OF PHOSPHORYLASE BY EPINEPHRINE IN PERFUSED CONTRACTING HEART, LIVER SLICES AND

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1 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright C i967 by The Willims & Wilkins Co. Vol. 156, No. 3 Printed in U.S.A. THE EFFECT OF VARIATIONS OF ph UPON THE ACTIVATION OF PHOSPHORYLASE BY EPINEPHRINE IN PERFUSED CONTRACTING HEART, LIVER SLICES AND SKELETAL MUSCLE ROBERT C. REYNOLDS AND NIELS HAUGAARD Deprtments of Anesthesi nd Phrmcology, School of Medicine, University of Pennsylvni, Phildelphi, Pennsylvni Accepted for publiction Jnury 16, 1967 ABSTRACT REYNOLDS, ROBERT C. AND NIELS HAUCAARD: The effect of vritions of ph upon the ctivtion of phosphorylse by epinephrine in perfused contrcting hert, liver slices nd skeletl muscle. J. Phrmc. exp. Ther. 156: 17-25, The effect of vritions of ph upon epinephrine ctivtion of phosphorylse ws studied in isolted perfused rt herts s well s in diphrgms nd liver slices incubted in vitro. The ction of epinephrine upon phosphorylse ws significntly enhnced in ll three tissues by n increse of ph from 6.9 to 7.8. In the perfused hert, the increse in the metbolic effect of epinephrine with rise in ph ws ssocited with n increse in the inotropic ction of the hormone. In diphrgm, n increse in ph lone cused rise in phosphorylse ctivity which could not be blocked by drenergic blocking gents but ws dependent upon the presence of clcium in the incubtion medi Pretretment of rts with dichloroisoproterenol blocked the ction of epinephrine upon diphrgm phosphorylse t ph 7.8 nd 6.9. In liver slices, epinephrine ctivity ws enhnced by n increse of PH, but the ction of cyclic 3,5 - denosine phosphte ws unffected by the sme chnge in hydrogen ion concentrtion. It is well known tht vritions in rteril blood ph cn produce ltertions in the ctions of ctecholmines. However, the ctul mechnisms by which sympthomimetic ctivity is frequently diminished with cidosis nd, in some cses, enhnced during lklosis hve never been understood. The present study ws designed to determine whether chnges in hydrogen ion concentrtion produced ltertions in biochemicl effects of epinephrine s well s in its physiologic ction. In recent yers, there hs been considerble interest in the reltionship between the physiologic ctions of the sympthomimetic mines nd the biochemicl chnges in the cell produced by these compounds (Ellis, 1956; Hugrd nd Hess, 1965). From the work of Sutherlnd nd his co-workers (Sutherlnd nd Rll, 196; Sutherlnd, ) it hs been estblished Received for publiction October 31, work ws supported by grnt (HE- 1813) from the Hert Institute of the Ntionl Institutes of Helth. 2Postdoctorl trinee in the Deprtment of Anesthesi Supported by U.S. Public Helth Service Reserch Trining Grnt 5-T1-GM tht the biochemicl ction of epinephrine in the cell involves n increse in the rte of synthesis of the coenzyme, cyclic 3, 5 -denosine phosphte (cyclic 3, 5 -AMP). Subsequently, in liver the rise in concentrtion of this compound cuses formtion of ctive phosphorylse from n inctive precursor. In skeletl muscle, hert nd brin, cyclic 3, 5 -AMP leds to trnsformtion of phosphorylse b to the more ctive form, phosphorylse The role tht these biochemicl events ply in the physiologic ctions of epinephrine still remins to be elucidted. In the hert stimulted by sympthomimetic mines, the ctivity of phosphorylse is incresed (Hess nd Hugrd, 1958; Kukovetz et ci., 1959; Myer nd Morn, 196). However, phosphorylse ctivtion is most likely not the immedite cuse of the increse in force of contrction of the hert produced by epinephrine. A positive inotropic effect without phosphorylse stimultion hs been observed with low doses of epinephrine (Myer et t., 1963; Drummond, 196). Seprtion of the mechnicl nd enzymtic effects of epinephrine on the hert hs been demonstrted lso by experiments in which 17

2 18 REYNOLDS AND HAUGAARD Vol. 156 force of contrction nd phosphorylse ctivity were followed t close time intervls. In such experiments, it ws found tht the inotropic ction of epinephrine commenced erlier thn the rise in phosphorylse ctivity (Willimson, 196; #{216}ye,1965; Robison et l., 1965). In experiments in which cyclic 3, 5 -AMP ws lso determined, the increse in concentrtion of this compound ws better correlted with the motropic effect of epinephrine thn ws phosphorylse ctivtion (Willimson, 196; Robison et ci., 1965). If the increse in the intrcellulr content of cyclic 3, 5 -AMP is the immedite cuse of the chnge in mechnicl ctivity of the hert, it ppers tht n ction of the coenzyme other thn phosphorylse ctivtion is involved in the initition of the increse in force of contrction. Horn et t. (1966) showed in experiments with the perfused rt hert tht incresed glycogenolysis ws not required for the initition of the inotropic ction of epinephrine but plys n importnt role in sustining the elevted force of contrction. Although n increse in phosphorylse ctivity my not be the cuse of the increse in force of contrction of the hert fter epinephrine dministrtion, phosphorylse ctivtion remins n importnt nd prominent ction of epinephrine in ll tissues studied nd is reflection of intrcellulr concentrtion of cyclic 3, 5 -AMP. In the study reported here, the effects of ph upon phosphorylse ctivtion by epinephrine in liver, skeletl muscle nd hert were investigted. A consistent effect of cyclic 3,S -AMP on phosphorylse ctivity cn be demonstrted in vitro in liver nd with this tissue the ction of cyclic 3, 5 -AMP s well s epinephrine ws studied. METHODS. Perfused rt herts. Wistr strin mle rts (2-25 g) were decpitted, the herts removed, weighed on torsion blnce nd ttched to Lngendorif perfusion pprtus. The herts were perfused with solutions of the following molr composition, described by Chenoweth (196):.12, NC1;.56, KC1;.2, CCl2;.2, MgCl2;.25 NHCO3; nd.1, glucose. The solutions were bubbled constntly with mixture of 5% COr- 95% 2; ph ws vried by ltering the content of NHCO, from. (ph 7.8) to.5 M (ph 6.95). The NCl ws vried to provide constnt osmolrity t ll ph vlues. Isometric force of contrction nd hert rte were mesured with force-displcement trnsducer (Grss, FT.3) connected to the pex of the hert with silk ligture. The herts were perfused for 1 to 15 mm prior to the infusion of epinephrine or.9% NC1 to permit the hert to rech stble level of contrctility. Freshly prepred solutions of epinephrine in.9% NCl or control injections of.9% NCl were infused through ctheters wrmed to 37#{176}C nd plced close to the coronry openings. All infusions were in volume of 2 ml injected over period of 13 sec. The effustes were collected, gssed with 5% C-95% nd the ph ws mesured. As soon s dose of epinephrine produced mximl effect, the hert ws dropped into slush of dry ice nd ethnol nd subsequently ground in mortr with cold solution of.1 M NF-.2 M ethylenediminetetrcetic cid (EDTA; s disodium slt). The extrct ws djusted to concentrtion of 1:15 nd nlyzed for phosphorylse nd totl phosphorylse ctivity by the method of Con nd Illingworth 1956). The finl concentrtion in the rection mixture ws 3.3 mg of tissue/ml. Results re reported s percentge phosphorylse of totl phosphorylse. Rt diphrgms. Albino mle rts (1-17 g) were decpitted nd the hemidiphrgms crefully removed, divided in hlf, weighed nd plced in ice-cold medium. The composition of the medium ws identicl to the bicrbontebuffered perfusion solution described for the isolted hert experiments. Segments of diphrgm were plced into 25-ml Erlenmeyer flsks contining 1 ml of medium, which hd been sturted with 5% C2-95%. After incubtion in Dubnoff incubtor t 37#{176}Cfor 5 mm,.1 ml of freshly prepred solutions of epinephrine or.9% NC1 ws dded nd the flsks regssed. At the end of 1 mm, the tissues were removed nd immeditely homogenized in mortr with 1 ml of cold.1 M NF-.2 M EDTA. The finl dilution ws 1 mg of tissue! 5 ml. Solutions remining in the incubtion flsks were regssed with 5% CO2-95% Os nd the ph ws mesured. Phosphorylse determintions were performed in mnner similr to tht described for the hert (Au et ci., 196). Results re reported s percentge of phosphorylse In one series of experiments with rt diphrgm, the nimls were pretreted with drenergic blocking gents 6 mm prior to decpittion. The rts received i.m. injections of either dichloroisoproterenol (DCI), 1 mg/kg, or dihydroergotmine (DHE), 5 mg/kg. Rt liver slices. Livers were removed rpidly

3 1967 EFFECT OF PH ON PHOSPHORYLASE 19 from mle Wistr rts nd chilled in bicrbonte-buffered solution. Outer slices,.5 mm thick, were prepred with Stdie-Riggs tissue slicer. The slices were blotted gently on filter pper, weighed nd plced in ice-cold Chenoweth s solution. The incubtion medi were identicl to the bicrbonte-buffered solutions employed in the perfused hert nd diphrgm experiments. After 5 mm of incubtion t 37#{176}C,.1-mi portions of epinephrine (1 g/ml), cyclic 3,5 -AMP (.1 M) or.9% NC1 (control) were dded to the flsks, which were regssed with 5% CO2-95% Os. After 1 mm of incubtion the slices were removed from the flsks nd immeditely ground in 1 ml of icecold.1 M NF-.2 M EDTA. Additionl cold NF-EDTA solution ws dded to the mortr to give totl volume of 3 ml/1 mg of wet tissue. Anlysis of phosphorylse ws performed s described by Brunner nd Hugrd (1965). Results re reported s units of ctive phosphorylse per ssy (16.6 mg of tissue). Drugs. Epinephrine ws prepred dily from smple of crystlline l-epinephrine bitrtrte generously supplied by Sterling-Winthrop Reserch Institute, Rensseler, N.Y. All doses or concentrtions re expressed s microgrms of free bse. Cyclic 3,5 -AMP ws obtined from Sigm Chemicl Co., St. Louis, Mo. DCI ws obtined s the hydrochloride slt from the Aldrich Chemicl Co., Milwukee, Wis. Ampules of DHE methnesuifonte (1 mg/mi) were from Sndoz, Hnover, N.J. Sttisticl nlysis of results ws performed using the Student s t test (Snedecor, 1956). When the P vlues were less thn.5, the difference between two observtions ws considered to be significnt. RESULTS. Effect of vrition of ph upon epinephrine ctivity in the perfused rt hert. Figure 1, A nd B, presents dt obtined from isolted rt herts perfused with solutions t ph 7.8 nd 6.9. In figure 1A the inotropic effects of epinephrtne re plotted s functions of the mounts of epinephrine dded to the perfusion fluid. Figure lb depicts dt obtined from the phosphorylse nlyses of the sme herts. The ph vlues presented in figures 1, A nd B, re those of the solutions prior to perfusion of the herts. The ctul ph of the solutions in the coronry vessels ws probbly slightly lower since mesurements of the effustes reveled tht the ph of the perfusion solutions ws.1 to.2 of ph unit below the initil vlues. The drop in ph ws the sme t ph 7.8 nd 6.9. The vrition of ph hd no significnt effect upon the strength of contrction of the isolted herts in the bsence of dded epinephrine. At ph 6.9, the men control mplitude of contrction ws.9 ±.29 mm nd t ph 7.8 the control mplitude ws 5.15 ±.8 mm. There ws significnt difference (P =.2) between the smll chnge in contrctile force produced by the control injection of 2 ml of.9% NCl t ph 6.9 (-.53 ± 15 mm) nd t ph 7.8 (-.7 ±.3 mm). There ws no significnt difference in the control levels of phosphorylse t ph 6.9 (3.6 ±.5%) nd t ph 7.8 (27.9 ± 1.2%). Chnges in the force of contrction produced by injections of epinephrine in mounts of.3 to 1. tg were significntly greter t ph 7.8 thn t ph 6.9 (fig. 1A). Only when the dose of epinephrine ws incresed to 3. g did the effect of the drug t ph 6.9 equl tht t ph 7.8. With this dose of epinephrine, the ction of the drug t ph 6.9 reched the mximl effect on crdic contrctility obtined t ph 7.8 with n mount of epinephrine s low s.1 g. As seen in figure 1B, the effect of epinephrine on phosphorylse ctivity ws greter t ph 7.8 thn t ph 6.9 t ll doses of the hormone. The dose-response curve t ph 6.9 for the effect of epinephrine on contrctility (fig. 1A) hs initilly sigmoid shpe. We hve no dequte explntion for the sudden increse in contrctile force when the dose of epinephrine ws incresed from 1 to 3 g. Effects of vritions of ph upon epinephrine ctivtion of phosphorylse in skeletl muscle. Dt obtined from rt diphrgms incubted with.1 to 3. g/ml of epinephrine t ph 7.8 nd 6.9 re presented in figure 2. As in the hert experiments, the percentge of ctive phosphorylse ws significntly greter t ph 7.8 thn t ph 6.9 over wide rnge of concentrtions of epinephrine. At both ph vlues, mximl effect of epinephrine ws produced by concentrtion of.3 p.g/ml. Incresing the concentrtion of epinephrine 1-fold bove this vlue did not significntly ffect the phosphorylse ctivtion t either ph. The effect of single, submximlly effective concentrtion of epinephrine on rt diphrgm phosphorylse ws determined t four different ph vlues (fig. 3). There ws no significnt difference in the ctivtion of phosphorylse

4 2 REYNOLDS AND HAUGAARD Vol. 156 I- z w U, 1 Cl) E E Li. U, -J I- I- z I EPINEPHRINE, gm U, U) -J I I EPINEPHRINE, gm Fm. 1. The effect of epinephrine upon contrctile force nd phosphorylse in perfused rt herts t ph 7.8 nd 6.9. Chnges in contrctile force (A) nd percentge phosphorylse (B) from the sme herts re plotted ginst the log dose of epinephrine. Ech point represents the men dt obtined from 8 to 1 herts, with S.E.M. represented by brckets. produced by epinephrine (.5 jag/mi) when the ph ws rised from 6.9 to 7.2; however, there ws significnt, brupt increse in epinephrine ctivtion of phosphorylse when the ph of the incubtion medium ws incresed bove ph 7.2 to 7.6 nd 7.8. It should be noted tht there ws lso significnt increse in the mount of ctive phosphorylse present in the control segments of rt diphrgm, without epinephrine, s the ph of the incubtion solution ws incresed bove 6.9. In spite of the increse in control phosphorylse, the effect of epinephrine (difference between the percentge of phosphorylse with nd without epinephrine) ws significntly incresed with rise of ph bove 7.2. Pretretment of rts with DCI, 1 mg/kg, completely bolished the effect of epinephrine (.5 jag/mi) upon the isolted diphrgm t ph 6.9 nd 7.8 (tble 1). However, the increse in phosphorylse ctivity produced by the chnge of ph from 6.9 to 7.8 ws unffected by DCI. DHE pretretment, 5 mg/kg, did not influence the level of phosphorylse ctivity under ny of the experimentl conditions (tble 1). The bicrbonte-buffered incubtion or per-

5 1967 EFFECT OF PH ON PHOSPHORYLASE 21 fusion solutions used in the experiments with diphrgm nd isolted herts reported thus fr contined 2 mm CC12. In the experiments with rt diphrgm presented in tble 2, the CCl2 content of the bicrbonte-buffered incubtion medi ws incresed to mm or ws omitted from the solutions. In ddition to the bicrbonte-buffered incubtion medi, phosphtebuffered medium (. M sodium phosphte,.87 M NC1,.5 M KC1 nd.2 M MgCl2), which could contin no more thn.2 m1 clcium in solution t 37#{176}C(Neumn nd Neumn, 1958), ws lso used. In the bsence of epinephrine, there ws no significnt effect of clcium on phosphorylse ctivity of diphrgm segments incubted t ph 6.9. In contrst, t ph 7.8 there ws significnt decrese in control phosphorylse ctivity 5 U, U) 3O U) 2C ii EPINEPHRINE.5 1JGRAMSIML IC EPINEPHRINE, blgrams/ml FIG. 2. The effect of epinephrine upon rt diphrgm phosphorylse t ph 7.8 nd 6.9. Ech point on the grph represents the men of to 8 experiments with S.E.M. represented by brckets. There is significnt difference between the effect of epinephrine t ph 6.9 nd the effect of the sme dose of epinephrine t ph 7.8. c OEPlNTRINE b.b Fic. 3. The effect of vritions of ph upon phosphorylse ctivity in the rt diphrgm with nd without epinephrine. Ech point on the grph represents the men of 7 to 11 experiments with S.E.M. represented by brckets. ph TABLE 1 The effect of pretretment of rts with drenergic blocking gents on phosphorylse ctivity of the rt diphrgm incubted with nd without epinephrine t ph 6.9 nd 7.8#{176} Percentge of Phosphorylse ± S.E.M. Pretretment N ph 6.9 ph 7.8 No epinephrine Epinephrine (.5,g/m1) No epinephrine Epinephrine (.5 5g/ml) Control (no pretretment) DCI (1mg/kg) DHE (5mg/kg) ±.6. ±.9 c 3.8 ± ± ±.86 (P =.23) 19. ± ± ± ± ± ± 1.9 (P <.1) 5.9 ± 6.58 #{176}Rts were given i.m. injections of drenergic blocking gents 6 mm prior to scrifice nd removl of the diphrgms. Diphrgm qurters were incubted in bicrbonte-buffered solutions with nd without epinephrine t ph 6.9 nd 7.8 for 1 mm, nd then they were nlyzed for phosphorylse ctivity. N = number of experiments. Ech P vlue derived by comprison to its corresponding control.

6 22 REYNOLDS AND HAUGAARD Vol. 156 TABLE 2 Effect of vrying concentrtions of clcium in incubtion medi t ph 6.9 nd 7.8 upon the ctivtion of rt diphrgm phosphorylse with nd without epinephrine The bicrbonte-buffered solutions were gssed with 5% C2-95% 2. The phosphte-buffered medi were gssed with 1% 2. Tissue segments were exposed to epinephrine (.5 pg/mi) or to.1 ml of.9% NCI (controls) for 1 mm t 37#{176}Cnd then were nlyzed for phosphorylse ctivity. Percentge of Phosphorylse S.E.M. Medium ph 6.9 ph 7.8 N#{176} Control Epinephrine Difference N Control Epinephrine Difference No clcium ±.6. ±.5 b 2.9 ±.9.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.65 (P =.12) ± 1.15 (P =.2) 38.5 ± ± ± ± ± 2.29 #{176} N = number of experiments. b For evlution of significnce ech figure is compred with the corresponding vlue obtined with bicrbonte medium contining 2. mm CCl2. TABLE 3 Effect of time nd ph upon epinephrine ctivtion of rt diphrgm phosphorylse Flsk 1 2 Bicrbonte-buffered incubtion medi 2.mM clcium.mm clcium Phosphte-buffered incubtion medium No clcium Di- No Procedure Control#{176} Incubtion Incubtion 2 jb Incubtion 3 Phosphorylse ph 7.8 ph 6.9 %*S.E.M. 9.1 ± ± ± ± ± ± 2.35 N.S.#{176} 23.3 ± ± 2.5 P -. 2C N.S. #{176}Forthe control,.1 ml of.9% NCI ws dded to the flsk. Incubtion time ws 1 mm. b In flsk no.2 three rt diphrgm segments were exposed to single dose of epinephrine (.5 pg/mi) for successive 1-mm incubtion periods in phosphte-buffered incubtion medi t ph 7.8 or 6.9. The gs phse ws oxygen. There were experiments t ech ph. Compred to Incubtion 1 t ph 7.8. d Compred to Incubtion 1 t ph 6.9. when clcium ws eliminted from the bicrbonte-buffered solution or when phosphte-buffered incubtion medium ws used. A significnt increse in phosphorylse ctivity ws produced by rising the ph from 6.9 to 7.8 with ll of the bicrbonte-buffered medi However, there ws no significnt difference between the phosphte-buffered controls t ph 6.9 nd 7.8. In contrst to the influence of clcium upon the control levels of phosphorylse t ph 7.8, the clcium ion hd no effect on the ction of epinephrine on diphrgm phosphorylse t either ph. Since chnges in stbility of epinephrine with ph could ffect our results, control experiments were performed to determine whether ltertion of ph influenced the rte of inctivtion of epinephrine. Three diphrgm qurters from the sme niml were incubted successively for 1-mm periods in the sme phosphte-buffered solution contining submximlly effective concentrtion of epinephrine (.5 jag/mi). A fourth segment of diphrgm ws incubted with the sme conditions in the bsence of epinephrine. Phosphorylse nlyses of these diphrgms re reported in tble 3. At ph 7.8 nd 6.9, the ddition of epinephrine (flsk no. 2) during the first incubtion period produced significnt increse in phosphorylse

7 1967 EFFECT OF PH ON PHOSPHORYLASE 23 TABLE Effect of vritions of ph upon epinephrine nd cyclic 8,5 -AMP ctivtion of phosphorylse in rt liver slices#{176} Phosphorylse Activity Vritions Control Epinephrine (1. pg/mi) f Differenceb Control I Cyclic (.1 3 im) 5 -AMP Derence ph 6.9 ph 7.8 P units/ssy.5 ± ± ± ±.31 I 7.22 ±.11 I 1.6 ±.23.2 <.1.6 units/ssy.28 ± ± ± ±.28 I 7.31 ± ±.19 <.1.3 N.S. #{176}Rt liver slices were incubted with.1 ml of.9% NCl (control), epinephrine or cyclic 3,5 -AMP t ph 6.9 nd 7.8 for 1 mm t 37#{176}C. Bicrbonte-buffered medi were used with 5% COr-95% in the gs phse. Twelve experiments were conducted for ech condition. Differences re clculted from the results of pired experiments. ctivity, compred to the controls without epinephrine (flsk no. 1). As noted in previous experiments, the ctivity of epinephrine t ph 7.8 ws significntly greter thn t ph 6.9. The second incubtion of diphrgm qurter in the sme epinephrine solution produced n ctivtion of phosphorylse tht ws not significntly different from tht produced by the first incubtion t ph 7.8 or 6.9. It ws only fter the third 1-mm incubtion tht significnt decrese in phosphorylse ctivtion by epinephrine could be noted t ph 7.8. The difference in phosphorylse ctivity in the first nd third incubted diphrgm qurters t ph 6.9 ws not significnt. Effect of vritions of ph upon ctivtion of phosphorylse in rt liver slices by epinephrine nd cyclic 3,#{2} -AMP.Tble presents dt from experiments with rt liver slices incubted in bicrbonte-buffered medi t ph 7.8 nd 6.9 in the bsence nd presence of epinephrine (1. /Lg/ml). In ddition, the effect of cyclic 3, 5 - AMP (.1 mm) ws lso studied. There ws significnt increse in phosphoryise ctivity in the control liver slices when the ph ws incresed from 6.9 to 7.8. Epinephrine produced ctivtion of liver phosphorylse t both ph vlues; however, the effect of epinephrine (difference between the level of phosphorylse ctivity with epinephrine nd control phosphorylse ctivity) ws incresed significntly s the ph of the incubtion medium ws rised from 6.9 to 7.8. In contrst to results with epinephrine, the effect of cyclic 3, 5 -AMP on liver phosphorylse ws not ffected by the chnges of ph employed in these experiments. DIsCussIoN. The effects of vritions of ph upon ctecholmine ctivity hve been studied by investigtors using mny different biologic preprtions. Studies with perfused herts (Snyder nd Andrus, 1919; Slnt nd Johnston, 192; Ahlgren, 193), in situ herts (Schipp, 1933; Virtue nd Simmons, 1955; Guzmn et l., 1959; Drby et l., 196; Keith et l., 196) nd studies on crdic excitbility in mn (Cmpbell et l., 1958) hve demonstrted tht sympthomimetic ctivity is incresed with lklosis nd decresed with cidosis. The type of cidosis or lklosis-i.e., respirtory or metbolic-seems to mke little difference in the results obtined. Other preprtions nd tissues in which ctecholmine ctivity hs been reported to be similrly ffected by vritions of ph include bronchil smooth muscle (Blumenthl et l., 1956, 1961; Mithoefer et l., 1965) nd nictitting membrne (Tenney, 1956). The experiments presented in this pper show tht the ction of epinephrine on biochemicl rections in the cell is lso influenced by chnges in hydrogen ion concentrtion. In the three tissues studied epinephrine becomes more effective in ctivting the enzyme phosphorylse s the ph is incresed. In considering the effect of ph chnges upon ny biologic ctivity of drug, it is importnt to tke into ccount the extent of ioniztion of the drug over the rnge of ph studied. Epinephrine hs two pk vlues ner the ph rnge utilized in this study. Lewis (195) hs reported tht one of the phenolic groups of epinephrine hs pk vlue of The pk vlue for the mine group ws reported to be 9.9. These

8 2 REYNOLDS AND HAUGAARD Vol. 156 results hve been confirmed by Kisbye (1958) who reported pk vlues for epinephrine of 8.7 nd 1.1. Using the pk vlues determined by Lewis, it cn be clculted tht epinephrine exists s pproximtely 95% ction, % zwittenon nd 1% nion nd undissocited molecule t ph 7.5. This reltionship of the four different ionic species of epinephrine does not chnge pprecibly unless the ph is rised bove 8.. The single pk of cyclic 3, 5 -AMP of 3.82 (Lipkin et l., 1959) is considerbly out of the rnge of ph exmined in this study. From these considertions, it cn be ssumed tht the ltertions of epinephrine ctivity with ph reported here re not the result of vritions in the ioniztion of epinephrine or cyclic 3, 5 -AMP but re reflections of chnges t the cellulr level. The control experiments, in which severl diphrgms were incubted consecutively in the sme epinephrine solution, showed tht deteriortion of epinephrine during incubtion ws unlikely to be significnt fctor in these experiments. It is of considerble interest tht the ltertion of the ctivtion of phosphorylse in the hert is ffected by the sme vrition of ph tht lters the inotropic response of the hert to epinephrine. These results re n dditionl illustrtion of the close reltion tht exists between the mechnicl effects of epinephrine nd the effects on the cyclic 3, 5 -AMP-phosphorylse system. Epinephrine ctivtion of phosphoryise in skeletl muscle ws ffected by chnges of ph in mnner similr to tht of hert muscle. Increses of ph of the perfusion nd incubtion solutions produced n increse in epinephrine ctivtion of phosphorylse in both crdic nd skeletl muscle. One mjor difference between the two tissues ws the fct tht n increse of ph lone ppered to ctivte skeletl muscle phosphorylse in the bsence of epinephrine. The fct tht this increse in phosphorylse ctivity could not be blocked by drenergic blocking gents suggests tht it is not cused by relese of ctecholmines. Experiments performed with vrying concentrtions of clcium in the diphrgm incubtion solution indicte tht the spontneous ctivtion of phosphorylse my be relted to the ction of clcium. Krebs et cii. (1959) hve shown tht clcium ions re cpble of ctivting the intrcellulr enzyme, phosphorylse b kinse, of rbbit skeletl muscle, nd it my be speculted tht clcium hs n effect t this site in the experiments reported here. Experiments with liver slices showed tht the effect of epinephrine ws influenced by ph in mnner similr to tht in crdic nd skeletl muscle. In ddition, it ws demonstrted in this tissue tht n elevtion of ph which cused mrked increse in epinephrine ctivtion of phosphoryise hd no effect on the ction of cyciic 3, 5 -AMP. This indictes tht the hydrogen ion concentrtion influences cellulr function prior to the formtion of cyclic 3, 5 - AMP. The site of ction my be the cyclse system, which is believed to be ssocited with cell membrnes (Dvoren nd Sutherlnd, 1963). A finl nswer to this problem must wit the results of experiments in which tissue concentrtions of cyclic 3, 5 -AMP re determined directly. Both in hert nd in diphrgm, the mximi effect of epinephrine ws much less t ph 6.9 thn t ph 7.8. When the epinephrine concentrtion t the lower ph ws incresed to produce its pek effect on phosphorylse, the mjor portion of the enzyme ws nevertheless in the b form. It ppers tht the bility of tissues to respond to the glycogenolytic ction of epinephrine is dependent on the hydrogen ion concentrtion. At low vlues of ph the extent to which the phosphoryise system cn be ctivted by epinephrine is much smller thn t higher ph vlues. One my speculte tht the resistnce to the crdiovsculr nd bronchil effects of high doses of ctecholmines seen in ptients with cidosis (Cmpbell et l., 1958; Blumenthl et l., 1961; Mithoefer et l., 1965) my be relted to similr biochemicl ltertions in the cells of the trget orgns. In cses of cidosis in vivo the chnges in ph re smller thn those studied here with the perfused hert. However, they re of sufficient mgnitude to expect tht significnt chnges would occur in the responsiveness of the cyclic 3, 5 -AMP-phosphorylse system to ctecholmines. REFERENCES AHLGREN, G.: Uber die Einwirkung von Kohlens#{228}ure, Bikrbont und H-ionenkonzentrtion uf #{252}beriebende Orgn und ihre Beeinflflssbrkeit durch Phrmk Em Beitrg zur Frge der Zusmmensetzung der Serumslzl#{26}sung. Sknd. Arch. Physiol. 59: 1-23, 193. ALl, H. I. E. S., ANTONIO, A. AND HAUGAARD N.:

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