 N NaOH, l’, 100 “C 5 Eliminates most of pro-

10% trichloroacetic acid, l’, 100

10% trichloroacetic acid, l’, 19 Reduction of some pro-

staining

tein staining

4 “C

100 “C tein bands, including

1 N HC1,30,37 “C 101 1 N HC1, l’, 100 “C 20 Reduction of some pro-

tein bands, including myosin and 165K

myosin and 165K.

1 M hydroxylamine in 0.16 M 97 sodium acetate, pH 5.5 (20 min, 4 “C)

incorporation and tension are very similar (Fig. 3). The half- maximal effect of isoproterenol on both tension and 32P incorporation occurs near IO-’ M isoproterenol.

The time course of 32P incorporation into the 165K-protein in response to isoproterenol is shown in Fig. 4. In this example, intact atria were incubated in M isoproterenol for various times. A statistically sigmfkant (p < 0.01) 2-fold increase is observed after a 15-s exposure to isoproterenol. Isoproterenol increases 32P incorporation and tension with similar time courses (Fig. 4). The maximum effect of M isoproterenol on both tension and 32P incorporation occurs within 30 s to 1 min.

Carbamylcholine-In the experiment shown in Fig. 5, we examined the ability of CCh to decrease the 32P incorporation stimulated by isoproterenol. Atria were incubated in M isoproterenol alone for 5 min and then in isoproterenol

400

Time (min)

FIG. 4. Time course of action of isoproterenol on 32P incorporation into the 165K protein and on tension. Atria, preincubated in 32P, were exposed to M isoproterenol for the times indicated on the ordinate and processed by Method B (see “Experi- mental Procedures”). Stippled burs are relative 32P incorporation into the 165K protein expressed as the percentage of 32P incorporation into the 165K protein exposed to HCOs-Ringer solution. Each point is the mean of 3 to 11 atria. Error burs are 1 S.E. Statistically significant difference from control is indicated by a star (p < 0.01). Tension is shown by the solid line.

soo/

Log E.4 FIG. 3. Effect of various concentrations of isoproterenol on

32P incorporation and tension. Atria were preincubated in 32P, exposed to isoproterenol for 5 min, and homogenized by Method B (see “Experimental Procedures”). Stippled burs are relative 32P incorporation into the 165K protein expressed as the percentage of 32P incorporation into the 165K protein in atria exposed to HCOa-Ringer for 5 min. Each point is the mean of at least three determinations. Error burs show 1 S.D. Statistically significant difference from control is indicated by a star (p < 0.01). Systolic tension (-1 was recorded as under “Methods.” The systolic tension developed after 5 min exposure to isoproterenol is plotted as the percentage of tension developed in control Ringer’s solution.

Ec4 FIG. 5. Effect of various concentrations of CCh on “P incor-

poration and tension. Incorporation of 32P (stippled burs): atria were preincubated in 32P, rinsed, exposed to M isoproterenol for 5 min, and then exposed to 10” M isoproterenol and various concentrations of CCh as shown on the x-axis. The atria were processed by Method B (see “Experimental Procedures”). Incorporation of 32P into the 165K protein is expressed as the percentage of incorporation into the 165K protein in atria exposed to HC03-Ringer solution. Each point is the mean of three determinations. Error burs are 1 S.E. Star indicates significant difference from control (p < 0.05). Tension (H): systolic tension was measured as described under “Meth- ods.”


and various concentrations of CCh for an additional 15 min. In this experiment, M CCh produces a statistically significant 0, < 0.05) decrease in 32P incorporation into the 165K protein. Micromolar concentrations of CCh were able to lower the 32P content of the 165K protein to control levels observed in Ringer solution alone. We also found that CCh decreases basal 32P incorporation into the 165K band with a dose-response relationship similar to that shown in Fig. 5.

The dose-response relationships for the effects of CCh on 32P incorporation and tension are similar, although not identical (Fig. 5). The dose-response curve for the effect of CCh on tension lies to the left of the dose-response curve for the effect of CCh on 32P incorporation.

The effect of CCh on 32P incorporation occurs more slowly than the effect on tension (Fig. 6). In this experiment, atria were incubated in M isoproterenol for 4 min and then exposed to M isoproterenol and M CCh for various times. Within 30 s of exposure to M CCh, tension declines

7 0 - 5 M l s 0 1 l O ‘ ~ M CCh

t T ‘ ’1

T 4

in

FIG. 6. Time course of action of CCh on “P incorporation and tension. 32P incorporation (stippled bars): atria prelabeled with 32P were incubated in M isoproterenol for 4 min and then in lo-’ M isoproterenol and lo-’ M CCh for the times indicated on the x-axis and processed by Method B (see “Experimental Procedures”). Incor- poration of 32P was expressed as the percentage of incorporation into the 165K protein in atria exposed to HCOs-Ringer solution. Each point is the mean of three determinations. Error bars are 1 S.E. Star indicates significant difference from 4-min time point (p < 0.01). Tension is shown by the solid line.

a b c d e f g h i I S 0

‘sp c,ch is0 c b PROP IS0 PROP RINGER Cch ON6 IS0 CUI ONB

2: :g . -j

4 _I..

c

”

FIG. 7. Autoradiograms of SDS gels showing effects of cholinergic and adrenergic antagonists on 32P incorporation into the 165K protein. To conserve space, only the region near the 165K band (arrows) is illustrated. Atrial pieces were incubated in 32P in Mn-Ringer solution alone (b, d, e, g, h) or in =P in Mn-Ringer solution containing 5 X M propranolol (a, c ) or 1 X M quinuclidinyl benzilate (f, i) for 3 h. The tissue was rinsed and then exposed for 5 min to a, M propranolol; b, M isoproterenol; c, loWs M propranolol and M isoproterenol; d, Mn-Ringer solution, or for 20 min to e, M CCh; f , lo-‘ M CCh and IO-‘ M quinuclidinyl benzilate; g, M isoproterenol; h, M isoproterenol and M CCh and I, lo-‘ M isoproterenol, M CCh, and M quinuclidinyl benzilate. The tissues were then homogenized and processed by Method A (see “Experimental Procedures”). Panels a to c, d to f, and g to i are from different experiments.

to zero and the atria cease beating. However, a significant (p < 0.01) decrease in 32P incorporation does not occur until 1 min in CCh, and this change in ”P incorporation is small. Within 3 min of exposure to CCh, 32P content of the 165K band has declined to a level which is not significantly different from control.

Pharmacology Because stimulation of 32P incorporation by isoproterenol is

blocked by M propranolol (Fig. 7, a to c), we conclude isoproterenol acts via P-adrenergic receptors. The effect of CCh in reducing the basal 3’P incorporation (Fig. 7, d to f ) or in inhibiting the isoproterenol-stimulated incorporation (Fig. 7, g to i ) is blocked by 1 PM quinuclidinyl benzilate (a potent muscarinic antagonist). This demonstrates that the effect of CCh is mediated by muscarinic ACh receptors.

Mediation of Phosphorylation by cAMP Since it is well established that isoproterenol increases

cAMP levels and protein kinase activity in heart (see review by Tsien, 1977), we wanted to determine whether phosphorylation of the 165K-protein was mediated by CAMP-dependent protein kinase. Several lines of evidence suggest that 165K-protein phosphorylation is mediated by CAMP-dependent protein kinase in intact cells. (i) incubation of cells with db-CAMP (Fig. 8, a and c) or 8-bromo cAMP (Fig. 10) usually produces an increase in 32P incorporation into the 165K-pro-

a b C d

Ringer Is0 dbcA AMP

-I

”.

e f

Ringer Is0

9

CTx

e

e

FIG. 8. Effects of CAMP and cholera toxin on 32P incorporation into the 165K protein. Autoradiograms of the 165K (arrows) region of SDS gels are shown. Tissue was incubated in 32P in Mn- Ringer solution ( a to f ) or in 32P in Mn-Ringer solution containing 10 pg/ml of cholera toxin (g) for 3 h. Tissue was then exposed for 20 in to Ringer’s solution (a , e) , M isoproterenol (b , f ) , 1 mM db-CAMP (c ) , or 1 m~ 5’-AMP (d) . The sample incubated in cholera toxih (g) was homogenized without additional incubation. Panels a to d and e t o g are from different experiments. Tissue was processed by Method A (see “Experimental Procedures”).


tein. This effect was seen in eight out of 10 experiments. Sodium butyrate, adenosine, and 5'-AMP at 1 m~ (Fig. 8) do not stimulate 32P incorporation into the 165K-protein. (ii) incubation with phosphodiesterase inhibitors (theophylline or R07-2956) increases incorporation of 32P into the 165K-protein (not illustrated). (iii) incubation of cells in 10 pg/ml of cholera toxin, which elevates cAMP levels in all cells tested (Gill, 1977), produces an increase in phosphorylation of the 165K- protein (Fig. 8, e tog). These results support the idea that the 165K-protein is phosphorylated in a CAMP-dependent manner.

Next, we wanted to know whether the 165K-protein could be phosphorylated by cyclic nucleotide-dependent protein kinases in homogenates. Homogenates of whole hearts were incubated in the standard phosphorylation reaction mixture containing 100 p~ [y-"'PIATP and various concentrations of cAMP or cGMP without added exogenous protein kinase. Under the conditions of the assay, the phosphorylation of the 165K-protein was linear for at least 15 s (Fig. 9a). Incorpora- tion of 32P into the 165K-protein band was stimulated 8-fold by 1 p~ cAMP (Fig. 9, b and d). Phosphorylation was small in the absence of exogenous CAMP. cGMP at concentrations below 1 m~ did not stimulate 32P incorporation into the 165K-

protein (Fig. 9, b and c). cGMP at concentrations of 1 m ~ , however, increased 32P incorporation into the 165K-protein 3.7-fold. 1 mM Ca2+ did not affect phosphorylation of the 165K band.

The 165K-protein can also be phosphorylated by purified protein kinases. Both purified catalytic subunit of CAMP- dependent protein kinase and purifled cGMP-dependent protein kinase will catalyze the incorporation of 32P into the 165K-protein in homogenates. We have not studied the kinet- ics of the reaction, but CAMP-dependent protein kinase and cGMP-dependent protein kinase at 3 p~ stimulate 32P incorporation into the 165K-protein -30-fold under standard assay conditions (Fig. 9, e and f ) . The proteins that are phosphorylated in the presence of cyclic AMP-dependent protein kinase and cAMP are similar (Fig. 9, d and e). Pudied cyclic GMP-dependent protein kinase, in addition, catalyzes 32P incorporation into several other proteins (Fig. Sf).

Mechanism of CCh-induced Decrease in 32P Incoporation Since it has been hypothesized that the negative inotropic

and chronotropic effects of cholinergic agonists on the heart beat may be mediated by cGMP (George et al., 1970; Lee et al., 1972; Watanabe and Besch, 1975; Gardner and Allen,

c d e f CGMP CAMP A-PK G-PK ."- .- -

1 I I I I 1 I

0 -0 -7 -0 -5 -4 -3 Log [cyclic nucleotide7

FIG. 9. Phosphorylation of 165K protein in homogenates. a, time course of :'lP incorporation into 165K protein in standard assay mixture. Reaction mixture ( 5 0 pl) contained 100 p~ [y-"PIATP and 1 p CAMP. The reaction was initiated by addition of homogenate and was carried out on ice at 4 "C for the times indicated. The reaction was stopped by addition of 10 pl of 10% SDS-IO% P-mercaptoethanol. b, effect of cAMP and cCMP on 3lP incorporation into

165K protein. Various concentrations of cAMP (0) or cGMP (A) were present in the reaction mixture. Reaction was carried out at 4 "C for 15 s. c to f, autoradiograms of SDS gels. Heart homogenates were phosphorylated in the presence of M cCMP (c), M cAMP (d), 3 p~ catalytic subunit of CAMP-dependent protein kinase (e), or 3 p~ purified cCMP-dependent protein kinase and 1 p~ cCMP ( f ) .


a

Ringer

b c d CCh +

Is0 CAMP cAMP

4 - -

).‘ . * -

e 1 , 8-Br-CAMP

.- v) j . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c

l i m e ( m i n ) FIG. 10. Effect of CCh on 8-bromo cAMP-stimulated phos-

phorylation and tension. Tissue was incubated in 32P for 3 h, rinsed, and exposed for 20 min to Mn-Ringer solution (a), lo-@ M isoproterenol (b ) , 1 m~ 6-bromo cAMP (c), and 1 mM 6-bromo cAMP and lo-’ M CCh (d). Arrows indicate the 165K band. Method A (see “Experi- mental Procedures”). e, tension: an atrium was exposed to 1 mM 6- bromo CAMP, which increased contractile tension -25%. Addition of 2 X IO-’ M CCh arrested the heartbeat.

Actin-

“ ”

Myofibrils

e f c-

FIG. 11. Subcellular fractionation of the 16SK phoephopro- tein. Frog heart was homogenized and fractioned as described under “Methods.” SDS-polyacrylamide gels stained with Coomassie bril- liant blue (a, c, e) and autoradiograms of these gels (b , d, f ) are shown. a, b, supernate from 5OOO X g, 10-min centrifugation. c, d, pellet from 5OOO X g, 10-min centrifugation. e, f , Triton-washed myofibrillar fraction. Arrows indicate myosin, actin, and the 165,000- dalton protein band.

1976a, 1976b), we tested the effects of db-cGMP on 165K- protein phosphorylation in intact cells. db-cGMP alone some- times produces a small but definite increase in 32P incorporation into the 165K-protein. This effect was not consistent.

Nevertheless, db-cGMP never mimicked the effects of CCh in reducing 32P incorporation (either basal or isoproterenol-stim- da ted) into the 165K-protein.

It has been suggested that some of the inhibitory effects of cholinergic agonists on the heart may be mediated by decreases in adenylate cyclase activity and intracellular cAMP levels (Murad et al., 1962; L a b i a and Sonnenblick, 1971; Gardner and Allen, 1977; Keeley et al., 1978; Jakobs et al., 1979). We found, however, that CCh was able to inhibit the increased 3’P incorporation, into the 165K-protein, that is stimulated by 8-bromo cAMP (Fig. 10, a to d). This suggested that CCh may be able to decrease phosphorylation of the 165K-protein by a mechanism independent of cAMP levels.

Identity and Subcellular Localization of 165K Protein Several experiments examined the phosphorylation of the

165K-protein in different tissues. Homogenates were made of heart, skeletal muscle, liver, testis, lung, and intestine. The homogenates were incubated with cAMP and [Y-~~PIATP and the phosphoproteins separated on SDS gels. In no case was there a distinct 165,000-dalton band which incorporated :v2P, except in cardiac muscle. In one experiment, when exogenous catalytic subunit of CAMP-dependent protein kinase at a concentration of 4 pg/ml was added to the phosphorylation reaction, a small amount of 32P was found a t 165K in skeletal

a b c d e f g h i Rabbit C Frog 165K Frog 165K

190: o 4SOue o SO 4 6 0 W o eooue o eooue

myosln- W“

” ”- 1) 0 1 6 5 K . . 150K- -

FIG. 12. Immunoprecipitation of frog 165K-protein and rabbit C-protein. Antibody was prepared against the 165-protein as described under “Methods.” a to g, Coomassie-stained gels; h, i, autoradiograms of gels f and g. a, b, rabbit heart C-protein: 2 pg of rabbit heart C-protein in EDTA-PO4 was incubated with 450 pg of IgG ( b ) or without IgG in a final volume of 100 p1 for 1 h at 22 “C (a). The solution was then added to an excess of protein A (S. aureus), vortexed, and incubated an additional hour. The mixture was centrifuged 5 min at 15,000 X g and the supernate removed and run on SDS-polyacrylamide gels. c to e, frog 165K-protein: Triton-washed myofibrils were prepared by the method of Stull and Buss (1977) and extracted with EDTA-PO4. Aliquots of the extract containing -2 pg of the 165K-protein were then incubated with 450 pg of IgG (e) , with 50 pg of IgG (d), or without IgG (c). The solutions were processed as in a and b. f to i, phosphorylated frog 165K-protein: frog atria were incubated for 3 h in 32P, rinsed, exposed to IO-’ M isoproterenol for IO min, and homogenized in 2 ml of EDTA-PO4 containing lo-@ M pepstatin A and lo-’ M phenylmethylsulfonyl fluoride. The extract was centrifuged at 5OOO X g for 10 min. The supernate, which contained -20 pg of 165K-protein, was split into two aliquots. One aliquot (g, i) was incubated with 900 pg of IgG and the other ( f , h) was incubated without IgG in a final volume of 1 ml. Incubations were 16 h on ice. The solutions were then treated with protein A as described above. Although these figures employ buffer controls, identical results were obtained when preimmune or nonimmune serum was used as a control.


muscle. The amount of incorporation, however, was less than 4% of that incorporated into the 165K band in cardiac muscle. These results suggest that the 165K-protein is phosphorylated in a CAMP-dependent manner only in cardiac muscle.

Subcellular fractionation studies were undertaken to local- ize the 165K-protein. The 165K-protein was found to be enriched in Triton-washed myofibrils prepared by a modifi- cation of the method of Stull and Buss (1977) (Fig. 11). Over 40% of the 165K-protein was recovered in the final myofibrillar fraction. Thirty-nine percent of the total myosin was recovered in the same fraction.

FIG. 13. Indirect immunofluorescent labeling of myofibrils. Myofibrils were prepared from frog heart by the method of Granger and Lazarides (1979). Myofibrils attached to cover slips were incubated 1 h at room temperature with antiserum or preimmune serum diluted 1:lOO with Ringer's solution containing 1 m EGTA and no calcium (EGTA-Ringer solution), rinsed 30 min with EGTA-Ringer solution, and incubated in fluorescein-conjugated goat-anti-rabbit IgG diluted 1:IOO with EGTA-Ringer solution for 1 h. The slides were rinsed 1 h and examined at 1250X with a IOOX neofluar objective. A, phase-contrast micrograph of a myofibril labeled with antiserum to the 165K-protein. B, fluorescence micrograph of fiber in A. C, fluorescence micrograph of a myofibril labeled with preimmune serum. D, phase-contrast micrograph of a myofibril extracted with EDTA- PO, prior to labeling with IgG. E, fluorescence micrograph of myofibril in D.

Solubility experiments suggest that the 165K-protein is C- protein (Offer et al., 1973). Both the 165K-protein and C- protein (a) are extracted from washed myofibrils by 10 mM EDTA, 15.2 mM NaH2P04, 135 m Na2HP04 (EDTA-PO4) (Fig. 12c), (b) are extracted from muscle by Guba-Straub solution (0.3 M KCl, 0.1 M KH2P04), and (c ) pass unretarded through DEAE-Sephacel equilibrated in EDTA-PO4 buffer.

Immunological evidence supports the conclusion that the 165K-protein is C-protein. Antibodies were prepared against the 165K band cut from SDS-polyacrylamide gels (see "Meth- ods"). The antibody which was produced binds both to the frog 165K-protein and to C-protein purified from rabbit cardiac muscle (Fig. 12). This was shown by incubating partially purified, frog heart 165K-protein (Fig. 12, c to e), "P-labeled atrial extracts (Fig. 12, a and b) , and purified rabbit heart C- protein (Fig. 12, a and 6 ) with antibody. The antibody (and antibody-antigen complexes) were then precipitated with IgG Sorb. When IgG from immunized animals was used in this procedure, the frog 165K-protein and rabbit heart C-protein were precipitated. IgG from preimmune serum was ineffective in precipitating these proteins.

This antibody was then used to label myofibrils by indirect immunofluorescence. The antibody binds to the region of overlap between thick and thin filaments (Fig. 13). This is the same distribution which has been found for C-protein (Offer, 1972; Moos, 1972; Rome, 1972; Pepe and Drucker, 1975). The protein responsible for the immunofluorescent staining can be extracted by EDTA-PO4 (Fig. 13), which is another charac- teristic of C-protein (Offer et al., 1973).

DISCUSSION

The main finding of this paper is that P-adrenergic and cholinergic agonists have opposite effects on 32P incorporation into a 165,000-dalton phosphoprotein in intact heart muscle. In this discussion we address three questions: (i) what is the 165K protein? (ii) what are the mechanisms of phosphorylation and dephosphorylation of this protein? (iii) does this protein play a role in the regulation of cardiac contractility by neurotransmitters?

Identification of the 165K Protein-Several lines of evidence suggest that the 165K-protein is C-protein. The 165K- and C-proteins (a) are both found in Triton-washed myofibrils, (b ) have identical solubilities and affinities for DEAE, (c ) are both located in the same region of the myofibril as determined by indirect immunofluorescence, and (4 share antigenic determinants as shown by the ability of antibody to 165K-protein to bind to rabbit heart C-protein. Recently, Jeacocke and England (1980) have reported that a 155,000- dalton protein in rat cardiac muscle becomes phosphorylated in response to epinephrine. They suggest this protein is C- protein on the basis of i ts solubility. C-protein is a protein which recently has been shown to be a component of both skeletal and cardiac muscle fibers (Offer et al., 1973; Yama- mot0 and Moos, 1981) and is responsible for the transverse stripes which are seen in high resolution micrographs of the A band (Offer, 1972). Both electron microscopic (Offer, 1972) and x-ray diffraction (Rome, 1972; Rome et al., 1973) studies have shown that antibody to C-protein is distributed along thick filaments in skeletal muscle with a 43-nm spacing. C- protein is not present along the entire length of the thick filament. however, but is found in two zones, each -400-nm wide, separated by -300 nm on either side of the M-line. The molecular weight of C-protein varies with the type of muscle it comes from. The SDS molecular weights of heart, red skeletal, and white skeletal muscle C-proteins from rabbit are 150,000,145,000 and 135,000 (Yamamoto and Moos, 1981).

The molecular weight of our 165K-protein is larger than the


molecular weight of rabbit heart C-protein (Fig. 12), but this is not too surprising considering the range of molecular weights found for different C-proteins (Yamamoto and Moos, 1981). The location of C-protein in the myofibril and the finding that it is reversibly phosphorylated and dephospho- rylated in response to neurotransmitters suggests this protein may play a regulatory role in the contractile activity of the heart (Offer, 1972). However, although C-protein binds to myosin and actin and has small effects on myosin ATPase activity, the function of C-protein is unknown.

Mechanisms of Phosphorylation-Many of the effects of p agonists have been shown to be due to CAMP-dependent 'phosphorylation (Robison et al., 1971; Entmann, 1974; Tsien, 1977). We believe that the stimulation of 32P incorporation into the 165K-protein caused by isoproterenol is also catalyzed by CAMP-dependent protein kinase for several reasons. (i) cAMP derivatives mimic the effect of isoproterenol on 165K- protein phosphorylation (Fig. 8) and on tension (Fig. 10). (ii) agents that elevate intracellular cAMP levels, such as cholera toxin (Fig. 8) and phosphodiesterase inhibitors, increase 165K- protein phosphorylation. (iii) phosphorylation of the 165K- protein in homogenates is stimulated by addition of cAMP to the reaction mixture (Fig. 9). The small amount of 32P incorporated into the 165K-protein in the absence of added cAMP is probably caused by endogenous cAMP formed by adenylate cyclase in the preparation. When the phosphorylation assay is run with concentrations of ATP (5 p ~ ) well below the K,,, of the endogenous adenylate cyclase, no incorporation of :32P into the 165K-protein is detectable without the addition of exogenous CAMP. Phosphorylation of the 165K-protein is not stimulated by low concentrations of cGMP or by Ca2+ ions. Exogenously added cGMP-dependent protein kinase, however, is able to phosphorylate the 165K-protein. This suggests that, although the 165K-protein is a substrate for cGMP- dependent protein kinase, the concentration of this enzyme in the frog heart is low.

Although it is' clear that at least some of the effects of ,fi agonists on the heart are mediated by activation of the adenylate cyclase system, the mechanism of action of ACh is not clear. Certain effects of ACh may be mediated by increases in myocardial cGMP levels (George et al., 1970; Lee et al., 1972; Watanabe and Besch, 1975; Gardner and Allen, 1976a; 1976b; 1977), others by decreases in cAMP levels (Murad et al., 1962; LaRaia and Sonnenblick, 1971; Gardner and Allen, 1977; Keeley et al., 1978, Jakobs et al., 1979), and some effects may be cyclic nucleotide-independent (Linden and Brooker, 1979).

The decrease in 32P incorporation into the 165K-protein produced by CCh in intact cells appears not to be mediated by a cGMP-dependent process for several reasons. (i) db- cGMP does not mimic the effects of CCh on 165K protein phosphorylation in intact cells. On the contrary, db-cGMP often stimulates 32P incorporation slightly. (ii) cGMP has no effect, or, a t high concentrations, stimulates :'2P incorporation into the 165K-protein in homogenates.

Because ACh can inactivate adenylate cyclase (Murad et al., 1962; LaRaia and Sonnenblick, 1971; Gardner and Allen, 1977; Jakobs et al., 1979; Brown, 1979), the ability of ACh to decrease basal or isoproterenol-stimulated 32P incorporation into the 165K-protein could, theoretically, be caused by an inactivation of adenylate cyclase and a subsequent turn-off of the CAMP-dependent protein kinase. This mechanism is excluded by the finding that CCh can rapidly and completely inhibit the effect of 8-bromo cAMP in elevating 32P incorporation into the 165K-protein in intact cells (Fig. 10). Further, since 8-bromo cAMP is resistant to phosphodiesterases, we conclude that ACh does not activate a phosphodiesterase that decreases cAMP levels. These findings suggest that CCh may

act by a mechanism which does not involve changes in cyclic nucleotide levels.

The 165K-protein described here is a candidate for a phosphoprotein that may play a role in regulation of the heart beat by autonomic neurotransmitters. We have shown that isoproterenol produces an increase in 32P incorporation of this protein. The increase in 32P content most likely represents an increase in phosphate content of the protein and is unlikely to be due to an exchange of labeled phosphates for unlabeled phosphates. Exchange is excluded because CCh produces a rapid decrease in 32P content of the 165K-protein (Fig. 6) despite the constant specific activity of the ATP pool. We have not measured accurately the stoichometry of phosphate incorporation into the 165K-protein because of the small amount of protein in frog hearts.

The changes in 3'P content of the 165K-protein are unlikely to be secondary to changes in tension because isoproterenol and CCh alter 32P incorporation, whether the hearts are bathed in HCOs-Ringer, in which they are beating spontaneously, or in Mn-Ringer or tetrodotoxin-Ringer, in which the beat is arrested.

Several findings suggest that C-protein may play a role in the regulation of cardiac tension in response to isoproterenol. (i) the concentration of isoproterenol required to produce half- maximal effects on tension and on 32P incorporation into the 165K-protein were similar (Fig. 3). (ii) the incorporation of "P into the 165K-protein is correlated with the contractile activity of the heart. All agents that increased tension also de- creased 32P incorporation. (iii) the time courses of action of isoproterenol on tension and 165K-protein phosphorylation were similar (Fig. 4).

Although the increase in "P incorporation produced by isoproterenol correlates well with the increase in tension, the decrease in 32P incorporation produced by CCh occurs at higher concentrations and more slowly than the decrease in tension (Figs. 5 and 6). There are several possible explanations for the lack of correlation between tension and "P incorporation in response to CCh. One explanation is that CCh reduces tension by a mechanism independent of C-protein. I t is well established that CCh reduces the slow inward Ca" current of the action potential and increases membrane K conductance (Giles and Noble, 1976). These effects may occur more quickly than the effect on protein phosphorylation and may be the main factors responsible for the inhibitory effects of CCh on the heartbeat. Alternatively, the relationship between phosphate content of C-protein and tension may not be a simple one. Indeed, tension may be related to the rate of phosphorylation of the protein and not to the steady state levels of protein phosphate present.

Acknowledgments-We are indebted to Dr. David Glass for his generous d t s of protein kinase and valuable discussions, to Dr. Carl Moos and K. Yamamoto for purified rabbit heart C-protein, and to Dr. Susan Lowey for antibody against skeletal muscle "protein. We thank Joanna Corley and Vicki Shadix for typing the manuscript, Jan Giles for problem solving, and Hoffmann-LaRoche for gifts of 3- quinuclidinyl benzilate and R07-2759.

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protein phosphorylation

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dalton myofibrillar

campdependent protein

p incor poration

p incorpora tion

bromo camp