EFFECT OF QUINIDINE, PROCAINE AMIDE AND LIDOCAINE ON CALCIUM TRANSPORT BY SUBCELLULAR PARTICLES OF THE RABBIT HEART
A Thesis Presenfed to the University of Mqnitobo
In Portiql Fulfillment of the Requirements for the Degree of Mosfer of Science in Physiology
by
Jqmes A.C. Horrow Deportmeni of Physiology Foculty of Medicine Augusf, 1973 ACKNOWLEDGEMENTS
I wish to extend my sincerest grotitude fo Dr. Noronion S. Dhollo for his potience ond guidonce fhroughout the course of my studies ond for providing me with the opportunity ond focilities to corry ouf this investigotion. I would qlso like fo thonk Dr. K.R. Hughes for his encourogement qnd odvice.
I would like ïo thonk my porents for the interesf, understonding ond qssistonce they hove given me during my university coreer.
I wish to offer my oppreciqfion to my colleogues ond friends of the
Deporfmenf of Physiology, especiolly Chorles Tomlinson, Ken Vorley, Irwin
Borowski ond Ben McNomoro whose help ond qdvice greotly benefited me. I would olso f ike to fhonk Chris Ring ond Jogot Singh for fheir ossistonce in the preporotion of this monuscript.
This study wos supported by gronfs to Dr. N.5. Dhollo from ihe Medicol
Reseorch Council of Conodo. TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
INTRODUCTION AND STATEMENT OF THE PROBLEM
REVIEW OF L]TERATURE
A. Current Concept of Regulof ion of Colcium in the Heqrf
B. Effects of Vorious Phormocologic Agents on Cqlcium Tronsporf in Heorf
METHODS
A. Isolotion of Sorcoplosmic Retîculum ond Mitochondrio t2
B. Isolqtion of Sorcolemmol Froction l2
C. Meosuremenf of Colcium Tronsport t3
I . Colcium binding 13
2. Colcium uptoke by microsomes t4
3. Colcium uptoke by mitochondrio 14
4. Cqlcium odsorpfion 14
RESULTS
A. Cqlcium Binding 16
B. Colcium Upfoke 2l
C. Colcium Adsorpfion 26
DISCUSSION 33
CO NCLUS IO NS 38
REFERENCES 39 LIST OF FIGURES
Figure
I Effect of vqrious concentrotions of quinidine on colcium binding by robbit heqrf heovy microsomes ond mitochondrio t7 {-f' Influence of Mg on the effecf of 1 mM quinidine on colcium binding by robbit heort heqvy microsomes ond mifochondrio 20
Effect of vorious concenirotions of quinidine on colcium uptoke by robbit heorf heovy microsomes ond mifochondrio 23 -4. Influence of Mg on fhe effecf of I mM quinidine on colcium uptoke by robbit heorf heqvy microsomes 27
Effect of vqrious concentrotions of quinidine, procoine omide ond lidocoine on colcium odsorption by robbit heort heovy microsomes 29
Effect of vorious concentrotions of quinidine, procoine omide ond lidocoine on colcium odsorption by robbit heori miiochondrio 3l
Effecf of vqrious concenfrqtíons of quinidine, procoine omide ond lidocoine on colcium odsorpfion by robbit heqrf sorcolemmq 32 LIST OF TABLES
Tqble Poge
Time course of colcium binding by robbit heori heovy microsomes in the presence of I mM quinidÏne, procoine omide qnd lidocoine 18
Time course of cqlcium binding by robbit heorr mifochondrio in fhe presence of Ï mM quinidine, procoine omide ond lidocoine 19
Influence of ATP on the effeci of I mM quinidine on colcium binding by robbit heorf heqvy microsomes ond mitochondriq 22
4 Time course of colcium upfoke by robbit heort heovy microsomes in the presence of I mM quÎnidine, procoine omide ond lidocqine 24
Time course of cqlcium uptoke by robbit heqrt mitochondrio in the presence of I mM quinidine, procoine omide ond lidocoine 25
6 Influence of ATP on the effect of I mM quinidine on colcium uptoke by robbit heod heqvy microsomes ond mitochondrio 28 ABSTRACT
Quinidine hos been reported to depress cqlcium uptoke ond colcium
sfimulqied ATPose of the dog heort sorcoplosmic reticulum, however, no informotion
concerning the oction of this ogent on other orgonelles such os mitochondrio ond
sorcolemmo, which qre olso considered to porticipote in colcium regulotion in heort,
is ovoiloble in the literqture. The present investigoiion wqs undertoken fo study
the effecfs of quinidine on colcium binding ond upioke by robbit heort sorcoplosmic
reficulum os well os mifochondrio. The ocfion of this ogent on Mg**- ATP
independenf colcïum odsorption by these orgonelles ond sorcolemmc¡ wos olso studied.
Two ofher ogents, procoine qmide ond lidocoine were used for comporison purposes.
Quinidine wos found to sfimulote colcium binding by heovy microsomes
(in fhe presence of ATP) ot initiol infervols of íncubotion ond oi low concentrotîons
of Mg . Procqine omide, but not lidocoine, olso showed stimulotion of colcium
binding by microsomes. On the ofher hond, quinidine wos found to depress colcium
binding by mitochondrio. This depressont effecf wos not opporenf ot initiql intervols
of incubotion, low concentrofions of ATP or Mn**. Lidocoine bui not procoine omide olso depressed cqlcium binding by mitochondrio ot lote time infervols of incuboTion.
Quinidine in concentrqtions of t0-4 ond l0-3 M produced o morked depressont effect on microsomol (in the presence of ATP ond oxolofe) ond miiochondriol (in fhe presence of ATP, P¡ ond succinote) cof cium uptoke. The depressonl effect of quinidine on microsomol ond mifochondriol colcium uptoke wos observed ot oll concenfrotions of ATP but wqs not opporent when low concentrqf ions of Mg** *ur. used in the incubotion medium. Procqine omide hod no effecl on microsomol or mitochondriol colcium upfoke whereos lidocoine showed o stimulotory effect only on microsomol colcium upfoke qf initiql infervols of incubotion.
Quinidine ot concenfrqtions of l0-4 to l0-2 M stimuloted colcium -l--l-'- odsorpf ion (Mg' ATP independeni) by microsomol, mitochondriol ond sorcolemmol frocfions. Procoine omide hqd no effecf on sorcolemmq, siimulqted microsomol cqlcium odsorption ond produced o biphosíc effect on mifochondriol colcium odsorpfion.
Lidocoine, on fhe other hond, hod o depressont effecf on these froctîons ot high concentrofions.
These results suggest fhot quinidine moy be ocfing of microsomol, mitochondriol ond sorcolemmo levels. The oction of quinidine in terms of colcium odsorption, binding ond upioke seems to be of o complex noture ond possesses some similoritíes ond differences from other ogenfs, procoine omide ond lidocoine. INTRODUCTION AND STATEMENT OF THE PROBLEM
Quinidine in low concenfrofions hos on onf iorrhythmic oction, whereos in high
concentrotions it is known to depress myocordiol confrocfility ond produce confrocture of skeletol muscle (l - 3). Vqrious investigotors hove ottempfed to exploin fhese phormocologicol effecfs of quinidine on cqrdioc ond skeleiol muscles on the bosis of its oction on colcium tronsporf by sorcoplosmic reficulum (4 - 8). Ii should be noted thot colcium is generolly believed fo serve qs o link between excitofion ond confrocfion of the cordioc ond skelefol muscles (1r 9 - 12). However, unlike skeletol muscle where sorcoplosmic reficulum ploys o predominqnt role in ihe regulofion of introcellulor colcium, the colcium movements in cordioc muscle ore considered fo be reguloted by sorcoplosmic reticulum, mitochondrio ond sqrcolemmo (13 - 21). Therefore, it wqs the purpose of this study to investigqte the obilities of both sorcoplosmic reficulum (heovy microsomes), ond mitochondrio fo bind ond occumulote colcium in the obsence ond presence of quinidine under vqrious experimentol condifions. In oddition, the effects of ihis ogent on fhe Mg*- ATP independenf colcium odsorption by these subcellulor frqctions ond sorcolemmo were invesfigofed. Preliminory resulfs reported in this study were presenfed before fhe Conodiqn Federotion of Biologicol Societies (22).
-l - REVIEW OF LITERATURE
A. Currenl Concept of Regulqtïon of Colcium in the Heort
Colcium ís now generolly believed to be the fínol octivotor of the controcfile
system of cordioc muscle (1, 10, 12,20,21,23,24). Currenf theory stotes fhqt
extrocellulor colcium enters the heqrt cell during depolorizofion qnd is olso releosed
from superficiol sifes on the sqrcolemmo. In qddifion, colcium is considered to be
releosed from internol storoge sites such os sorcoplosmic reliculum. The introcellulor
level of ionized cqlcium is thus roised obove lO-7 lrrt. This cof cium binds to îroponin,
fhe colcium receptive protein ot the confroctile opporotus ond stimulofes octomyosin
ATPose. Binding of cqlcium to Ìroponin releoses its inhibition upon fhe scfin ond
myosin system whereos stimulof ion of ATPqse ond subsequent hydrolysis of ATP provides
fhe energy for sliding of the myofilomenfs. The intrqcellulor level of colcium is fhen
lowered to opproximotely lO-7¡¡t by the sorcoplosmic reticulum os well os ocfive
efflux of cofcium ocross fhe sorcolemmo thus resulfing in reloxofion of the cordioc
muscle. Although mitochondrio ore obundqnt in ihe cqrdioc cef I ond ore oble to
fronsport colcium, their role in roising ond lowering fhe intrqcellulqr concentrotion of
ionized colcium during confroction ond reloxotion, respecTively, hos nof yef been
cleorly defined.
While colcium tronsporf properties of sorcoplosmic reficulum ond mitochondrio hsve been extensively studied, the sorcolemmo of cqrdioc muscfe hqs nof received much oftenfion. This is porfly due to the foct fhot fhere ore difficulties in isoloting this frocfion in o relotively pure form. However, colcium hos been shown fo be required for electricol ocfivify of the sorcolemmo ond its influx hos been shown fo
-2- -3-
contribute, olongwith Nq+ ond K+, to fhe mognitude qnd durotion of the oction
potentiol (20, 2l). Recenily o number of invesf igqtors hove demonstrofed the presence
of q Co#- stimuloted ATPose in heorf sorcolemm a (25 - 27). This enzyme cqn be
considered fo be involved in fhe movement of colcium ocross the sorcolemmo. Although
some of the invesfigotors hove reporfed the isolofion of heort sorcolemmo with octive
No+- K+ ATPoru ond odenylote cyclose (28 - 30), no informotion concerning its
obility to tronsport colcium is ovqiloble.
The bosemenf membrone ihot coofs fhe heort sorcolemmq is o mucopoly- socchqride or mucopolysocchoride - protein complex contoining negotively chorged sites (31). This bqsement membrone olso lines fhe surfoce of the T-sysfem. It hos been demonstroted by Scott (32) thot the mucopolysocchorides hove o strong offinity for cofions notobly colcium. Recently, Modeiro ut g!_. (33) were oble io show thot sorcolemmol vesicles isoloted from skeletol muscle hqve Ìhe obility to bind ions
-|_|-'++' ' ' notobly Co' , Mg' ond Zn' . These workers hove proposed thoi onionic Aroups in the sorcolemmo, either phosphoric sifes of phospholipids or coråoxyl groups of profeins moy bind these cotions. If the phospholipids were removed by phospholipose C, fhere wos o decreose in cotions binding. Zinc ions showed the highest offinity, whereos Co# ++ ond Mg" hod fhe some offinity. Cordiqc sorcolemmo con olso be conceived to hove similor colcium binding sites which mc¡y serve os o source of trigger colcium in the process of exciiofion-confroction coupling (34, 35).
Since fhe work of Ebqshi ond Lipmonn (3ó), in which they were oble to show fhe obility of frogmented sorcoplosmic ref iculum vesicles to occumulote rqdio- -4- ocfive colcium in fhe presence of ATP ond Mg#, it is generolly occepied ihot colcium qvoilqble for binding to froponin is derived olmost enfirely from the sorcoplosmic reficulum. This colcium is mode ovoiloble by possive releose from the sorcoplosmic reficulum qfter the orrivql of the wove of depolorizotion from fhe T-system (12).
The role of sorcoplosmic reticulum in the control of colcium levels in ihe muscle cell is olso indicoted by the very close onotomicql relqtionship of the sorcoplosmic
reficulum ond myofibrils (37, 38). Furthermore, in vifro studies hove shown thof heorf , os well os skeletol muscle sorcoplosmic reticulum con bind qnd occumulote cqlcium
(17 , 18, 39 - 41). In ihe presence of colcium precipitoting ogents such qs oxolofe or
ínorgonic phosphote, cordioc sorcoplosmic reticulum hqs been shown to occumulote micromolor quonfifies of cqlcium. Ikemoto ut g!. (42) hove shown the presence of
insoluble colcium sqlts within the microsomol vesicles. In the obsence of the
precipitoting ogent, the infrovesiculor precipifofion of colcium solts does not occur.
The ferm "colcium binding" is commonly opplied to the obility of microsomes to foke up
cqlcium in the obsence of the precipitoting ogents. On rhe other hond, the term
"cqlcium uptoke" is usuolly employed to signify colcium occumulotion by microsomes
în the presence of the precipitoting ogents.
A number of experimenfol condifions hove been shown fo influence colcium
tronsport by cordioc sorcoplosmic reticulum. The concentrofions of No+ ond K* hove
been found to offect colcium upfoke (43r 44). High concenfrotions of NoCl ond KCI
were demonstrofed to inhibit colcium upfoke, whereos low concentrofions of these ions
cqused on enhoncement of initiql rote ond moxÌmol upfoke; K* *o, more effective thon
No+. Both ATP ond Mg# were olso found fo be importont for colcium uptoke, which is now well esroblished to be qn energy dependent process. When ATP ond Mg* were omitted from fhe incubotion medium only o smoll omount of cqlcium wos occumulqfed by the frogments of sorcoplosmic reticulum ond this process hos been referred to os possive cqlcium odsorption which is primorily due to o physicol interocfion of colcium with the membrone (40). Lee (45) hos demonsfroted thqf isoloted sorcoplosmic reticulum could releose colcium in vitro following electricol stimuloiion. This cqlcium releose wqs found io be dependent upon fhe frequency of sïimulotion.
The controversy of whether sorcoplosmic reticulum ploys the predominont role os o storoge site for colcium in the heorf hqs been roised by morphologicol ond biochemicol evidence. Ii should be recognized thot most of the work concerning fhe mechonisms of excitotion-controcfion coupling hos been done using sorcoplosmic reticulum from skelefql muscle. Although the properfies of sorcoplosmic reticulum from heort hos been shown to be similor to thot of skeletol muscle, cordioc muscle hos o sporse sorcotubulor system (38). Furthermore, fhe qbsence of tronsverse fubules ond the poucity of sorcoplosmic reticulum in the frog venfricle suggesf the existence of other mechonisms for fhe regulotion of introcellulor cqlcium in cordioc controction (46).
Also kineiic evidence provided by Kofz ond Repke (47)høs shown thot colcium uptoke by cordioc sorcoplosmic reiiculum wos too slow fo occount for relqxotion.
From the originol work of Clelond ond Slofer (48), in which they showed thot mitochondrio could toke up colcium, qttention is being gîven to this event occuring in heort miiochondrio os o possible mechonism for the regulotion of introcellulor cqlcium.
Vorious workers (44,49 - 52) hove shown thot mifochondrio in the presence of ATP con occumulofe Co**. When mifochondrio were equilibroted with inorgonic phosphote -6- ond cqlcium, elecfron dense gronules, possibly due to ihe precipitofion of cqlcium solts, were observed under fhe elecfron microscope (50). The hydrolysis of ATP by mitochondrio hos been shown to occur concommiffonily wíth colcium occumulotion + os well os H eiection inlo the incubotion medium (52). The energy dependent cqlcium upioke by mitochondrio is inhibited by oligomycin (53) ond ozide (44). Divqlenf cofions such os Mg* ond Mn** stimulote colcium upfoke by mitochondrio whereos
ADP, orfhophosphote ond high concentrof ions of Nq+ ond K+ reduce colcium occumulotion (51).
Thot mitochondriq do ploy q role in heort cqlcium metobolism hos been supported by the work of Pofriorco ond Corofol¡ (13) in which these workers hove shown 45Co* thot mosf of the iniected in onimols wos crccumulqted by heort mitochondrio qnd thot the specific octivity of colcium in mitochondrío wos higher thon thot of the sorcoplosmic reticulum. Similor results were found by Dhollo el ol. (18) in the isolqted perfused heort preporofions. Also mifochondrio hqve been found to inhibit super- precipitotion by myofibrils (54). Mitochondrio ore obundonf in heort cells ond ore sifuoted in close proximity to the myofibrils (14, 16, 17). Furthermore, fhe heorts were unoble fo relox when perfused with oligomycin, o well known inhibitor of colcium upioke by mitochondrio (ló). These sfudies tend to support fhe role of mitochondriq in fhe reguloiion of infrocellulqr colcium in fhe heort. Hougoord ond his ossociotes
(15) hove olso implicoted this role of mitochondrio in the controction-reloxotion cycle of the myocordium. -7 -
B. Effecfs of Vqrious Phormocologic Agenfs on Colcium Tronsport in Hecrt
A number of phormocologicolly octive ogenfs hove been shown fo influence the cqlcium fronsporf ing sysfem of both sorcopfosmic reficulum qnd mirochondrio of the heort" Confliciing reports in fhe literofure hqve not settled the mode of ocfion of the cordioc glycosides. Mony invesiigotors hove reporfed fhot ouqboin ond strophonfhidin depress colcium uptoke in mitochondrio ond sorcoplosmic reiiculum (55 - 58).
However, o number of ofher investigotors clqim thot the cordiqc alycosides hqve no effect on colcium qccumulotion by fhe subcellulor porficles (17, 47, 59, 60). In view of fhese results, it is difficult io exploin the positive inotropic effecfs of the cordioc glycosides on the bosis of fheir oction on the colcium tronsporf by The sub- ce llu lor porf ic les .
The effect of the cofecholomines ond cyclic 3'r5'-odenosine monophosphofe
(cyclic AMP) on colcium fronsporf hos olso received much ottention in order to exploin the positive inotropic octions of these ogents. However, conflicting reports hove oppeored in fhe liferqture. Some investigotors hqve reporfed thof epinephrine, norepinephrine ond cyclic AMP ougment cqlcium occumulotion by sorcoplosmic reticulum
(61 - 64). Sorcoplosmic reticulum hos olso been shown to contoin odenylote cyclose
(65 - 67). The stimulqtion of this enzyme by cotecholomines with increosed formotion of cyclic AMP is considered to occount for fhe qctíon of cofecholomines (ó3). Thus the increosed cqlcium content of the sorcoplosmic reticulum ollows more cqlcium to be mode qvoiloble to the conÌroclile opporotus duríng depolorizoiion (ó3). The hypoihesis thot increosed levels of cyclíc AMP cquse on ougmenfotion of the sqrcofubulor cqlcium pool is interesting in the sense thot it helps fo exploin the positive inotropic -8- effect of vorious inierventions ot lhe moleculor level. However, other workers hove foiled to confirm the existence of such qn effect of cyclic AMP (47, 68). Likewise, bofh glucogon ond cotecholomines hove been reported fo be ineffective in enhoncing colcium upfoke by the heort sqrcofubulor vesicles (47, 59, 68). These sfudies suggesf thof The proposed mechonism of hormonol oclion on colcium fronsport ocross sorco- tubulqr vesicles should be token with some coution.
Recenily cordiqc sorcofubulor membrones possessing odenyloTe cyclose ond cqlcium occumuloting octivities hove been shown io contoin cyclic AMP stimulofed protein kinose (ó9). If hqs been suggested thqf cyclic AMP stimuloies the formofion of o membrone phosphoprotein which mediqtes fhe cycf ic AMP induced chonges in colcium fronsport. The experimenfs described by Kirchberger.t g!. (70) reveoled fhof ihe oddition of exogenous protein kinose in high concentrotions wos necessory fo show the effeci of cyclic AMP on colcium uptoke. Since Ìhese workers foiled to observe on ocfion of cyclic AMP - protein kinose on cqlcium binding by sorcofubulor vesicles, fhe significonce of their reporfed effeci on cqlcium upfoke is subiecf to some serious questions. In this regord, it should be mentioned thqt Gertz uf gl-. (7I) hove ottributed the slimulofory effect of cyclic AMP fo q non-specific profecfive effect of fhe nucleofide to retord fhe in vifro deferiorotion of cordioc sorcotubulor colcium uptoke. Furfhermore, cyclic AMP hos been shown lo be wifhout on effect on fhe phosphoryl tronsfer reoction, which moy represent fhe formotion of o corrier system ond fhus focilitote the influx of colcium info fhe sqrcotubulor vesicles (72).
The borbiturotes hove been found to depress colcium uptoke by sorcoplosmic reticulum ond mitochondrio. Noyleret ql. (73) ond Loin ef ol. (74) hove reported -9 - thqf millimolor concenfrofions of sodium pentoborbitql interfered with the obility of cordioc microsomes to occumulote colcium. Briggs uf gl. (75) found fhot omytol
inhibiied colcium uptoke in microsomql vesicles of the dog heort buf the inhibition could be prevenfed by strophonfhín. However, Dronsfield et ql. (76) foiled io detect ony effecf of I to ó mM pentoborbifol on cqlcium uptoke by microsomes but hove reporfed inhibiiion of colcium uptoke by mitochondriq.
The beto-recepfor blocking ogents hove been found fo depress myocordiol controclility ond this ocfion hqs been ottributed to the effecls of ihese ogenfs on the colcium tronsporfing system of the heort. Vorious investigotors (6, 77 - 79) hove shown thoi propronofol, olprenolol ond pronetholol qre copoble of depressing colcium uptoke by heorf mitochondrio os well os sorcoplosmic reficulum. The results of these experiments ore difficult fo inferpref becouse of the high concentrqf ions of drug used qs compored to thot in vivo.
The effects of the ontiorrhythmic drugs on heoÉ function hove usuolly been interpreted on the bosis thof these ogents ore oble to olter the electrophysiologicol properties of the myocordium (2r 3, 80). However, o number of reports hove oppeored recenTly in the literqture with respecf fo the effects of these ogents on coicium tronsporf in heort ond sJceletol muscle subcellulor systems. In both cqses, quinidine qt concentrofions of l0-4 ond l0-3 M hos been shown fo depress cqlcium uptoke ond # Co - octivoted ATPose of sorcoplosmic reficulum (4 - fl. These concentrofions of quinidine couse tw¡fch potentiofion qnd confrocture in skeletol muscle (81 ,82) whereos cordiqc muscle in ihe presence of this ogent shows o significont depression of tension development (80). Isoocson ond Sondow (82) oftributed fhe effect of quinidine on skeletol -10- muscle such thof this ogent is oble to either depress cqlcium occumulotion by sorco- plosmic reticulum or releqse colcium from this orgonelle fhereby roising the free myoplosmic concentrqtion of this ion ond prolonging the ocfive stote. However, Fuchs et ol. (4) otfribuied the reduction of myocordiol fension development io depression of colcium occumulotion by heqrt sorcoplosmic reticulum but the increose in myoplosmic colcium would be quickly reduced by ofher cqlcium tronsport systems such os mitochondriq ond sqrcolemmo. Some impetus wos given fo fhis concept by
Corvolho (5) when he showed thof skeletol muscle sorcoplosmic reficulum in the presence of the opticol isomer of quinidine, quinine, wos oble to releose the possively bound colcium, thof is, in fhe obsence of ATP ond depress the selective binding of colcium induced by ATP. Although quinidine depressed cqlcium upfoke in vitrg, lhis is not definitive evidence for the ontiqrrhythmic effecf of ihe drug in rl"". This is portly due fo the foct fhot lhe concentrofions used ore greofer thon the theropeufic ronge. However, Conn (84) hos shown thot quinidine binds to sorcolemmo qnd mitochondrio ond Bolzer (7) using frifium lobelled dihydroquinidine, hos shown binding of on omouni of quinidine fo skeletql muscle sorcoplosmic reficulum comporoble to fhe concentrofion required to elicii 50olo inhibirion of cqlcium uptoke.
Severol reports hove oppeored in the literofure fhoï ore contrqdictory with observotions of other workers. The work of Scqles ond Mclnfosh (85) hos shown thqf quinidine of o concentrotion of 2 x lO-4 tvt coused q smqll but significqnt increose in fhe torol ATP dependent colcîum binding ond upfoke by skeletol muscle sorcoplosmic reticulum. These workers hove olso reported fhoi quinidine sfimulqted toiol ,ATPose ocfivity of this frqction. These resulfs ore in disogreemenf wifh Fuchs et ol. (4) -il-
qnd Bolzer (7) who hove reported o depression of fhe ATPose octivity of sorcoplosmic
reticulum from bofh heqrt ond skeletol muscle. Quinidine hos olso been shown fo ocf
on colcium tronsporting systems ofher thqn sorcoplosmic reiiculum. Noock (77) hos
shown thof quinidine decreqsed the velociry of colcium upfoke qnd sfote 4 respirotion
by heort mitochondriq without influencing the efficiency of oxidotive phosphorylofïon.
Groco ond Von Zwieten (8ó) hove shown fhof 5 * t0-5 M quinidine reduced ihe rote of 4tco* cellulor ond exfrocellulor exchonge in electricolly driven isoloted guineo pig
qlrio. They ottribute fheir results io o diminished membrone permeobility for colcium
during excifqtion due fo quinidine. Modeiro ond Cqrvqlho (33) working with skelefol
muscle sorcolemmo hqve shown fhot quinine compefitively inhibited colcium binding
in o medium contoining opproximotely 100 to 200 FM colcium. Quinidine (l mM)
olso hqs been shown fo reducu No*- K+ ATPor" octivity in bovine heori sorcolemmo
by 5O/o (87); fhis inhibition wos not Mg# dependent becouse vorious concentrqtions -t-+' of Mg (3 to 4 mM) showed no significont chonge in ihe inhibitory effecf . These
results showing fhe effecf of quinidinu on No*- K+ ATPor" con be interpreted fo reflecf the qction of fhe drug of sorcolemmo. METHODS
A. Isolotion of Sorcoplosmic Reticulum qnd Mitochondrio
Heolthy mole olbino robbits were socrificed by cervicol dislocotion ond the
heorfs quickly excised ond ploced in ice cold homogenizing medium contoining
0.25 M sucrose, I mM EDTA ond 20 mM Tris-HCl, pH 7.0. The oirio were dissected out ond fhe ventricles trimmed of fqf ond connective tissue. The ventricles were weighed qnd minced with scissors. The tissue wos homogenized in l0 volumes of medio in o Woring Blendor for 2x 20 seconds with o 1 minute infervol. The homogenofe wos filtered through 2loyers of gouze ond centrifuged ot 1,000 x g for
20 minutes to remove cell debris. The supernotont wqs cenfrifuged of 10r000 x g
for 20 minutes to obtoin the mitochondriol pellef . This pellef wos woshed qnd suspended in the homogenizing medium, spun of 11000 x g for l0 minufes, the residue discorded ond the supernofont further centrifuged ot 8r000 x g for l0 minufes to obtoin mitochondriol froction. The posf 10,000 x g supernofont wos centrifuged ot 401000 x g for 45 minuies, the pellel woshed, resuspended in 0.ó M
KCI contoining 20 mM Tris-HCl, pH ó.8 - 7.0 ond centrifuged ot 40,000x g for
45 minutes to seporote heovy microsomes. Both mitochondriol ond microsomol froctions were suspended in 0.25 M sucrose, l0 mM Tris-HCl, pH 7.0 ot o profein concentrotion of 1 to 2 ng/ml. This procedure for isolqting mitochondriq ond heovy microsomes is simifor to thot used previously in this loboroiory (88).
B. Isolotion of Sorcolemmo Froction
Robbit heort ventricles were woshed thoroughly, diced with o poir of scissors ond homogenized in l0 volumes of 50 mMTris-HCl, pH 7.4, contoining
-12- -13- t mM EDTA in o Woring Blendor îor 2x 30 seconds with on intervol of I minute. 'l1000 The homogenote wos filtered ihrough gouze ond centrifuged ot x g for'10 minutes. The sedimenf wos suspended in 25 volumes of l0 mM Tris-HCl , pH7.4 ond stirred in the cold room for 30 minutes qnd centrifuged ot 11000 x g for l0 minutes. The residue wos then suspended in 25 volumes of l0 mM Tris-HCl, pH
8.0, siirred for 30 minutes qnd centrifuged ot 11000 x g for l0 minutes. The sediment wqs suspended in l0 mM Tris-HCl , pH 7.4, stirred for 30 minules qnd centrifuged of 11000 x g for l0 minutes; this srep wos repeoted ogoin. The sedimenf wos suspended in 25 volumes of l0 mM Tris-HCl, pH 7.4, exlrocled with 0.4 M LiBr for45 minutes ond centrifuged ot 1,000 x g for l0 minuies. This sedimenT wqs woshed with l0 mM Tris-HCl, pH 7.4, ond suspended in I mM
Tris-HCl , pH7.4, ond immediotely used. The froction isoloted in the obove monner will be referred to os sorcolemmql froction. This method is the some os thof described by McNqmorq (89) ond is o modificofion of thot described by Kono ond Colowich (90).
C. Meosurement of Cqlcium Tronsport
I . Colcium binding
Colcium binding by mitochondrio, microsomes ond sqrcolemmo wos meosured in o medium consisting of 100 mM KCI ,2mM MgClr, 20 mMTris-HCl, pH ó.8to 45coclr,2 7.0, o.l mM mM No2-ATP in o fotol volume of I to3 ml. The mitochondriol protein concenfrofion in the incubqtion medium wos 0.2to 0.3 mg/ml.
The sorcolemmol ond microsomol frqctions were incubqted of o profein concentroiion -14-
of 0.1 to 0.2 mg/ml. The froctions were pre-incuboted for 3 minufes ot 25"C in scocl, the presence of ATP ond drug. The reqcfion wos storied by the oddiiion ol
(New Englond Nucleor, Dorvol, Quebec) ond stopped by millipore filtrqf ion 45Co (Millipore Corporotion, pore size 0 .45 ¡t). The qmount of in 0.1 ml of filtroie
wos onolyzed in I0 ml of Broy's solufion in q Pockord Tri-Corb scinfillqtion
spectrometer.
2. Colcium uptqke by microsomes
Colcium uptoke by microsomes wos meosured by the method described for
binding except thot 5 mM potossium oxolofe wos qdded fo fhe incubofion medium ond
0.02 to 0.05mg/ml microsomol protein ot o temperqfure of 37oC wos employed.
3. Colcium uptoke by mitochondrio
Colcium uptoke by mitochondrio wqs defermined qt 37"C in the colcium
binding medium in the presence of 4 mM inorgonic phosphote (Pi) ond 5 mM succinqfe.
A mitochondriol protein concentrotion of 0.t to 0.2 ng/ml wqs used
4. Colcium qdsorption
Colcíum odsorption by mícrosomol, mitochondriql, ond sorcolemmol froctions
wos studied by the method described for colcium bindîng except thot Mg# ond ATP
were excluded from the incubotion medium. The protein concenfrof ion in fhe
incubotion medium wqs 0.1 to 0.2 mg/ml. The temperoture of the medium wos 25oC.
All of the qbove procedures for isolofing subcellulqr froctions were corried out in o cold room ot 0 to 4oC. Either o Sorvoll RC2-B or on Infernqtionol 820-A refrigeroted centrifuge wqs used. The proiein concenfrof ion wos defermined occording -t5-
To the method of Lowry et ol. (9,|'). The results were onolyzed stotisficolly by the conventionql student "t" tesf.
Quinidine gluconote U.S.P. wos obtoined from El¡ L¡lly ond Compony,
Indionopolis, Indiono, procoine omide hydrochloride, U.S.P. (Pronestyl) from E.R.
Squibb ond Sons Ltd., Monfreol, Quebec qnd lidocoine hydrochloride, U.S.P.
(Xylocoine) from Astro Chemicqls Ltd., Mississougo, Onforio. RESULTS
A. Colcium Binding
Colcium binding by heorf heovy microsomes ond mifochondrio wos determined
in the presence of vqrious concenfrofions of quinidine qnd the results ore described
in Figure l. No significont (P > 0.05) chonges în cqlcium binding by heovy microsomes wqs observed. However, mitochondriol colcium binding wos significontly
(P < 0.05) depressed by l0-4 ond lO-3 ¡¡t quinidine. The time course of colcium binding by heort heovy microsomes in fhe presence of I mM quinidine, procoine omide ond lidocoine is shown in Tqble l. Quinidine ond procoine omide hod o significont
(P < 0.05) stimulotory oction ot the eqrlier fime infervols of incubotion. On the other hond, lidocoine exerted no significont (P > 0.05) effect ihroughout fhe course of incubotion. The time course of colcium binding by heort mitochondrio in ihe presence of I mM quinidine, procoine omide ond lidocoine is shown in Toble 2.
Quinidine ond lidocqine, unlike procoine omide, showed o significont (P ( 0.05) depression of colcium binding oi the lqter time intervqls of incubotion.
Cqlcium binding by heort heovy microsomes qnd mitochondriq in the obsence ond presence of t mM quinidine wos defermined of vqrious concenfrqtions of Mg*
in fhe incubotion medium. The resulfs concerning the effect of quinidine ore shown
in Figure 2. At low concentrofions of Mn** quinidine exerfed o signÌficonf
(P < 0.05) stimulotory effeci on microsomol colcium binding. On the other hond, quinidine did not influence mifochondriol colcium binding ot concenfrotions of ..j+' Mg up to I.5 mM; o significonf (P < 0.05) decreose in colcium binding wos opporenf qi 2 mM Mg*. Colcium binding by heort heovy microsomes qnd
-16- -17 -
J o ú. zl- o o àe r.+ --r I r9z x------{,, o z MITOCHONDRIA m- =2 o J O
765 - LoG EQUlrrllolruelvr
FIGURE I Effecf of vorious concentrotions of quinidine on colcium binding by robbit heort heovy microsomes ond mifochondrio. The incubotion medium wos the sqme os thoi described for colcium binding in the Meihods section except ihot l0 mM Mg# ond 4 mM ATP were used. The time of incubotion of the subcellulor frqctions with 45Co* *o, 5 minufes. The control volues for mïcrosomol ond mitochondriol colcium binding were 49 + ó ond 43 t3 nmoles/mg protein respectively. Eoch volue is o meon t S.E. of 5 experiments. -t8-
TABLE I
Time Course of Colcium Binding by Robbif Heort Heovy Microsomes in the Presence of I mM Quinidine, Procoine Amide qnd Lidocqine
Heovy microsomol colcium (nmolesr/mg protein)**
Incubolion time Control Quinidine Procoineomide Lidocoine
30 sec 37 !3 ól t3* 4s !3 4t !2 1 min 47 +3 64!1* 66!4* 54 !3 2 min ss !2 ó3 13 óó13* ó3 13 5 min ó0+3 71 +4 76!2* 72+4 l0 min 72+2 72!4 74!4 75+3
P < 0.05 +& Eoch volue is q meon t S.E. of ó experimenfs. The incubotion medium wos the sqme os thof described for colcium binding in the Methods section. _19 _
TABLE 2
Time Course of Cqlcium Binding by Robbit Heort Mitochondrio În the Presence of I mM Quinidine, Procoine Amide ond Lidocqine
Mitochondriol colcium (nmoles/mg profein)**
Incubotion time Conf rol Quinidine Procoine omide Lidocoine
30 sec 39+3 47+4 52!4 3ó+3 1 min 45 !3 46 !3 s6!4 55+2 2 min 64 !2 48!2* 59 13 5313* 5 min 69 t4 54+3* 76t4 53 !2* l0 min 73+3 58+4* 7e !4 54+4*
P < 0.05 Eqch volue is o meon tS.E. of ó experiments. The incubotion medium wos the some os lhot described for colcium binding in fhe Methods secfion. -20-
J o É. F z o HEAVY U MICROSOMES I s -.J I () Tr-_ / z L\- õ 120 z m E f 100 o MITOCHONDRIA c)
0.8 1.2 [vrs.] mNt
FIGURE 2 Influence of Mg'' on the effect of I mM quinidine on colcium
binding by robbit heorf heovy microsomes ond mitochondrio. The incubqtion medium wqs the some os thot described in the Methods
section excepf vorious concentrqtions of Mg* were employed. The 45ao++ time of incubotion of the subcellulor froctions with wos 5 minutes. Eqch vqlue is o meqn + S.E. of 5 experimenfs. -21 - mifochondrio of vorious concenlrotions of ATP in the obsence qnd presence of I mM quinidine is shown in Toble 3. Heovy microsomes in ihe presence of quinidine over the concenfrotion ronge of ATP tested showed no significont (P > 0.05) differences in colcium binding from fhot of microsomes incubqted in the obsence of the drug. The depression due to quinidine on cqlcium binding by mitochondrio wos nof observed qt low concentrotions of ATP whereos quinidine (l mM) significonfly (P < 0.05) depressed mitochondriol colcium binding ot higher concenfrqtions (l to 5 mM) of ATP.
B. Cqlcium Upfoke
The effect of vorious concentrqf ions of quinidine or cqlcium upfoke by heort heqvy microsomes ond mifochondrio is shown in Figure 3. Quinidine qf low concenfrofions exerfed no effecl (P > 0.05); however, ot high concentrofions
(lO-4 qnd l0-3 M) quinidine significontly (P < 0.01) depressed colcium uptoke by both heovy microsomes ond mifochondrio. The time course of colcium uptoke by heovy microsomes in the presence of I mM quinidine, procoine omide ond lídocoine is shown in Tqble 4. Significonf depressont oction (P < 0.05) of quinidine become opporenf ofter 2 minufes of incubotion. Microsomql colcium uptoke in the presence of procoine omide wqs unoffected (P > 0.05) whereos lidocoine significontly
(P <0.05) sfimuloted uptoke during the firsi 2 minufes of incubotion.
The time course of colcium uptoke by heorf mitochondrio in the presence of I mM quïnidine, procoine qmide ond lidocqine is shown in Toble 5. Mitochondriol colcium upfoke wos significontly (P < 0.05) depressed by quinidine during the l0 minule incubqtion period. Procqine qmide qnd lidocqine exerted no significont _22_
TABLE 3
Influence of ATP on the Effecf of I mM Quinidine on Colcium Binding by Robbit Heort Heovy Microsomes ond Mitochondrio
Colcium binding (nmoles/mg protein)**
Heovy microsomes Mirochondriq
(ATP) mM Control Quinidine Control Quinidine
0.05 38 13 30 !2 35+2 39+3 0.10 47 !2 53!4 40 !2 48!2 0.50 53 13 s7 !4 s7 !3 53+3 I .00 5et3 55 13 ó0 13 45!4* 2.00 7t !4 65 !2 68!4 50 !2* 5.00 35t2 31 +2 59+2 47 !3*
P < 0.05 Eoch volue is q meon t S.E. of 5 experiments. The incubotion medium wqs the some os thqt described for colcium binding in the Methods secfion except fhof vqrious concenfrqtions of ATP were employed. -23-
MITOCHONDRIA
d1 É. zF o o è€ uJ Y F o- f HEAVY E f MICROSOMES õ o
FIGURE 3 Effect of vqrious concentroiions of quinidine on colcium uptoke by robbit heort heovy microsomes qnd miiochondrio. The incubofion medium wos fhe some os described for cqlcium uptoke in fhe Methods seciion excepf ihot l0 mM Mg*ond 4 mMATP were used. The time 45ao++ of incubotion of the subcellulqr froctions with wos 5 minufes. The control volues for microsomol ond miiochondriol colcium uptoke
were 1342 + 162 ond 110 + 4 nmoles/mg protein respecfively. Eoch volue is o meqn + S.E. of 5 experimenfs. -24'
TABLE 4
Time Course of Colcium Uptoke by Robbit Heort Heovy Microsomes in the Presence of I mM Quinidine, Procoine Amide ond Lidocoine
Heovy microsomql colcium (nmoles/mg protein)**
Incubofion time Control Quinidine Procoine omide Lidocoine
30 sec 355 + 34 457 + 22 476 t 35 675 ! 35* I min 529 !37 4e0 ! 21 585 + 38 l0ó8 t 80* 2 min 813 + 24 586 ! 42* 7t8 ! 35 1280 1 I02* 5 min 1272 + 79 ó48 I 53* 1047 ¡ 70 1385 t I l0 l0 min 1291 + 85 801 + 45* 1162 + 104 1414 + 107
P < 0.05 Eoch volue is o meon 1S.E. of ó experiments. The incubofion medium wos fhe some os fhoT described for cqlcium uptoke in the Meïhods secfion. -25-
TABLE 5
Time Course of Colcium Uptoke by Robbit Heqrf Mitochondriq in the Presence of I mM Quinidine, Procoine Amide ond Lidocoine
Mitochondriol colcium (nmoles/mg protein)**
Incubotion time Confrol Quinidine Procoine qmide Lidocoine
30 sec 128!4 8l 13* l3Btó 127 !6 I min ls8t2 112 t 6* r4013 129 t3 2 min 155t3 125 ! 4* 143!4 1s6!4 .l30 5 min 169!5 1 5* 153 + 3 18s t7 10 min 20318 142 ! 6* 179 !6 194+7
P < 0.05 ++ Eoch volue is o meon tS.E. of ó experiments. The incubotion medium wos fhe some qs fhof described for colcium uptoke in fhe Methods section. -26-
(P > 0.05) effeci on mitochondriol colcium upfoke during the course of incuboiion.
The effect of vqrious concenfrof ions of Mg* on colcium upfoke by heorf
heovy microsomes in the obsence ond presence of I mM quinidine ís shown in ++ Figure 4. At Mg concentrofions less thqn 0.75 mM quinidine exerted no
significont (P > 0.05) effect on microsomol colcium upioke. At higher concentrotions {-+ of Mg" quinidine significontly (P < 0.05) depressed colcium occumulotion by the
microsomql froction. Preliminory results showed o similor trend of quinidine ocf ion
on mitochondriol colcium uptoke. Colcium uptoke by heort heovy microsomes ond mitochondrio in the obsence ond presence of I mM quinidine of vorious concentrotions of ATP is shown in Toble ó. Both mícrosomol ond mitochondriol cqlcium uptoke were significontly (P < 0.05) reduced in fhe presence of quinidine ot oll ATP concenfrotions fesfed.
C. Cqlcium Adsorpiion
Heovy microsomol colcium odsorption (Mg*- ATP independent bindíng) in fhe presence of vorious concentrqtions of quinidine, procoine qmide ond lidocoine is shown in Figure 5. Low concentrqtions of quinidine (10-7 to l0-4 M) ond procoine omide (10-7 to l0-3 M) d¡d nof influence odsorption. At high concentrqtions these drugs significonily (p < 0.05) sfimuloied cqlcium odsorpfion by heovy microsomes.
Lidocoine, on the ofher hond, showed no effect of low concentrofions (l O-7 to lO-3 ¡¡t) but ot higher concenfrofions if significontly (P < 0.05) depressed colcium odsorption.
Mitochondriol colcíum odsorption in fhe presence of vqrious concenfrqtions -27 -
lmM QUINIDINE
tt 1.4 2.0 [*n + l.vl
^t--L FIGURE 4 Influence of Mg" on the effect of I mM quinidine on colcium uptoke by robbit heort heovy microsomes. The incubotion medium wqs the some os fhot described in fhe Methods section except vorious concentrotions of Mg** were employed. The fime of 45Co* incubotion of the heovy microsomes with wos 5 minutes. Eqch vqlue is o meon t S.E. of 5 experiments. -28-
TABLE 6
Influence of ATP on the Effect of I mM Quinidine on Colcium Upfoke by Heorf Heovy Microsomes ond Mitochondrio
Cqlcium uptoke (nmoles/mg prolein)"*
Heovy microsomes Mitochondrio
(ATP) mM Control Quinidine Conf rol Quinidine
0.05 563 t 2s 394 ! 19* 85 15 63 +,4* 0.10 620 !22 433 !26" 88 14 69 !4* 0.50 844 J 30 534 !20* r0l t4 77 !5* I .00 1059 r 32 739 !35* I13tó 87 !4" 2.00 1209 ! 42 860 ! 44* 133 t 5 94!5* 5.00 440 + 39 322 t28" 9l t3 67 +3*
P < 0.05 +ù Eoch vqlue is o meqn t S.E. of 5 experiments. The incubqtion medium wos fhe some os thof described for colcium uptoke in the Methods section except fhot vorious concentrotions of ATP were employed. -29 -
HEAVY MICROSOMAL CALCIUM ADSORPTION 80 ._QUINIDINE O-_PROCAINE AMIDE c---LIDOCAtNE z l¡J F o 860 0- o) € + + ord u,4o o õ \J_ITT I------r-Ilifl\r
20 \ I t76s4g2/ -uoc [onuc] vr
FIGURE 5 Effect of vorious concentrotions of quinídine, procoine omide ond lidocoine on colcium odsorption by robbit heort heovy microsomes. The incubofion medium wos fhe some qs fhot described for colcium binding in the Methods secf ion except thoi Mg# ond ATP were excluded from the medium. The time pf incubqfion of the heovy microsomes with 45Co* wos 5 minutes. The control volues for
microsomol colcium odsorption wos 42 + 3 nmoles,/mg protein. Eoch vqlue is q meqn t S.E. oî 4 experiments. -30- of quinidine, procoine omide ond lidocqine is shown in Figure ó. Quinidine ot concentrotions greoter thon lO-4 ¡¡t morkedly (P < 0.01) stimulofed colcium odsorption. Procoine omide hod o slight but significonr (P < 0.05) depressont effect ot concenfrotions less thon lO-5 tt¡ but showed o significont (P < 0.05) sfimulotion ot o concenfrqtion of l0-2 m. On the ofher hond, lidocoine produced o slighi but signifícont (P < 0.05) depressont effecf ot high concentrotions (10-4 -2 fo l0 - M).
Sqrcolemmol colcium odsorption in the presence of vorious concentrof ions of guinidine, procoine omide ond lidocoine is shown in Figure 7. Quinidine significontly (P < 0"05) enhonced colcium odsorption buf only or high concentrofions -2 -t (10 " qnd l0 'M) whereos procoine omide exerted no effect over fhe ronge of concenlrotions Tested. Lidocoine, on fhe other hond, significontly (P < 0.05) depressed colcium odsorpfion of o high concentrotion (lO-2 tut). -31 -
MITOCHONDRIAL CALCIUM ADSORPTION
.-QUINIDINE AMIDE l¡J ,__PROCAINE =ts o---LlDocAlNE o-IYJ o- o) E + + orú Ø3 o o E
654 - Loc fonuc] vr
FIGURE ó Effect of vorious concentrotions of quinidine, procoine omide qnd
lidocqine on colcium odsorption by robbit heort mitochondriq. The incubofion medium wqs the some qs thot described for colcium binding in ihe Methods section except thot Mg++ ond ATP were excluded from the medium. The time of incubqtion of the heovy 45Co* microsomes with wos 5 minutes. The control volues for mitochondriol colcium odsorption wqs 2l !2 nmoles/mg protein. Eoch volue is q meon t S.E. of 4 experiments. -32-
SARCOLEMMAL CALC IUM
150 ADSORPTION .-QUINIDINE 4 __PROCAINE AMIDE [¡ o---LIDOCAINE o É. 0-110 o) E + + ()rü a 9zo o E
tf ? 30
'l-r-¿ r o" 7 6 5 4 3 z -Loc fonuc] rvr
FIGURE 7 Effeci of vorious concentrofions of quinidine, procoine omide ond lidocoine on colcium odsorpfion by robbit heqrf sorcolemmo. The incubqÌion medium wos ihe some os ihof described for cqlcium binding in the Methods secfion except thot Mg# ond ATP were excluded from the medium. The fime of incubqlion of the heovy microsomes w¡th 45Co* *o, 5 minutes. The control volue for sorcolemmol cqlcium odsorptÌon wos 90 t 5 nmoles/mg profein. Eoch volue is q meqn 4 S.E. of 4 experiments. DISCUSSION
The colcium tronsport system by subcellulor porticles of lhe heqrt oppeors to be o complex process. Cqlcium con be conceived to inferoct with biomembrones fhrough two processes which ore energy independent ond energy dependent. The energy independent mechonism reflects possive odsorption of colcium on the membrones due to the presence of phospholipids (92). On the other hond, the energy dependent mechonism requiring ATP is usuolly believed to be of two types, nomely colcium binding ond colcium uptoke. Recently, colcium binding ond uptoke by cordioc microsomes hove been found to be two seporote processes which moy initiolly shore common sifes on the membrone (93). Colcium binding hos been considered to occur of on introcellulor releqse sire thot iniiiqies systole by delivering colcium to the controctile opporofus whereos colcium uptoke hos been suggested to represent lhe occumulofion of colcium into on intrqcellulor sforoge site whose colcium content indirectly determines fhe qmount of colcium thot is delivered to ihe.contrqctile opporotus (47). Il is likely thot colcium odsorpfíon, bindíng ond uptoke processes olso occur in mitochondriol membrones, however, iheir role in myocordiol funcfion is not cleorly opporent. Since the sorcolemmol preporotion employed in this study did not show ATP dependent colcium binding or uptoke (89), it is suggested fhot these processes moy be obsenf in the cell membrone. If is olso possible thot mechonisms for ATP dependent colcium binding ond uptoke ore inqctivoted during the isolotion procedure. At ony rofe, binding ond uptoke processes by microsomes ond mitochondrio qnd cqlcium odsorption by these froctions os well os sorcolemmo of the heqrt con be considered to poriicipote in the regulotion of introcellulor colcium.
In this study quinidine hos been demonstroted to depress colcium uptoke by
-33- -34- cord¡oc heovy microsomes. This is in ogreement with the findings of other ínvesfigorors with both cordiqc ond skeletol muscle frogments of sorcoplosmic reticulum (4, 6r 7).
Since colcium uptoke os meosured in this study is o result of octive influx ond possive efflux of cqlcium ocross the vesiculor membrones, fhe observed decreose in colcium uptoke due to quinidine moy be o consequence of fhe oction of this drug on these processes. This is subsfontioted by ihe foci fhot quinidine hos been reporied to decreose the octivity of microsomol ATPose whích is considered to be involved in the ocfive tronsport of colcium (4r 7). Since the depressont effect of quinidine wos not opporenf ot low concentrotions of Mg*, it is likely thot quinidine moy be ocfing on the breokdown of o phosphoprofein, on intermediofe in ihe tronsport of colcium (94).
This suggestion is further supported by o recently proposed mechonism of quinidine on rhe inhibition of fhe cordioc sorcotub ule -l - ¡t32p reoction (95). Although quinidine hos been reported to be copoble of releosing cqlcium from the microsomol
frocfion (8), convincing dofo is required to drow o firm conclusion in this regord.
The observed depression of colcium uptoke by quinidine moy not be due to
chonges in colcium bindíng obility of cordioc microsomes. This is borne by the foct fhot under conditions when colcium uptoke wos depressed, colcium binding wqs nof olfered. On fhe other hond, cqlcium binding wos stimuloted ol initiol sfoges of
incubotion or ot low Mg* concentrqtions. Quinidine hos olso been found fo hove o slight stimulotory effect on ATP dependent colcium binding by skeletol muscle sorcoplosmic reticulum (85). This stimulotory effect of quinidine on colcium binding oppeors to be mosked oi high concentrotions of Mg+ under conditions when quinidine
is inhibiting fhe colcium uptoke sife. In the obsence of Mg# ond ATP colcium -35- odsorption by heovy microsomes wos morkedly siimuloted. Thus it oppeors ihot quinidine hos o complex mode of oction on fhe microsomol membrone ond Mg# seems fo ploy on imporfont role in determining ihe locus of its qction.
Unlike quinidine, procoine omide hod no effecf on microsomol colcium uptoke while lidocqine showed on initiol stimulotory effecf . Shinebourne et gl. (ó) were unoble to observe sn effect of lidocoine on colcium uptoke by cordioc microsomes. In this sfudy lidocoine, like quinidine, did noi influence colcium binding whereos procoine omide sfimuloted it. Furthermore, quinidine qnd procoine omide were found lo stimulofe microsomol colcium odsorption whereqs lidocoine hod o depressont effect. These resulls suggest thot quinidine, procoine omide ond lidocoine ocf on different sites involved in the process of colcium fronsport by the heort sorco-
plosmic reticulum. Although these drugs hove been shown to hove vorying degrees of
cordiodepressqnt oction (3) it seems unlikely fhof these ogents exert their cordioc
effects solely due to their influence on microsomol colcium tronsport.
It wos demonslroted in this study fhof quinidine depressed mitochondriql
colcium bindîng ond upfoke ond stimulqted mitochondriql colcium odsorption. Noock
et ql. (77) hos olso shown thof quinidine decreosed the rote of colcium uptoke by
heort mifochondrio. The depressonl effecr of quinidine on colcium binding by
mifochondriq wos found to be Mg+ ond ATP dependeni whereos colcium uploke wos + only Mg' ' dependent. On fhe bosis of the ovoiloble informofion concerning
mitochondriol colcium fronsport it is difficult to stote with certointy the exqct site of
drug ociion qnd therefore further studies ore cleorly needed lo elucidofe this
mechqnism. However, it should be nofed thof ihe mechonism of quinidine oction on -36-
mitochondrio seems to be different from lidocoine ond procoine omide. For exomple,
lidocoine, buf not procoine omide, depressed colcium binding. Both procoine omide
ond lidocoine hqd no influence on mitochondriql cqlcium uptoke. Procoine omide
produced o biphosic effect on mifochondriol colcium odsorption whereos lidocoine
depressed it. Lidocoine ond procoine qmide hove qlso been reported To hqve no effect
on heort mitochondrio colcium uploke (77). The significonce of these drugs on
mitochondriol colcium tronspori with respecf to rheir cordiodepressonf ocfions is o
mqtter of speculotion ot presenf .
We hove found thot quinidine stimulqfed sorcolemmql colcium odsorpfion.
Since colcium is o well-known membrone stobilizer (92), it is possible thot the cordio-
depressonf effect of quinidine moy porfly be medioted through its oction on the sorcolemmo. In contrqst, quinidine hqs been shown To decreose colcium odsorption by skeletol muscle sorcolemmo (5). This difference in fhe ocfion of quinidine on the sorcolemmo of cordioc ond skeletol muscle moy portly exploin ihe difference in fhe effects of this drug on fhe mechonicol properfies of fhese muscle. The results concerning
the effects of quinidine on colcium movements in qfriol muscle ond No*- K+ ATPor. of the ventriculor muscle con olso be interpreted os fo reflecf the sorcolemmol site of quinidine oction (86, 87). Since lidocoine, unlike quinidine, depressed sorcolemmol colcium odsorption whereos procoine omide wos withouf effect, these results suggest differences in the mode of oction of these ogents on cordiqc muscle.
In this study we hove shown thot quinidine offects colcium tronsport properties of sorcoplosmic reticulum, mifochondrio ond sorcolemmo. The in vitro concenTrotions fhof were used to elicit fhese effects oppeor to be higher fhon the doses of ihis drug -37 -
which ore employed theropeuticolly for the treotment of common orrhythmios.
However, it is possible thot the locol concentrotion of Ìhis drug ot the mícrosomol,
mitochondriql ond sorcolemmol sifes moy be different thon thot in the circulofion. If
should be noted thot quinidine hos been shown to bind with sorcoplosmic reticulum,
mitochondrio ond sorcolemma(7,84r 96). Thus it oppeors ihot fhe cordiodepressonf
effecf ot high doses of quinidine moy be due to the modificqtion of colcium fronsport
properties of these orgonelles. This study does not in ony woy rule ouf the modificotion of ofher functions of fhese membrone systems by quinidine. CONCLUSIONS
In this siudy the effects of quinidine, procoine omide ond lidocqine on the colcium fronsport properÌies of heort sorcoplosmic ref iculum, mifochondriq ond sqrcolemmo under vqrious experimentql conditions were investigofed. From fhe dqto obtoined in the sfudy, the following conclusions ore drqwn:
o) Quinidine wos found to inhibit colcium tronsport by heorf mifochondriq os well os sorcoplosmic reticulum.
b) The mode of quinidine oction on cqlcium tronsport seems to be complex
in noture in which Mg*ploys o cruciol role in determining its effecis.
c) In qddition to its ocfion on sorcoplosmic reticulum ond mifochondrio, quinidine hqs been demonsfrqted fo influence sorcolemmol colcium odsorpf ion.
d) The site of quinidine ocfion on heort membrqnes oppeqrs to be differenf from ofher ogents such os procoine omide ond lidocoine.
e) The cordiodepressont effect ot high doses of quinidine moy be due to o modificofion of colcium tronsport properfies of cell orgonelles buf fhe doio does not rule out fhe modificqtion of fhe other functions of these membrone sysfems by quinidine.
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