Effect of Epinephrine on Phosphorylase b Kinase in Perfused Rat Hearts*

Transcription

1 THE JOURNAL OF BOLOGC.~ CHEMSTRY Vol. 241, No. 24, ssue of December 25, PP , 1966 Prined in U.S.A. Effec of Epinephrine on Phosphorylase b Kinase in Perfused Ra Hears* (Received for publicaion, July 8, 1966) GEORGE. DRUMMOND, LOVERNE DUNCAN, AND ELEEN HERTZMAN From he Deparmen of Pharmacology, School of Medicine, Universiy of Briish Columbia, Vancouver 8, Canada SUMMARY Phosphorylase b kinase was assayed in exracs prepared from perfused ra hears reaed wih epinephrine. The amine produced modes bu significan acivaion of he enzyme in exremely small doses. The acivaion was never as grea as would be expeced from sudies in viro of he purified enzyme. ncrease in aciviy in proporion o he conrols was greaer when he assays were conduced a ph 6.8 han a ph 8.2, so ha ph 6.8 o 8.2 raios were increased. sopropylnorepinephrine also acivaed he enzyme in perfused hears, bu mehoxamine was wihou effec. Acivaion produced by a maximal dose of epinephrine (0.1 pg) was blocked by reamen of he hears wih he adrenergic blocking agens, dichloroisoproerenol and nehalide. When he dose of epinephrine was varied, acivaion of he enzyme closely paralleled he inoropic response. When a maximal dose (0.1 pg) of epinephrine was adminisered, phosphorylase b kinase was maximally acivaed in 1 se, well ahead of he inoropic response which reached a peak in 10 sec. Acivaion of phosphorylase b kinase was almos idenical in ime wih he rise in adenosine 3,5 -cyclic phosphae levels. The levels of his cyclic nucleoide increased g-fold in 1 se afer 0.1 pg of epinephrine was adminisered. Epinephrine has a profound inoropic acion on he myocardium and simulaes he conversion of phosphorylase b o he a form in beaing hears (l-4). is known ha epinephrine simulaes he formaion of adenosine 3,5 -cyclic phosphae (cyclic adenosine monophosphae) in a variey of issues. Robison e al. (4) and Cheung and Williamson (5) have shown ha when epinephrine was adminisered o perfused ra hears, cyclic AMP levels rose wih sriking rapidiy and reached maximum levels before he conracile response developed fully. has been generally assumed ha increased phosphorylase a levels resul from he acivaion of phosphorylase b kinase by cyclic AMP. Posner, Sern, and Krebs (6) have already shown ha large doses of epinephrine significanly increased ra skeleal muscle cyclic AMP levels, which were accompanied by increased * This work was suppored by grans from he Life nsurance Medical Research Fund, Philadelphia, he Briish Columbia Hear Foundaion, and he Medical Research Council of Canada. phosphorylase b kinase aciviy. Hammermeiser, Yunis, and Krebs (7), however, were unable o demonsrae a significan acivaion of phosphorylase b kinase in perfused rabbi hears by doses of epinephrine which increased cyclic AMP levels. n he preceding communicaion (8) we have shown ha a supramaximal dose of epinephrine produced a highly significan increase in phosphorylase b kinase in perfused ra hears. Acivaion of he enzyme was a a maximum wihin 3 se afer injecion. The conracile response reached a maximum considerably laer, a 10 sec. was of ineres o examine he naure of phosphorylase b kinase acivaion more closely wih he use of submaximal or physiological doses of epinephrine. n paricular, i was of ineres o examine he rae of enzyme acivaion wih respec o rise in cyclic AMP levels and wih respec o developmen of he inoropic response. Such a sudy is he subjec of his repor. EXPERMENTAL PROCEDURE MaerialsEpinephrine and isopropylnorepinephrine were prepared as sock soluions conaining 1 mg per ml in 0.1% sodium meabisulfie. Mehoxamine, dichloroisoproerenol, and nehalide (-(2.naphhyl)2-isopropylaminoehanol) sock soluions conained 10 mg per ml. Such soluions were sored a -18. Diluions were prepared in Tyrode s soluion immediaely before adminisraion in such a manner ha he desired dose was available in 0.1 ml. Fresh diluions were prepared for each hear. Perfusion, Sampling, and Assay Mehods--Female Wisar ras weighing 180 o 200 g were used. Hears were perfused and conracile aciviy recorded as previously described (8). All agens were delivered in a volume of 0.1 ml hrough hin polyehylene ubing (void volume, 0.03 ml) ino he glass cannula immediaely above he hear. Soluions of adrenergic amines were injeced over a 5-se inerval. Dichloroisoproerenol and nehalide soluions were delivered over a O-se inerval. When epinephrine was subsequenly adminisered, i was injeced 5 se afer he blocking agen and was delivered over he usual 5-se inerval. For dichloroisoproerenol and nehalide conrols, 0.1 ml of Tyrode s soluion was injeced 5 se afer he blocking agen. For normal conrols, 0.1 ml of Tyrode s was adminisered. Hears were frozen a he appropriae inerval (measured from he insan of compleion of injecion), and sored in liquid Nz unil use. Venricular issue was homogenized, and a 30,000 X g supernaan fracion was prepared (8). The cenrifuged exracs were dilued 1:4 wih 15 mu neural cold cyseine soluion, and 0.2-ml aliquos were used for he assay of phosphorylase b kinase,

2 5900 Epinephrine on Phosphorylase b Kinase in Ra Hear Vol. 241, No F KNASE ph CONTRACTLE FORCE Q- T 1 PH &S/8.2 RATO KNASE ph lO EPNEPHRNE.cg g.14 0 =.12 2 (Y cd.08 2 d.04 a 3 and he ube was simulaneously immersed in a boiling waer bah. The mixure was sirred for 5 min, and afer cooling, he exrac was cenrifuged a 30,000 x g for 10 min a 0. The supernaan fluid was carefully removed and he residue was reexraced wih anoher 5 ml of waer and cenrifuged. The combined supernaan fluid was passed hrough a DEAE-cellulose column (0.7 x 6 cm) in he carbonae form (generaed wih 10 ml of 1 M riehylammonium bicarbona.e, ph 7.5, and horoughly washed wih waer) a a flow rae of abou 0.3 ml per min. The column was hen washed wih 30 ml of waer. Cyclic AMP was elued wih 40 mm riehylammonium bicarbonae, ph 7.5. Thiry milliliers of effluen were colleced a a flow rae of 0.5 ml per min. This soluion was aken o dryness on a roary evaporaor and residual riehylammonium bicarbonae removed by re-evacuaion of small volumes of waer. The residue was dissolved by carefully washing down he walls of he flask wih 2-ml volumes of waer, he washing being repeaed hree imes. This soluion was lyophilized, and he residue was dissolved in 1 ml of 25 mm poassium phosphae, ph 7.5. Recovery of lop3 pmole of cyclic AMP was quaniaive. Assay of cyclic AMP was performed by he mehod of Posner e al. (10) excep ha he acivaion of phosphorylase b kinase was carried ou a 4 for 30 min raher han for 10 min a 30. We found ha his modificaion gave more reproducible resuls and a wider spread of acivaion due o cyclic AMP. Three sandard concenraions of cyclic AMP (1, 4, and 8 X 10e8 M) were carried in each assay. All exracs were assayed in riplicae. Care was aken o ensure ha he yield of cyclic AMP was proporional o he volume of exrac used in he assay. This was necessary because inhibiory maerial (10) could be deeced, especially in exracs from epinephrine-reaed hears, when amouns were used which gave near maximal acivaion. Resuls are expressed in millimicromoles per g of venricle, fresh weigh. FG. 1. Effec of epinephrine dose on phosphorylase b kinase acivaion and conracile force. Seven ra hears were used for RESULTS he conrol as well as for each dose of epinephrine. Hears were frozen when he conracile response reached a maximum (average, Assay of phosphorylase b kinase in crude hear exracs is 9 se). All assays were performed in duplicae a ph 6.8 and 8.2. echnically difficul, bu he previous daa (8) make i clear ha The verical bars represen he sandard error of he mean. he enzyme exiss essenially in he nonacivaed form in perfused ra hears (ph 6.8 o 8.2 raio of 0.06). A supramaximal dose of which was sared wihin 5 min afer cenrifugaion. Phospho- epinephrine (2 pg) caused a 5-fold increase in enzyme aciviy as rylase 6 kinase assays were conduced a ph 6.8 and 8.2 (8, 9). measured a ph 6.8 and a a-fold increase in ph 6.8 o 8.2 aciviy Each experimenal ube was accompanied by a conrol in which raios (8). When submaximal doses of epinephrine were adhear exrac and all componens of he kinase assay excep phos- minisered, assay of he resuling exracs revealed ha phosphorylase b were presen during he kinase incubaion inerval phorylase b kinase was significanly acivaed (Fig. 1) by doses (phosphorylase was replaced by 0.1 ml of 15 mm cyseine solu- of epinephrine as small as pg (P = 0.001). As he dose was ion). Afer incubaion, a O.l-ml aliquo of each conrol ube increased, he aciviy increased o a maximum and leveled off was added o 1.8 ml of diluion buffer conaining 200 pg of ade- when he amoun adminisered was 0.1 pg. Acivaion of he nylic deaminase insead of 1.9 ml as for experimenal ubes (dilu- enzyme followed very closely he conracile force response, ion effecively erminaes kinase aciviy). To he dilued which also rose o a maximum wih a dose of 0.1 pg and hen conrol ubes, phosphorylase b in an amoun equivalen o ha leveled off. n proporion o conrol values, acivaion was more presen in he dilued experimenal ubes (0.1 ml of a 1:6 dilu- marked a ph 6.8 han a ph 8.2 so ha he ph 6.8 o 8.2 aciviy ion of he sock enzyme soluion which conained 100,000 unis raios also increased in a manner very similar o he conracile per ml) was added. A 0.2.ml aliquo of each ube was hen response (Fig. 1). A dose of 1.0 pg (seven hears), no shown on assayed for phosphorylase a. All assays wih appropriae con- he graph, produced no furher increase in conracile force or in rols were performed in duplicae. Phosphorylase b kinase ac- phosphorylase b kinase acivaion. Thus, a dose of 0.1 pg proiviy was expressed as unis per ml of undilued exrac. duced a degree of acivaion almos idenical wih ha obained Assay of Cyclic AMP-The mehod of exracing frozen issue wih 2 pg under similar circumsances (8), i.e. a 5-fold increase was a modificaion of ha described by Hammermeiser, Yunis, in aciviy a ph 6.8 and a a-fold increase in ph 6.8 o 8.2 acivand Krebs (7). Frozen venricle from wo hears was carefully iy raio. weighed (1 g) and ground o a fine powder in a sainless seel ube Because of he sriking associaion beween he increase in under liquid Ns. To he powder were added 5 ml of waer a 90 kinase aciviy and he increase in he.conracile response, an-

3 ssue of December 25, 1966 G.. Drummond, L. Duncan, and E. He&man 5901 oher experimen was performed in which a dose of 0.1 pg (now aken as maximal) was adminisered. The hears were frozen a various imes during he developmen and decline of he conracile response and assayed for phosphorylase 6 kinase aciviy. Oher hears were perfused in an idenical manner and assayed for cyclic AMP conen. The firs hears were frozen 1 se afer compleion of he epinephrine injecion. A his ime he rise in conracile aciviy was barely eviden. The response rose o a maximum in 10 se and hen receded (Fig. 2). n conras, phosphorylase b kinase had reached essenially maximal acivaion a 1 se and was definiely maximal a 3 se as evidenced by he assays a ph 6.8. Again he naure of he acivaion was such ha he ph 6.8 o 8.2 aciviy raios increased S-fold and his increase was maximal a 1 sec. Cyclic AMP levels rose almos insananeously, were a a peak a 1 see, and were already decreasing a 3 sec. One migh anicipae from he daa of Robison e al. (4) ha cyclic AMP levels reach a maximum earlier han 3 se afer epinephrine adminisraion. The rise in levels recorded here is srikingly similar o ha of he above auhors. The increase above he conrol a 1 se was g-fold. Levels of cyclic AMP declined rapidly and had decreased 50% afer 10 sec. Phosphorylase b kinase aciviy began o decline afer 6 se and followed a decay paern similar o ha of he conracile response. A 180 se (four hears, no shown in Fig. 2), conracile aciviy had reurned o normal, and phosphorylase b kinase aciviy was no differen from he conrols. One mus conclude ha acivaion of phosphorylase b kinase precedes he conracile response and is, in fac, closely associaed in ime wih he rise in cyclic AMP levels. Again, he degree of acivaion of he enzyme was highly significan saisically. Differences beween all experimenal values up o 16 se and he conrol, when evaluaed by he Suden s es, yielded P values less han is widely acceped ha he inoropic and glycogenolyic acions of epinephrine resul from simulaion of p recepors in conras o Q recepors which consric vascular smooh muscle. would be logical o assume ha he acivaion of phosphorylase b kinase would resul from p adrenergic aciviy. f such were he case, acivaion of phosphorylase b kinase in perfused hears should be simulaed by oher,b-adrenergic amines such as isopropylnorepinephrine, and no by a-adrenergic amines such as mehoxamine. The laer amine produces no inoropic acion even a high doses. To es his, hears were perfused and injeced wih 0.1 pg of isopropylnorepinephrine. Anoher series of hears was injeced wih 100 pg of mehoxamine. Exracs were prepared and assayed for phosphorylase b kinase in he usual manner. sopropylnorepinephrine caused an acivaion of he enzyme (Table ) very similar o ha produced by 0.1 pg of epinephrine, while producing a maximal inoropic response. The a-adrenergic amine, mehoxamine, even a he high dose of 100 pg, produced no inoropic response. also produced no significan change in phosphorylase b kinase aciviy (Table ) as compared wih he conrol series done a he same ime. These daa sugges ha he acivaion of phosphorylase b kinase is a characerisic p acion of adrenergic amines. A consequence of his is ha he acivaion by epinephrine should be prevened by such fi-adrenergic blocking agens as dichloroisoproerenol and nehalide. These blocking agens are known o block he inoropic response o,!%adrenergic amines and o block he glycogenolyic acions as well. The rise in cyclic AMP levels caused by epinephrine in working ra hears is characerisically blocked by dichloroisoproerenol (4). Perfused hears were reaed wih dichloroisoproerenol or nehalide, followed by epinephrine (0.1 pg). Oher hears were reaed wih a blocking agen followed by Tyrode s soluion o serve as conrols. Ten seconds afer compleion of epinephrine injecion, he hears were frozen and CONTRACTLE KNASE ph 6.8 PH KNASE ph 8.2 RATO TME N SECONDS FG. 2. Time course of phosphorylase 6 kinase acivaion by epinephrine in perfused ra hears. Seven hears were used for each poin in ime for phosphorylase b kinase deerminaions. Eigh hears (exraced in pairs) were used for each poin in ime for cyclic AMP deerminaion. The dose of epinephrine was 0.1 pg. Verical bars represen he sandard error of he mean. TABLE Acion of isopropylnorepinephrine and mehoxamine on phosphorylase b kinase in perfused ra hears Hears were frozen 10 see afer compleion of injecion when he conracile response o isopropylnorepinephrine was a a maximum. Four hears were used in each experimen and all assays were performed in duplicae. Aciviies are expressed as unis per ml of undilued exrac f sandard error of he mean. Experimen Phosphorylase b kinase aciviy ph 6.8 U%ilS/Fd Conrol f 0.56 sopropylnorepinephrine (0.1 pg) 34.9 f 1.4 Mehoxamine (100 pg) f 0.45 ph 8.2 U?AS/d.06 ph 6 8 o 8.2 aciviy raio f f f iO f f 0.006

4 5902 Epinephrine on Phosphorylase b Kinase in Ra Hear Vol. 241, No. 24 TABLE Effec of adrenergic blocking agens on epinephrine acivaion of phosphorylase b kinase Five hears were used for each group. Adminisraion of blocking agens is described under Experimenal Procedure. The dose of dichloroisoproerenol was 20 pg, and of nehalide, 10 pg. Phosphorylase b kinase aciviy is expressed as in Table. Experimenal condiions Tyrode s conrol Epinephrine (0.1 pg ) Nehalide conrol. Dichloroisoproerenolconrol Nehalide followed by epinephrine (0.1 rd Dichloroisoproerenol followed by epinephrine (0.1 Pd Phosphorylase b kinase aciviy ph 6.8 ph 8.2 unis/ml U?dS/Wd 7.84 f f f f f f S f & f f f 5.3 ph 6.8 o 8.2 aciviy raio 0.072f f zo f f0.004 worked up in he usual manner. Boh dichloroisoproerenol and nehalide a he doses given produced sligh increases (abou 10%) in conracile aciviy above he normal conrol. Under he condiions of he experimen (see Experimenal Procedure ) epinephrine produced no inoropic response a he ime when he hears were frozen. The blockade of he inoropic response was hus complee. The values for phosphorylase b kinase are shown in Table. Nehalide and dichloroisoproerenol, when adminisered alone, appeared o cause very sligh increases in phosphorylase b kinase aciviy as compared wih he normal Tyrode s soluion conrol. Adminisraion of epinephrine, afer eiher blocking agen, produced no significan change in enzyme aciviy as evidenced by he ph 6.8 o 8.2 aciviy raios. Clearly, acivaion of he enzyme by epinephrine was blocked by hese wo agens under condiions in which he inoropic acion of epinephrine was also abolished. DSCUSSON The daa indicae ha phosphorylase b kinase is acivaed in perfused ra hears by low doses of epinephrine. The acivaion is especially eviden when conrols are used in he assay which compensae for side reacions occurring when ATP is incubaed wih crude hear exracs (8). A characerisic feaure of he acivaion process in perfused hears is ha i was never complee as deermined by he increase in ph 6.8 o 8.2 raios. Tha is, ph 6.8 o 8.2 raios never approached 1, bu were always near 0.22 as maxima. On he basis of sudies in viro (8, 9), he enzyme would seem o have considerably greaer poenial for acivaion, bu his was no achieved even wih doses as high as 2 and 5 pg (8). No reason for his is apparen. One can only surmise eiher ha a significan porion of he enzyme in he cell is no acivaed by epinephrine adminisraion or ha acivaion in he cell is quaniaively differen from ha in viro. Acivaion in perfused hears, however, followed he same general paern as would be expeced from he sudies in viro. ncreased aciviy relaive o conrol values was greaer a ph 6.8 han a ph 8.2, a consequence of which was increased ph 6.8 o 8.2 raios. should be poined ou also ha, in our sudies wih purified phosphorylase b kinase, acivaion by ATP and cyclic AMP was never complee eiher, i.e. ph 6.8 o 8.2 raios approaching 1 were no obained alhough considerably higher raios han 0.22 were found. The acivaion, alhough modes in degree, was generally ypical of fl-adrenergic aciviy, as would be expeced. Thus isopropylnorepinephrine, an adrenergic amine wih pure fl aciviy, caused an acivaion of he enzyme in perfused hears essenially idenical wih ha of epinephrine, while causing a maximal inoropic response. On he oher hand, mehoxamine, an (Yadrenergic amine, which is unable o excie he hear, failed o acivae phosphorylase b kinase in perfused hears even a a dose of 100 pg. Furhermore, he acivaing effec of a maximal dose of epinephrine (0.1 pg) was eliminaed by he P-adrenergic blocking agens, dichloroisoproerenol and nehalide, under condiions in which he inoropic response of epinephrine was abolished. A sriking feaure of he acivaion of phosphorylase b kinase was he exreme rapidiy wih which i occurred following epinephrine injecion. The enzyme was essenially maximally acivaed wihin 1 se afer he adminisraion of 0.1 pg of he amine. Furhermore, in experimens in which hears were frozen a peak inoropic response, acivaion of phosphorylase b kinase increased wih increasing dose of epinephrine in a manner ha closely paralleled he ino,ropic response. We have shown (11, 12) ha phosphorylase acivaion in perfused ra hears reaed wih epinephrine did no seem o be associaed wih he inoropic response. An increase in conracile force of abou 50% above he conrol occurred before a rise in phosphorylase a levels was eviden, i.e. wih doses above 0.06 pg. Such doses of epinephrine in he presen sudy gave maximal acivaion of phosphorylase b kinase. Oher invesigaors (4, 5, 13) have shown in ime sudies ha he rise in phosphorylase a levels lags significanly behind he inoropic response. This, and oher evidence (3), has led o he feeling ha acivaion of phosphorylase is dissociaed from he inoropic response. would seem likely from he presen sudies ha phosphorylase 6 kinase is acivaed considerably before phosphorylase a levels begin o rise. This would no be unreasonable if he wo enzymes were srucurally separaed in he cell. The acivaion of phosphorylase b kinase followed very closely he ime sequence of cyclic AMP formaion, boh evens being essenially maximal in 1 sec. Perhaps phosphorylase b kinase is delicaely associaed in some way wih membrane srucures so ha i is acivaed insananeously by he cyclic AMP produced by adenyl cyclase, a membranebound enzyme. Phosphorylase being free in he cyoplasm could hen be acivaed afer a ime lag. The presen resuls would lend suppor o he general hesis ha phosphorylase acivaion is caused by acivaion of phosphorylase b kinase-which is firs acivaed by cyclic AMP formed by epinephrine simulaion of adenyl cyclase. The precise relaionship of hese meabolic evens o one anoher and paricularly o he conracile evens mediaed by epinephrine is no clear. From he presen sudies i can now be said ha cyclic AMP levels are increased and phosphorylase 6 kinase is acivaed by

5 ssue of December 25, 1966 G.. Drummond, L. Duncan, and E. Herxman 5903 physiological levels of epinephrine which mediae he conracile aciviy. Cyclic AMP levels rise and phosphorylase b kinase becomes acivaed ahead of he conracile response, and hese evens mus surely be relaed in some unknown way o he mechanical response o epinephrine. REFERENCES 1. KUKOVETZ, W. R., HESS, M. E., SHANFELD, J., AND HAUOAARD, N., J. Pharmacol. Expl. Therap., 137, 122 (1959). 2. BELFORD, J., AND FENLEB, M. R., J. Pharmacol. Expl. Therap., 127, 259 (1959). 5. CHEUNQ. W. Y., AND WLLAMSON, J. R.. Naure (1965): 6. POSNER, J. B., STERN, R., AND KREBS, E. G., J. Biol. Chem., (1965). 7. HAM&RM&STE& K. E., YUNS, A. A., AND KREBS, E. G., J. Biol. Chem., 240, 986 (1965). 8. DRUMMOND, G.., AND DUNCAN, L., 241, 5893 (1966). 9. DRUMMOND, G.., AND DUNCAN, L., J. Biol. Chem., 241, 3097 (1966). 10. POSNER, J., HAMMERMESTER, K., BRATVOLD, G., AND KREBS, E. G., Biochemisry, (1964). 11. DRUMM~ND, G.., VALADARE;, J.. R. E., AND DUNCAN, L., Proc. So. Exwl. Biol. Med (1964). 3. MAYER, S. E., COTTEN, M. DEV., AND MORAN, N. C., J. Phar- 12. DRUMMOND, G.-., VALADARE;, J. k. E.1 AN; DUNCAN, L., in macol. Expl. Therap., 139, 275 (1963). W. M. PAUL, E. E. DANEL, C. M. KAY, AND G. MONCKTON 4. ROBSON, G. A., BUTCHER, R. MORQAN, H. E., (Ediors), Muscle, Pergamon Press, New York, 1964, p AND SUTHERLAND, E. W., Molecular Pharmacol., 1,168 (1965). Aca Physiol. Sand., 66, 251 (1965).