EFFECT8 OF CHRONIC VERÀPÀ¡,ITIJ ÃDMINISTRÀTION ON EIIE BIOCHE¡,ÍICAL CIIÀRACTERISTICS OF TTÍE IJ-TYPE CAIJCIUM CHÄNNEI.¡

BY

BIJAIR BURTON IJONSBERRY

A Thesis Subnitted to the Facul-ty of Graduate Studies in Partial- Fu1fílnent of the Requirenents for the Degree of

Masters of science

Departnent of Physiol-ogy Faculty of Medicine University of Manitoba Winnipeg, Manitoba

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tÂRTH SCttN([S Biôoêô

BIOCEEMICAL CEARACTERISTICS OF THN L-TYPE CÂ],CIUI'Í CEÄNNEL

BY

BLAIR BTIRTON LONSBERRT

A Thesis submitted to the Faculfy of G¡aduate Studies of the University of Manitoba in partial fuUillment of the requirene-nts for the degree of

ÈIASTER OF SCIENCE

@ 1993

Pen¡rission has been granted to the IIBRÄRY OF TI{E TINTyERSITY OF MA\TTOBA to lend or seII copies of ii.ís thesis, to the NAIONAL LIBRåRY OF OINADÄ to microfilm this thesis ¡nd to lend or sell copies of the 61m,. and LINTT¡ERSITY MIG'OEIL¡VS to publish ar ÂbsEact of ihis thesis. The autåor ¡ese¡r¡es other publications rights, and neithe¡ the thesis nor ertensive exbacts Êom it may be printed o¡ otherwise rtproduced without the autho/s pe¡srjssíon rÀBLE OF CONTENTS Page Acknowfedgements. i Abstract. ii List of Tabl-es. lv List of Figures. v

I. INTRODUCTION. . . . .

II. REVIEI{ OF I.IItrER.ATURE

A. EXCÏTATION-CONTRACTION COUPLING. 7 l-. Cardiac muscle. 7 2, SkêIeta] muscLe. 15 3. Smooth muscle. 2e

B. CAIJCIUM AND THE CALCTUM CHÀNNEI.,. 26 1. Calciun (i) Rol-e of calcium. 26 2. Calcium Channels (i) Types and Location. 29 (a) T-type caLciu¡n channels. 29 (b) N-type calciun channels. 30 (c) P-type caLciu¡n channels. 31 (d) L,-type cafciun channels. 32 (e) Skel-etal nuscle. JJ (f) cardiac nuscle. 36 (S) Snooth muscLe. 38 (ii) Ion movement through cafcium channels.... 39 (iii) Regulation of cal-cj-urn channel activity.,. 4L (iv) Rêgulat.ion of calciurn channel density.... 45 (a) Circulating drugs/hornones. . . (b) Disease states. 50

c. CALCIIIM CHANNEI-J ANTAGONTSTS 1. History. bl- 2. Classification. , . 53 3. Mêchanism of action. 56 4. Clinícal- application 64

XII. I'ÍATERIÀI.,S Àì¡D METITODS A. Methods l-. Drug treatnent protocol..... 70 2. Radioligand binding. 7L 3. Verapamil quantitation. 4. Statistical anaLysis. 73

B. Materj-als. 73 IV. RESULTS 1. Plasma verapamil and metaboLite quantitation and mortality data for the various drug adrninstratj-on protocols. 75 t Analysis of t3HlPN 2oo-11o binding characterj-stics in cardiac and brain tissues fron 2-week veraparnil implanted and injected rats (17.9 n.g/kg/day) ...,.. a7 3. Anafysis of [3H]PN 2oo-110 binding characteristics ín cardiac, brain and skeletal nuscle tissues and plasna veraparnil concentrations after a constant s.c. vêrapamíI injection from 2-16 $reeks. 90 Analysis of [3H]PN 2oo-110 binding characteristics in cardiac, braín and skeletaL muscl-e tissues and plasna veraparnil concentrat.ions after varying s.c. veraparnil injections (2.5 to 30 ng/kg/day)... 90 .Analysis of [3H]PN 2oo-l-10 binding characteristics in cardiac tissue and pLasna veraparnil ::::::::::l:::. :::::. ::::. ::il:.i:::?::. il.rîilfiT";^ 6. Influence of veraparnil treatÌnent on al-losteric interactions between the dihydropyrídine and phenylalkyla¡nine binding sites. 99

vr. DrscussroN. ... 106

VII. REFERENCES. ".. 1.18 ACKNOWIJEDGE¡'ÍENTS The years that went into the completion of this thesis vras not an indívidual- effort, but a col-l-aboration of several peoples hard

\^rork and tal-ents. There are severaL individuals r¡/ho have helped me enornously in the last several- years. I r^rould like to extend my gratitude to Deborah Dubo, Mike Czubryt, James Gilchrj-st, John Docherty, Shoba Thomas and Thane Maddaford for their help and friendship. To my committee rnernbers Dr. Parvan Singal, Dr. Paul Ganguly and Dr. Angel Zarain-Herzberg r I hrould like to thank the¡n for their encouragement and acade¡nic expertise throughout ny years here. .Forenost, I would like to thank rny advisor and friend, Dr. Grant Pierce. crant allowed ne the opportunity to start, continue and finish the years necessary to put together rny thesis, He took a chance on ne that others might not have, and he was afways there, providing support and encouragement. Final-1y, I v¿ould like to thank ny parents, farnily and friends who have always encouraged me to continue and strive for something better. Thanks for being there. ABSTRACT The Ca2+ channef antagonists are an inportant group of drugs used in the treat¡nent of a variety of clinicaf diseases. Patients are often on long-term treatment regimens in order to treat their particular disorder. The purpose of this study was to determine if chronic adrninistration of verapamil 1a ca2+ channel antaqonist) could ålter the biochemical characteristícs of the L-type Ca2+ channêl- as deternined ly ¡3H1rN 200-110 binding. Various modes of drug administration such as inplantable sl-ot¡-release pellets, s.c. injection and oral dosages vtere tested as means of raising plasma veraparnil l-evels. Circulating pl-asna verapamil fêvêls obtained from rats irnplanted with slo\^r-release verapanil pellets often reached levels l-0 fofd higher than s.c. injections and oraL dosages. In addítion, large quantities of the drug lvere rel-eased within the first 24 hours post-irnplantation, resulting in a high rnortality rate in these animals. It was concluded that inpfantable slol^r-release pellets are an unreliabÌe means of veraparnil ad¡ninistration. The bioche¡nicaI characteristics of cardiac, brain and skeletal muscle tissue all appear to be very resistant to al-teration by chronic veraparnil treatment. Variations in drug dosage by s.c. injection

(2.5 to 75 mg/kg/day) and duration of treatnent (24 hours to 16 weeks) had littl-e effect on altering the Ca2+ channel biochemical- characteristics . However¡ a decrease in B*u* and KD was observed in cardiac tissue obtained fro¡n rats irnplanted with a high dosage (50 ng) sl-ow-reLease pel-Let after 2 weeks duratj-on. In addition,

.l]. a significant increase in B*.* and KD v¡as observed in skeletal muscle v¡ith increasing veraparnil concentration administered by s.c.

injection. This increase observed in skel-etal muscle rnay be a consequence of high Local vêrapanil concentrations as a result of the injection protocol. ïn summary, our results d.ernonstrate that the irnplantable pel-l-ets are not a reliable adrninistration rnethod for veraparnil.

Further, cardiac (in addition to brain and skeletal rnuscle) Ca2+ channefs are highly resistant to change during chronic verapamil adninistration. our data suggest that the beneficial_ action of ca2+ antagonist therapy in different cardiac pathologies may not invol-ve a change in the biochernical characterj_stics of the channeL. Our data also questÍon the vafidity of previous studies which have described signifícant changes in receptor density aft.er ca2+ antagonist therapy.

1l- t IJIST OF TABÍJES Table Number Page

l_. Specific binding characteristics of t3HlPN 20o-l-i-o to crude nembrane fractions of heart and brain tissue in control, veraparni 1- inj ected and verapami 1- irnplanted (z weeks) rats...... 89 2. Specific binding characteristics of t3HlPN 2oo-110 to cardiac, brain and skeletaf nuscle mernbranes of rats treated for varyinq times with veraparnil inj ections. . . . 91 Pl-asma verapamil and norverapamil concentrations in rats ínjected for I weeks with varying concentratj-ons of verapamil. ....92 Specific binding characteristics of [3H]PN 2oo-i-10 to cardiac, brain and skeleta1 ¡nuscl-e ¡nernbranes of rats treated for I \,reeks with varying concentrations of veraparnil. ....93 5. Tnhibition of o.25nM t3HlPN 2oo-11-o binding by l-o uM verapamil. Inhibition is a percent of control, vrhere 100U is binding of IrH]PN 200-110 in the absence of verapamil. . . .105

tv IJIST OF FIGURES

F igure Nunber Page

1. Percent mortality as a functíon of verapamil- concentration ad¡ninistered (mg/kg/day) via sl-ow-release inplant pellets or subcutaneous injection. The theoretical vaLues for dail-y release of vêrapamil presented above were calcul-ated for the 5t Lo, 25, 50 and 75 ng pel-Iets...... ,,. ".76 2A. Representative tracing of a High Performance Liquid Chrornatography (HPLC) chronatograrn showing verapanif and its ¡netabolites (norveraparnil, D-617 and D-620) as a function of tirne. The tracing is fro¡n a control plasna sarnple spiked with l-00 ng each of verapamil. norveraparnil-, D-6L7 and D-620 standards...... "..7a 2F . Representative tracing of a High Performance Liquid chromatography (HPLC) chrornatograrn showing verapamil and íts netabolites (norverapamil, D-6L7 | D-620) as a function of tirne. The tracing ís from a blood sanpl-e obtained from a veraparnil irnplanted rat (50 ng) I hours post-implantation...... 79 3. Plasna verapanil concentratíons durÍng the 1st day of treatnent with different rnodes of veraparnil adrninistration (11.9 ng/kg/day). Va1uês represent the nean J S.E.M. for four to eight separate animals. *P<.05 1/s. other treatment nodalities ...... 80 4. Plasna norverapamil- concentrations during the first 24 hr after treabment r¡¡ith different types of verapanil adninistratíon (11.9 ng/kg/day). Values represent the nean t s.E.M. for four to eight rats. *P<.05 vs. other treat¡nent modalitíes...... 82 5. D-6f7 concentrations over ti¡ne in plasma fron rats treated with different. types of veraparnil adrninistration (11-.9 ng/kglday) . Values represent the mean + S.E.M. for four to eight rats. *P<.05 vs. other treatment ¡nodalitíes. ,.,.....83 6. Concentrations of the veraparnif netabol-ite D-620 over the course of 1 day in plasrna fro¡n rats treated with different modes of veraparnil ad¡ninistration (i-1.9 ng/kg/day). Values represent the nean + S.E.M. for four to eight rats, :tP<.05 vs. other treatment nodal-ities...... 84 7. Plasma concentrations of verapamil- (upper graph) and norverapanil (lower graph) after 1, 7 and 14 days of treatment with verapanil injections (11.9 ng/kglday) or the irnplanted verapamil pel,let (calculated to release 11.9 ng/kglday). Values represent the nean + S.E.M. for three to six rats. *P<.05 vs. injected group. ....85 8. Repr-esentative data demonstrating scatchard plot analysis of [3H]PN 2oo-110 binding to a crude membrane fraction of ventri-cular tislue. Bnâx:6.067 x l-0-11 M, KD: "onlroi3.g4a x 1o-10 M and proteiä-^ concentration: o.1ã4 mg/nl . . '...... 86 9. Repr-esentative data dernonstrating saturation of binding of IrH]PN 2oo-1-1-o to a crude nembrane fraction of control ventricufar tissue, The specifíc binding is a function of different concentrations of IrH]PN .200-L10. 8.""i 1.648 x to-13 lfrnotTng), KD3 3.g4i x 1o-1o M ana proËäÎn concentration3 0.184 ng/nl...... 88 10. specific bindíng characteristics of t3HlPN 2oo-l-10 to crude membrane fractions of heart tissue from control- and veraparnil inplanted (24 hours) rats. VerapaÍìil- peI1et size ís ín rng and irnplanted ínto 200 gram fenale Sprague- Daw1ey rats. Data are expressed as nean t S.E.M. of five to nine ani¡nal-s. .,,.96 Ll-. specific binding characteristics of t3HlPN 2oo-11-o t.o crude membrane fractions of heart tissue from control and verapanil irnplanted (7 days) rats. Verapanil pellet size is in rng änd irnplanted into 200 gran fernale sprague- Dawley rats. Data are expressed as nean f S.E;M. of fíve animals...... 97 L2. Pfasma verapamil concentrations (ng/nl) over tirne frorn rats irnplanted q¡ith different veraparnil peltet sizes (¡nS) . Data represent mean t s.E.M. of five to eight animal-s. ,. "....98 13. Specific binding characteristics of [3H]PN 2oo-110 to crude nembrane fractions of heart tissue from control- and verapamil injected (24 hours) rats. Verapanil injections were in ng/kq/day and injected into 200 gran fenale Sprague-Dawley rats. Data are expressed as mean + S.E.M. of six to twelve aninals. . . . .100 !4. Analysis of blood sarnples obtained from verapanil injected ani¡nals at 4 and 48 hours. Plasna veraparniJ- concentration is in ng/m1 and analyzed by High Performance Liquid chronatography. Data are expressed as mean t S.E.M. of two to tweLve animals. ...101 va 15. rnhibition of [3H]PN 2oo-l-10 binding to crud.e nembrane preparations of ventricufar tissue by veraparnil. Ventricular membranes were incubated with 0.25 nM IrH]PN 2oo-110 v¡ith various concentrations of unl-abel-led verapamj-I in the presence of 1 ¡nM ca2+, Values reprêsent nean + s.E.M. of eight experirnents ...... 1o3 L6, rnhibition of [3H]PN 200-L1-0 binding to crude membrane preparation of ventricuLar tissue by verapamil. ventricular nembranes were incubated t¡ith 0.25 nM IrH]PN 2oo-110 with various concentrations of unlabelled veraparní1 in the absence of ca2+. valuès represent mean + s. E.M. of eight experirnents , . . . . .104

v].L Int,roduction.

Cal-ciun channel antagonists, since their conceptíon sorne :O years ago, have received considerable attention both clinically and êxperinental- l-y. Their unifying characteristic of blocking the L- type Ca2+ channel in excitable and non-excitabfe cell_s has l-ed to their extensive use in treating . .rurr.a" of clinicat disorders. Researci is nov¡ being carried out in order to further el-ucidate their nol-ecular binding properties, mechanisn of action and continuing therapeutic considerations.

Four distinct types of ca2+ channels exist in excitabfe and non-excitable cells.121 The T-type ca2+ channel_ has been isolated fron hèart, skeletal muscle, s¡nooth nuscfe and neuronal tissue.123 rt is opened by rnild depolarization and their ionic currents are fast and transient.s T-channels are thought to play an irnportant rol-e in the activity of the sinoatrial node in the heart and are generaLly thought to be insensitive to Ca2+ channel- antagonists,l23

The N-type ca2+ channel is tocated primarily in neuronar tissue ancl

is thought to be responsible for controlling the ínf1ux of Ca2+ necessary for neurotransrnitter release.130 N-channeLs are insensitive to ca2+ channel antagonists and are inhibited by a group of snail toxins, r,l-conotoxíns. 130 A recently discovered ca2+ channel v¡as isolated frorn cerebelrar purkínje cell-s and was denoted

P-type ca2+ channel.139 rt was found to be insensi-tive to both ca2+ antagonists and ú)-conotoxin but was inhibited by a spider venorn toxin.138 Very littte is knoi,¡n about these channels, including r,Jhether the P-type ca2+ channel is a distinct channel_ or a group of several refated channels. The L-type Ca2+ channel has been isol-ated from cardiac, skeLetal, smooth and neuronal tissue.124 It is the primary means of ca2+ inf l-ux during the cardiac action potential and provides the necessary ca2+ for the rrca2+ induced ca2+ releaserr mechanisrn fron the sarcoplasrnic reticulum.161 The organic ca2+ channel antagonists preferentially bind to this channel and block the inward flux of ca2+ across the plasrna ¡nernbrane.5 The ability of Ca2+ channel antagonists to block thê slow inward Ca2+ rnovernent was identífied by Dr. A. Fleckenstein and his colleagues in the early 196ots. At that time, two prototype Ca2+ channel blockers were being studied; prenylamine and verapami1.2o9 These two compounds were shown to have a cardiopressant effect sirnilar to that seen by the removal- of Ca2+, resulting in the inhibition of excitation-contraction coupling and cardj_ac contracti Ì i ty .2rt Further research identifíed the abitiby of ca2+ channel antagonists to selectively bJ-ock the L-type Ca2+ channel, cruciaf in the excítation-contraction coupling process in cardiac tj-ssue. In 1966, a new cfass of drugs was designated, the Ca1ciu¡n Antagonists.2l0 At that tirne, two major groups of Ca2+ antagonísts existed, the croup A, or highly specific antagonísts and croup B, the less specific antagonists,2l6 It has been the Group A Ca2+ antagonists that have received the majority of clinical and experirnentaL focus. Group A consists of three rnajor subgroups: the dihydropyridines (e.g. nifedipine), the phenylaJ.kytarnines (e.g. verapanil-) and the benzothiazepines (e,g. dittiazem¡.217 The ca2+ channel antagonists are being used c1Ínically to treat a variety of disorders. Their prirnary use has come in the treatnent of cornplications related to the cardiovascufar system incLudi.ng arrhythnias, angina, hypertension and cardionyopathy.219 Due to the underlyj.ng nature of these disorders, treatrnent regirnes incorporating Ca2+ antagonists can be administered over prolonged periods of tine, fron nonths to even years, This raises the question of what effect these prolonged treatnìent regimês are havíng at a celLular Level. Studies looking at the effêcts of long-tern treatments with certaín conpounds have shown that, there is a relationship between alterations at a cellutar levef and prolonged drug treatment. Certain drugs, Like Ca2+ channef antagonists, mediate their action by binding to a specific receptor on the tj_ssue plasrna menbrane. Thê receptorrs actì-vity and density can be regulated by circulating drug concentrations and by particular physiological and pathophysiological states.187 A classic exanpLe of a circulating drug regulating irnportant characteristics of its receptor comes frorn studíes looking at the ß-adrenergic receptor and treatment with ß-receptor agonÍsts and antagonists. prolonged treatrnent with the ß-adrenergic blocking agent propanalol may lead to the deve]-oprnent of an increase in the nurnber of ß-receptor nurnbers in certain tissues like the heart.334 conversery, prolonged treatment with a ß-adrenergic stinulating agent 1ike epinephrine may 1ead to a decrease in the number of functional ß-receptors present in a tissue.266 The resulting increase in functionar receptor nunber following prolonged treatnent with an antagonist is referred to as an rrup-regulationrr , whil-e the decrease follov¡ing agonist treatment ís a rrdown-regulationrr . The change in the functional receptor numbers following agonist/ antagonist treatnent foLlows this general pattern, but it should be noted that such regulation is not an automatic consequence to drug exposure and in some systerns drug application nay result in no change or a change in the opposite direct i on33s. This study was undertaken to examine the effects on the L-type ca2+ channel after chronic exposure to the ca2+ channel- antagonist,

veraparnil. Previous studies have exa¡nined the effects on ca2+ channel density in a variety of tissues using different members of the ca2+ antagonist group. A reduction ín the number of nouse brain Ca2+ channels was noted. after 28 days of oral adrninistration of nifedipine and verapanil.2?5 A sirnilar d.ecrease in ca2+ channel number was obsêrved in rat brain and heart after ZO day intravenous nifedipine treatrnent2T6, v/hile no change was observed in rat heart after 14 day oral- nifedipine treatrnent,2TT It is apparent that there are a variety of experimental factors that may play a role in the regul-atíon of channel nurnber. such factors rnay include the modê of drug adrninistration (e.9. intravenous, subcutaneous or orally), type of drug (e.9. nifedipine, veraparnil or diltiazern), species and tissue specific differences and the duration of drug treatment . . Tn order to address sone of these factors a variety of experirnental protocols $rere introduced. An initiat study was undertaken to address lrhether alterations in ca2+ channel_ density were j-nf Luenced by the duration of drug adrninistration and/or drug concentration. To assess the influence of drug ad¡ninistration duration, a constant drug dosage was ad¡ninistered for varying periods of ti¡ne (ranging fron l- day to 16 weeks). Concentration dependent changes were assessed by adninistering a range of verapamif concentrations for a fixed period of time, The mode of drug adrninistration r,¡as addressed by utilising subcutaneous (s.c.) inplantable slow-release pel1ets, s.c. injection and oral adrninistratÍon r¡ia a p.o. intubator. ca2+ channel density and affinity \^rere deter¡nined usíng a radioligand binding assay ernploying t3HlPN 2oo-110 as the radioactive ligand. pN 2oo-110 is a nembér of the dihydropyridine group and has a high specific binding to the L,-type ca2+ channel, aLlowing for accurate quantitation of density (B^.*) and activity (K¡) . Presentl-y, it is unclear v¡hat the relationshíp is between ca2+ channel density and circulating leve1s of ca2+ channef antagonists. Thê exact circulating concentration of verapamil, for example, which is required to alter ca2+ channel characteristics is unknown. Verapamil is 87-93å protein bound, and has a first pass clearance of approximately 8Oå by the 1iver.219 The elimination hal-f-life of verapanil- in the blood is usually betlreen 3-7 hours, but does increase during chronic adrninistration and in conditions where there is liver or renal darnage.219 Therefore, in order to quantitate the anount of veraparnil that was actually reaching the different tissues, blood sanples were taken at varying times and the plasrna veraparnil and it.s ¡netabofites were quantitated by High Perfornance Liquid chromatography (HpLc). pl-asna quantitation of veraparnil and its metabol-ites aLso enabl_ed us to conpare the various neans of drug adrninistration with respect to circulating plasna fevels.

The binding sites for the three major classes of Ca2+ channel antagonists (dihydropyridines, phenylalkylarnines and

benz othiaz epines ) are located on the d1 subunit of thè Ca2+ channe1.164,234 These nol-ecular binding sitês are alfosterically linked to one another and binding of the antagonists to their respective sites is modífied by the presence of other bl_ockers and divalent catíons (e.g. c¿2+¡3ee. rn order to assess whether chroníc verapamil treatnent atters these allosteric j_nteractions or the response to ca2+, radioligand binding assays were carried out in the presence of varying verapamil concentrations and in the presence/ absence of ca2+ . The purpose of this study was to obtain a better understanding of the nolecular consequences of l-ong-tern therapy with Ca2+ channef antagonists. This infor¡nation is currentl-y lacking in the literature and vitar to the understandíng of the long-terrn effects of prolonged clinicaf treatnents with Ca2+ channel antagonists. REVTEW OF IJITERÀTURE

Excit,ation-contraction coupling in cardiac Muscle The E-C coupling process within cardiac muscfe can bèen separated into four stages: (i) action potential depoJ-arization of the sarcorennar (sL) membrane, (ii) ca2+ rerease from stores in the sarcoplasrnic reticulun (SR) induced by extracelLular transsarcol-e¡nmal Ca2+ novenent, (iií) binding of ca2+ to the thin fila¡nent troponin c protein and vi-a a series of reactions a]lowing interaction between actin and rnyosin to forrn cross-bridges resulting in muscle shortening (contraction), and (iv) relaxation of the muscle fíbres by lowering of the intracelluÌar ¡Ca2+1 via uptake by the SR and Ca2+ extrusion through the sarcolemma. il DeÞotarization of the nyocardlial celt The restíng membrane potential of myocardial cells is approxirnately -85nv to -9snv. Myocardial deporarization results in a rapid but brief Na+ inward movernent through tetrodotoxin sensitive sodiurn channels in the T-tubule/SR junctional "pace.3 Further depolarizatíon of the sL nembrane results in the opening of voltage dependent ca2+ channers. Tno types of ca2+ channels exist in cardiac ce1ls; T channel-s (transient) and L (long-lasting) . a T- channels are found primarily in pacenaking cel1s but are also found in ventricul-ar tíssue, These channers are activated rapidfy at polarization potentials nore negative than -5onv to _6onv, peak at -3omv, and inactivate quickly (5-30 nsec). These channels appear to contribute littre to the ca2+ current. L-channets activate at - 4OnV to -3onv, inactivate sfower than T-channels and carry approxinately three to fourfol-d more current. These channels appear . to be the major contributor to the forrnation of the characteristic rnyocardiaL action potential plateau and provide the necessary ca2+ flux to initiate contraction. The L-type channefs are the rnain route for transsarcolemmal ca2+ inffux.5

With the discovery of the SL., Na+-Ca2+ exchange system, it v¡as thought that this nay be another route for ca2+ j-nfl-ux during the E-C coupLing process.6 Much research over the past couple of decades has focused on discovering the role of the Na/ca exchanger. Electrophys iological and frux neasurenent studies have shor+n that the exchanger moves 3 Na+ for every L ca2+ ions, and is 7 I electrogenic. ' The Na/Ca exchanger nay contribute l_itt1e to Ca2+ influx.during depolarization in cardíac cel1s, but rnay act as a high capacity ca2+ efflux rnechanis¡n.9 The Na/ca exchanger may pLay a more import.ant role v¡ith respect to ca2+ infLux ín situations where the sL nembrane has been depolarized to pot.entials around OnV, or vrhen therê is an el_evation of intracellular ¡Na+1 as $rould be observed after blocking of the Na+7K+ purnp (eg. diqitalis).10 iil Calcium release f,ron sarcoplasmic retÍcu1um. Thê sarcopr-asrnic reticurun (sR) of cardiac celrs is a tubular, lipid bilayer netv,/ork of menbranes analogous to the endoprasrnic reticulum of non-contracting cef r.s.11 The sR is composed of t\^ro norphologicalì-y distinct conponents; (i) the junctional SR, and (ii) the longitudinat sR.12 The junctional sR cornes into cr.ose apposition to the T-tubuLê system of the sarcolenma. The junctionar sR contains feet that. span the gap between the sR and the T-tubules. These feet are believed to be the mediators of ca2+ refease fron the junctional SR.13 The 1ongitudinal- sR is responsibte for the purnping of Ca2+ ions fro¡n the cytopl-as¡n into the SR via the ATP dependent Ca2+ purnp contained within the

longitudinal SR membrane.14 The prinary functions of the SR include: (i) release of stored ca2+ via the ryanodine-sensitive ca2+ release channêl to provide the f inal- signal in the contractile protein activation, and (ii) relaxation of the muscLe by re- accumulation of ca2+ by the caLciurn punp.11 Other sources of ca2+ necessary for activation of the contractile apparatus have been proposed including direct transsarcolemmal ca2+ influx15 and ¡nitochondrial6 acting as a reversíbl-e Ca2+ storage site. At present, ¡nitochondria appear unlikely to participate in regulation of cytosolic Ca2+ on a beat to beat basis because it,s affinity for ca2+ and rat,e of ca2+ uptake are too l-ow.17 Insuffiqíent data are available presentJ-y to âdequately ans\,rer the questíon of transsarcolemmaf influx of ca2+ directJ-y activating myofilarnents. It appears that the relatíve contribution of direct transsarcolemrnar ca2+ infrux varies bethreen species. Rat is afrnost totally dependent upon SR Ca2+ rel-ease for contract,ion, while rabbit and guinea piS can develop ful1 contraction (at a slower rate) in the absence of SR ca2+ release and by direct t.ranssarcolemmal infl-ux. r4,79 t2o sR is alrnost nonexistent in frog ventricle, therefore, the frog is cornpletely dependent upon extracetlular Ca2+ for contraction.2l It appears that all cardiac nuscl-es are dependent upon sone transsarcolenmal Ca2+ inftux, if only for activation of ca2+ refease fron the sR. The current hypothesis for the activation of Ca2+ release from

the SR is via ca2+ induced ca2+ refease.22 Transsarcolemnal- ca2+ influx resul-ts in an increase in the myoplasmic free ¡ca2+1 on the outer surface of the SR, inducing a rel_easê of Ca2+ from the SR via the ryanodine sensitive ca2+ rel-ease channels. currênt

experirnental- data shows that part of the transsarco lernrna I influx actuafly foads the SR r,¡ith Ca2+ avail-abl-e for release during subsequent contraction s.22 t24,2s It appears that there is a tirne dependent and ca2+ dependent component to activation of ca2+ induced ca2+ rel-ease. A fast increase of free ca2+ on the outer surface of the SR appears to result in rel-ease of Ca2+ fron stores in the sR, white a sfow increase in the same ¡ca2+¡ resufts in a 1oading of the sR with ca2+. This loading of the sR vrouLd make that Ca2+ availabLe for release on subsequent cont.ractions. 26 Transsarcore¡nmal ca2+ currents have both a fast and stov¡ conponent which would appear to confirn the above observations.25

Na/Ca exchange may aLso play a role in infl-ux of SR Ca2+. Recent investigations into ca2+ rel-ease fron the sR have revear-ed. a possible triggering role by Na+. Na+ influx into the T-tubule/SR junctional space during the upstroke of the action potential. rnay result in the activation of the Na/Ca exchanger, extruding Na+ while ¡noving ca2+ inward. The resulting inward ca2* frux via the exchanger results in the triggering the release of Ca2+ from the SR.3'27

A difficulty raised in the hypothesis of Ca2+-induced Ca2+ rel-ease was whether the ca2+ released fron the sR was an all-or- none process. If this were the casef then the knov¡n gradation of contraction seen j-n cardiac muscle with varying transsarcolemmal ca2* fLux \^rould not be possible.2S Activation of SR ca2+ release would resuLt in all of the stored ca2+ being released, or none of it, and graded contractions would not be possible. Experiments perforned on skinned cardiac cells dernonstrated a negative feedback system operatíng on the ca2+-induced ca2+ release from the sR.22 At hígh free ¡ca2+¡ there was inhibition of further rel-ease fron the SR. These skinned cardiac ceIJ. experirnents denonstrated that the amount of ca2+ released via ca2+-induced ca2+ release is graded dependíng upon the ¡ca2+1 that triggers i¡.22,26 iiil MvofiLament interactions The principle role of ca2+ released frorn the SR is to inítiate the contraction of the nuscle. ca2+ initiates the contracti-on of cardiac nuscle at the l-evel of the myofilament, which is the contractile apparatus of the nuscle cell-. Myofilaments responsible for cardiac contracture include thick and thin fila¡nents which interact v/íth one another resulting in nuscle short.ening (contracture) and relaxation. There is a paral_tel- arrangement of the thick and thin filarnents v¡hich are interdigitated throughout the cel-l. During contraction there is an energy dependent sliding between the tvro filaments resulting in ¡nuscle ceI1 shortening. ReLaxation occurs when the sliding rnotion is passivety reversed (i.e., there is no energy expenditure by the f il_a¡nentous array). The thick/thin filaments are grouped to form fibrils, v/hich when arranged in parallel, form a sarconere. Bundles of fibriLs cooperate as a functional unit in the whole muscle.29 The thick f il-arnents consist of myosin molecules which are specif ical-J-y arranged. to forn the fifa¡nent. The rnyosin molecufe is a dimer of tr¡/o identicar heavy chains arranged in an alpha-helical fashion with each chain ending in a gfoburar head. The heads are arranged so that both heads protrude at thê same end.. The overalL structure of the filanent resurts in the globular heads protruding in a staggered pattern at each end of the filanent while the nidregion is devoid of heads. Contained within the heads is a rnyosin ATPase which inÈeracts with the thin filanent (specifically the actin conponent of the thin filament) . The rnyosín ATpase requires rnagnesium as cofactor and with its association v¡ith actin is referred to as actomyosin ATpase. ATp (adenosine triphosphate) is hydrol-ysed to ÀDp (adenosine diphosphate) and inorganic phosphate by actornyosin ÀTpase to provide the biochenical energy required for muscle contraction. 29

The thin filarnent is conposed of filamentous actin (a constituent of the actomyosin complex), tropornyosin and troponin in a ratio of 7:l-:1.3o The actin is arranged in a double-stranded helix fashion, while tropomyosin is a coiled helical dimer. Tropomyosin appears to associate in a head-to-tair series ar.ong the actin molecufe within or near the herical groove of the actin double strand. This placenent of troponyosin appears to regul_ate actin/rnyosin interaction during contraction.2g Tropomyosin novement ín/out of this groove is regulated by the thin fifanent associated troponin, r^¿hich is a compLex of three functionarly distinct proteins in a ratio of 1:L:1.30 Troponin I (TnI) is the subunit that acts to inhíbit actornyosin ATpase activity. Troponin

C (TnC) mediates calciu¡n sensitivity by binding cafcium in a specific manner and acting to decrease the inhibitory action of Tnr. Tnc will infLuence Tnr only when in the presence of the third troponin subunit, Troponin T (TnT), TnT appears to rnediate the inhibitory action of Tnc on Tnr as r¿erl as anchoring the troponin cornplex to tropornyosin. rn order for contraction to proceed, all three cornponents rnust be present.29 Excitation of the rnyocardiar celr and subsequent increases in the freê intracellular Ica2+] reach a threshold point at which time Tnc undergoes an i-nteractive change v/ith Tnr. Addítionar changes occur between Tnr' TnT and tropomyosin. The result of these alterations in proteín-protein interactions is a positional shift of the tropornyosin molecule relative to the actin morecule, rernoving the inhibition of actin/rnyosin interaction. Myosin/actin interaction resurts in activation of actomyosin ATpase, hydrolysation of ATp' and the resurtant biochernícar energy is transformed into ¡nechanical energy resulting Ín contraction,2g Alterations in the rever.s of cytosoric free ca2+ avairabrê for binding v¡i11 result in differing 1evels of contractility. For example, i-ncreased levels of ca2+ will resuft in a positive inotropic effect.3l AdjustÍients in the activity of the regulatory troponin subunits, such as occurs when rnagnesiurn binds to the Tnc subunit34 or v¡hen Tnr is phosphoryrated by cAMp dependent protein

13 33 kinase upon ß-adrenergic stimur-ation32 ' , wilt also alter the f ever_ of contraction. ivl Relaxation Refaxation of cardiac muscle occurs upon a decrease in the teveL ôf cytosolic free Ica2*]. Decreases in the fevel of cytosolic free ca2+ causes a dissociation of rnc bound ca2+, which rêsul-ts in the inhibitory effect of Tnr being re-estabfished,

Tropornyosin shifts back into the groove on the actin ¡nolecuf e preventing interact,ion betv¡een actin and rnyosin. Active cross bridge for¡nation is no longer possible and the ¡nuscle relaxes. The fevel of free ca2+ which was criticaf Ín the initiation of contraction Ís also irnportant in the relaxation process.29

The reduction in the cytosolic free Ca2+ levels occurs by several- means and at. different levels of the ceIl. At the lever- of the sarcol-ernmal membrane, two extrusion ¡nechanisrns are present: (i) Na/Ca exchangeg, and (ii) ATp-dependent Ca2+ pu¡p.36,s1 The prinary route of efflux of free ¡ca2+1 at the sarcolernrna is via the Na/Ca exchange systen. The Na/ca exchangerrs role in the nornaf infl_ux of ca2+ during E-c coupl-ing is still controversial, but ít does have a doninant rore in ca2+ extrusiott.9135 The Na/ca exchanger moves 3 Na+ ions into the cytosol for every L ca2+ Íon extruded. T Experirnental data in r¿hich the sR was inhibited by caffeine so that transsarcolenmar- transport was the onry neans of cerr relaxation showed that relaxation r+as considerabr.y sr-oÌ^red in the absence of externar Na+ which wourd greatry decrease the effectiveness of the Na/ca exchanger.g It is now apparent that the Na/ca exchanger and

L4 not the SL Ca2+ purnp contrÍbutes to the beat-to-beat relaxation. The afternative neans of reducÍng cytosolic free ¡ca2+¡ is via re-uptake by the SR. The longitudinal rnernbrane of the SR contains an ATP-dependent calciun puÍtp, functioning to transport ca2+ frorn

the cytosol- back into the SR stores.14 With each cycle of the Ca2+ punp, two Ca2+ ions are rnoved for every ÀTp hydrolysed to ADp plus inorganic phosphate.2l The SR ca2+ purnp competes with the Na/ca exchanger as the main relaxíng systern of rnyocardium. It is usually the SR.Ca2+ pump that is the nore significant in the movement of ca2+ necessary for relaxation.38 The Na/ca exchanger appears to êfflux ca2+ equivalent to that which entered via the ca2+ channeLs during the action potential plateau, Most of the free intracetlular Ca2+ that v¡as used for act,ivation of the rnyofiJ.arnents during contraction originated from the SR, and it is the SR ca2* pump that re- accunulates the sarne quantity of ca2+.39 Excitatíon-contraction Coupting in Skeletat ¡fuscle The E-C coupling process in skeletal muscle is very sirniLar to that of cardiac muscl-e. Skel-etal- nuscle E-C coupling can also be separated ínto four stages: (i) actíon potential depolarization of the sarcolernnaJ. rnembrane, (ii) Ca2+ release fron stores in the sarcoplasrnic reticufun (sR), (iii) binding of ca2+ to the thin filarnent troponin C protein and via a series of reactions allowing ínteract.ion between actín and myosin to form cross-bridges resulting in rnuscle shortening (contraction) , and (iv) relaxation of the nuscle fibres by lowering of the intracell-ular ¡ca2+¡ via uptake by the SR and Ca2+ extrusion through the sarcolenïna.

15 Fundamentaf differences do, hov¡ever, exist between skeLetal and cardiac tissues. The nost evident differences are in the action potential and the mechanis¡n of Ca2+ refease from the sR. Because the processes are very similar betr,reen the two types of tissue, the discussion here will be lirnited to describing the differences which exist. The action potential in skeletaL nuscle is significantly different frorn that seen in cardiac tissue. The duration of the action potential in skeletal muscLe is approxinately 1-5 ¡nil-líseconds, which is a rnagnitude of 30 to b0 times shorter than the action potential produced in cardiac tissue (2bO-3OO rnilliseconds).41 The depolarization upstroke of the skel_etaf musclê ,is a result of the opening of tetrodotoxin sensitive sodium channels, resulting in a brief inv¡ard movement of Na+ ions (rfastl sodiu¡n current) j-nto the t-tubul-e/sR junctional space. These sodium channels inactívate and close t,he channel within a few 10,000ths of a second, not alJ.owing any further Na+ infl_ux.42 The same membrane potential changê that activated the sodiurn channels al-so activates voltage-gated potassium (K+) channels. These K+- channefs are slolrer in opening then the Na+ channel_s. They open at the sa¡ne tine that the Na+-channels are inactivat,ing. The K+- channels permit K+ íon efffux fron the ceLl-, resulting in the downward portion of the action potential (repolarization) totrards the resting rnernbrane potential (approxiinately -9onv) .43'44 Missing in the skeLetal rnuscle action potential that is seen in the cardíac action potential. is the plateau phase, resulting frorn the opening

L6 of voltage-gated Ca2+ channels. As described previously, depolarization of the cardiac sarcoLemmal menbrane resufts in the activatíon of voltage-dependent L-type Ca2+ channels, which allow transsarcolernmal influx of ca2+ ions.4 This inv¡ard ca2+ current produces the long plateau phase characteristic of the card.iac action potential and necessary for induction of Ca2+ refease from the SR. 5

An additionaL difference in the E-C coupling process betr^reen skeletal and cardiac tissues is the means by which Ca2+ rel-ease fron the SR stores is triggered. In cardiac tissue, a Ca2+-induced

ca2+ release rnechanism resulting fro¡n an increase in the free ¡ca2+1 by transsarcolenmaf influx ís the current poputar hypothesis.22 The precise rneans by v¡hj-ch an action potential in the t-tubule system of the skefetal muscl-e produces a reLease of Ca2+ stored in

the terninal cisternae of the SR is controvers ia1 . 45 Several different hypotheses for this triggering rnechanism have been

proposed .

The triggering mechanism for Ca2+ release from the SR that is currentfy favoured in the literature was devel-oped by Frank and Bianchi, and involves the use of "trigger ca2+ ionsl to induce ca2+ rel-ease fro¡n the sR stores.46'47 A twitch actíon potential enters the t-tubule systen of the skefetal nuscle fibre and causes a rel-ease of the ca2+ ions that are bound to the t-tubule intracellular surface. These ca2+ ions are released. into the t- tubule/SR junctional space and diffuse across to the surface ¡nembrane of the terminaf cisternae (junctional snj . The released

L7 ca2+ ions are referred to as "trigger ca2+ ionsfi and they induce Ca2+ release fron the terrninal cisternae of the SR via the ryanodine-sensitive ca2+ refease channels. ca2+ from the terminal cisternae is released into the rnyoplasm of the muscl-e cell,48 The increased Ica2*] produces a ¡nechanj-cal response sirnilar to the process observed in cardiac tissue. Skeleta] muscl-e and cardiac rnuscLe differ in their requirements for extracellular ca2+ in the E-c- coupling process, In the absence of extracelluJ.ar ca2+, cardiac nuscle fibres wil_l- cease to contract as the necêssary ca2+ required for ca2+-induced Ca2+ release fron the SR comes from transsarcole¡nrnaf influx via the L-type calciurn channeLs.22 Ske1etal rnuscle fibres that are bathed in Ca2+ free sofutions wifl continue to eLícit twitch contractions for several ¡ninutes. The cessation of twitch contractions appears to be dependent upon the time required for the Ca2+ ions bound t,o the t-tubule intracellular ¡nembranes to diffuse out (2-s rninutes) . s0 The SR ca2+ stores require several hours to diffuse out a sufficient quantity of ca2+ to interfere with the contraction process and, therefore, are not the reason for terrninat,ion of contractions.49 Blockage of the tv¡itch contractions appears to be due to the rernoval of Ca2+ frorn the t-tubular membranes. Studies using organic channel blocking drugs (veraparnil and gatlopanil)s1 and blockers of calciun dependent processes (TMB-8) 52 have added support to this hypothesis. There are tv¡o additional proposed nechanisrns for E-C-coupling during the twitch contraction. The first proposes that inositol 1,4,5-triphosphate (IP3) produced by the hydrolysis of mernbrane bound phosphotidyl inositol 4,5-biphosphate is the chernicaf transrnitter in the skêl-etaf nuscIe.53.54 Recent investigations into IP3rs action have shown that IPs couLd act as a chemical transmitter in smooth nuscfe but in skeletal muscle the response to IP: is far too slow (a magnitude of 3) and the concentration required far too high for it to play a prínciple rol-e in the coupling process.55 1P3 nay pLay a rofe in the nodulation of cel-l- function rather than a chemical messenger role in the E-C coupling process . 56' 57 The second proposal involves a rnechanical link between the t- tubul-ar menbrane and the terminal cisternae of the junctional SR" This ¡nechanical- connection j.s proposed to be via the junctional. feet. The ¡nechanical Iínk invol-ves the use of voltage-dependent charge movenents to activate ca2+ release frorn the SR. There appears to be far rnore dihydropyridine binding sítes than actual calcium channel-s in skefetal muscl-e.58 If these dihydropyridÍne receptors are not linked to functionaL catciurn channels, thêy are thought to possibly act as rrvoltage sensorsrr59 and be associated, anatomicalLy and functionalJ.y, vrith the junctional feet processes that span the t-tubule/SR gap. These dihydropyridine rrvoltage sensorsrr are suggested to couple the electrical activity of t- tubule depolarization with Ca2+ release from the terrninal cisternae at thê junctional SR. 60 Whatever the rnechanism of triggering the release of Ca2+ from the junctional ryanodine-sensitive Ca2+ reLease channels, the resul-tant increase in rnyoplasrnic free ¡caz+3 activates the sketetaL muscl-e rnyofilanents to contract, The process of nyofilanent activation, cross bridge forrnation and relaxation are sirnilar to that seen in cardiac tissue,61 Mechanis¡ns involved in the J.owering of intracellular ¡Ca2+1 necessary for rêlaxation are al-so sirnilar betv/een the t\^/o tissues and include Na/Ca exchange, SR ATP- dependent Ca2+ purnp, and sarcolemnal ÀTP-dependent Ca2+ pump. The rel-ative irnportance of each of these mechanisrns also appears to be similar: to cardiac tissue, with the SR ATP-dependent Ca2+ pump contributing the nost to the relaxation process.62 Excitatíon-Contraction coupling in Snooth Muscle The E-C coupling process in s¡nooth musclê differs significantly frorn that of either skeletaL or cardiac nuscle. A rnajor reason for this difference is the structuraf nakeup and functionaL roLe of smooth ¡nuscle celIs. Unlike cardiac and skeletaL muscle which are fast contracting fibres, srnooth nuscl-e contraction is tonic in nature and may rernain in a contracted st.ate for prolonged periods of ti¡ne.63 The structural makeup of srnooth muscl-e ce1ls is unlike either cardiac or skeletal- muscl-e. In skeletal nuscl-e the organization of the contractile apparatus is highly ordered r¡¡ith a \'¿e11 def ined SR. In smooth muscl-e cel-ls, the organization is less ordered and ambiguous. The contractile apparatus in snooth nuscle consists of thin (actin), thick (nyosin) and inter¡nediate f ilarnents. The thin filanents are compromised prirnarÍly of actin and tropomyosin and insert. into dense fusifor¡n bodies on the pJ-asrna nembrane or within

20 the cytoplasrn.64 These d.ense bodies are comprised of interrnediate filanents whích are thought to function in a cytoskeletaf role and provide mechanical support for the myofilanents. The intermediate filarnents are comprised of virnentin and desrnin.6s Myosin is composed of two heavy chains and tv/o sets of J.ight-chain subunits, one of which (LC26) is 20,000 Da in size and regulatory in function.66 The ratÍo of thin:thick filanents differs betv¡een snooth muscle types and nay range from 5:t up to 27 2L,67 The SR of snooth nuscle, though present, is not as defineable a structure as it is in skel-etal or cardiac tissues. A1l snooth muscl-e ce1ls have a systern of sarcoplasmic reticuLun, though the volune is variable between smooth muscle types and rnay range frorn 2tuo 7.5? of the ceIl vol-urne.68 The SR of srnooth muscle cells is often a diffuse structure, v¡ith no rrdefineabler location v¿ithin thê ceIl. Certain regions of the SR do, hotrever, show structural specialization. For exampLe, the junctional SR is connected via bridging structures to the surface membrane. This structurè resenbles the tríadic formati_on found in skeletal_ rnusc1e.69 The coupling of the surface nenbrane r+ith the junctional SR a1lows for the possible transfer of surface electrical activity or the action of drugs on surface receptors to trigger ca2+ rel-ease.70 The longitudinal SR contains an ATp-dependent Ca2+ pump which Ís structurally similar to the Ca2* purnp found in both skeletal- and cardiac ¡nuscle SR.71 In addition, snooth nuscle cells have a si¡niLar ryanodine-sensitíve ca2+ release channel 1ocated in the junctional SR, responsíbIe for release of SR Ca2+ stores during

2L contraction. 72 The action potential ín smooth muscle differs from the action potentials seen in either skeletal or card.iac tissue. The resting menbrane potential of vascuLar srnooth nuscle celIs is approximately -40 to -55 mV.73 Vascufar smooth nuscfè cells fack tetrodotoxin- sensitive Na+ channels at the sarcolemmal menìbrane, and possess a lower permeability to K+ ions as cornpared to cardiac tissue.74 In the absênce of Na+ channeLs, the inward current is carried by ca2+ ions via voltage-dependent ca2+ channefs. T5 The repolarization phase of the smooth muscle cell is carried by K+ channels. Several types of K+ channel-s have been identified, including Ca2+-activated

K+ channels, delayed rectifier K+ channels, and ATp-sensitive K+ channels . 7 6

There arê two E-C coupling nechanisms active ín s¡nooth nuscl-ê ceIIs. Electrocheníca1 coupling depends upon depolarization of the sarcol-e¡nmaI mernbrane by the opening of voltage dependent Ca2+ channeLs, resulting in an Ínward transsarcol-emnal- novenent of ca2+ ions.77. Pharnaconechanical coupling results in contractions without polarization of the sarcolennal- membrane and rnay be a resul-t of the ca2+ released fron sequestered stores or ca2+ entry through channels opened by receptor occupation, or both.?7 Electrochenical coupling is dependent upon depolarization of the sarcolem¡nal mernbrane to open voltage-dependent ca2+ channels allowing inv¡ard transsarcolem¡naf flux of Ca2+ ions. The opening of voltage-dependent Ca2+ channefs occurs at varying leve1s of nembrane depolarization in the different srnooth muscle tissues.

22 For exampl-e, depolarization above -3omv will- result in opening of voltage-dependent ca2+ channels in rabbit intestine.lol This inward ca2+ fLux resul-ts in an increase in the myoplas¡nic free lCu2*1, inducing release of Ca2+ fron junctional SR stores via the ryanodine-sensitive Ca2+ release channels. This is equivalent to the ca2+-induced. ca2+ rel-ease observed in cardiac tissue.72 Contraction of smooth muscle is also possíble without the necessity of sarcolemmal menbrane polarization. In addition to those Ca2+ channefs that are opened upon nenbrane depolarization, there is a set of ca2+ channels that are not opened by depolarization but opened in response to receptor occupation by an appropriate agonist, for exarnple acetylchol ine. 78 This type of smooth muscle contraction is via pharmacornechanical coupling in v¡hich bínding of an appropriate ligand to the ca2+ channel_ rêceptor results in opening of the channêl- and an inward ca2+ fl-ux. Contraction using this process is dependent upon extracel_Iular ca2+.79 stimutation with certain other conpounds (pcE1- prostaglandin 81, angiotensin If) can elÍcit contractions in srnooth ¡nuscle cef l-s without initiating any ca2+ inward current and appear to act in releasing Ca2* from sequestered stores in the sR via a second nessenger systern 1rp3).80 The hornone receptors on the sarcol-emma are coupled via c-proteins to phosphatidyl inositol hydrolysis. The activated G-protein complex is speculated to target phospholipase C v¡hich converts phosphatidylinositol-4,5- diphosphate to diacylglycerol and Ipr.81'83 tp3 diffuses across to the junctional SR and sti¡nulates Ip3-responsive Ca2+ channel-s, thus

23 causing release of ca2r stores and contraction,82 ït is evident that there are severaf ways in which to trigger the necessary Ca2+ release from the SR stores. The triggered release of Ca2+ ions results in an increase in the myoplasmic free [ca2*]. This increase in the ¡ca2*1 can regul-ate contraction of s¡nooth nuscle in a variety of ways. One of the nore important ways involves the phosphoryJ.ation of the nyosin myofilament. Myosin contaíns a regulatory light chain subunit denoted Lc2o, LC2o is phosphorylated (at Seríne-19) by nyosin light chain kinase (MLCK), MLcK is activated by ca2+ and calmodulín (a ca2+-binding protein¡.84 The phosphorylation of LC2o by MLCK is the signal that initiates the cycling of cross-bridges necessary for contraction. LC2ors phosphoryl-ation appears to ínitiate contraction by turning on actonyosin ATPase activity8s as wel-l- as inducing conformational changes v¡ithin the thick fiLarnent facilitating cross-bridge formation. E6 Additional- regulation of srnooth nuscle contraction has been shorvn to come fron two actin-binding proteins, caldesmon and calponin. These tv,¡o proteins have been identified in smooth muscle cefl-s and are thought to regulate the thin-filament. Caldesnon from vascular s¡nooth muscle inhibits actomyosin ATpase activityST and enhances the binding of myosin to actin.88 Alterations in actornyosin binding or ATPase activity by caldesmon nay slor+ crossbridge detachrnent. SS catponin binds both myosin and actin and inhibits actomyosin ATpase activity.s9 This inhibiti.on is reversed by phosphoryLation by prot,ein kinase c, ca2+ and calmoduLin.go

24 Relaxation ín smooth nuscle appears to be dependent upon the state of phosphoryLation of the inyosin filanent. phosphoryfation of myosin LC2o is crucial in initiating contraction, and the reversal of Lc2o phosphorylation is a prerequisite for relaxation. The state of nyosin phosphoryl-ation can be regulated by the opposing actions of myosin phosphatase and by modulation of the activity of MLCK. Myosin phosphatase activity resul-ts in dephosphorylation of Lc2o causing a decrease in actomyosin ATpase activity and reduced cross-bridge cycling. Inhibitors of nyosin phosphatase (okaidaic acid toxingl¡ induce contraction or prevent relaxation of fibre preparationsg2, híghlighting rnyosin phosphataser s role in the rel-axation process. MI_.,CK phosphorylates LCzo and requires Ca2+ and caL¡nodul-in for activation. Phosphoryl-atíon of MLCK reduces its affinity for Ca2+, resulting in decreas.ed actívíty and reduced phosphoryLation of LCzo.93 MLCK can be phosphoryl-ated by several protein kinases including cyclic ÄMp- dependent kinase94, Ca2+/ calrnoduL in-dependent kinase II95, and protein kinase c96.

The relaxation process in s¡nooth nusc1e is also dependent upon lowering the rnyoplasmic free ¡ca2+¡ that was the trigger for the contraction process. The principl-e ¡node for this reduction is via the SR ATP-dependent Ca2+ punp, which is structural_ty sirnilar to the ca2+ purnp of skeletal and cardiac rnuscle SR.9? Àl_terations in the pump's activity are nediated by phosphofa¡nban. 98 In addition, Na/Ca exchangegg and sarcolemmal ATp-dependent Ca2+ purnploo have been identified in smooth muscle and may participate in the

25 relaxation process by transsarcolenmal- efflux of ca2+.

CAIJCIU]'Í AND THE CALCIUM CHÀNNEL

ca Lc íum: RoIe of Calcir¡m Calciurn plays an essential roLe in the normal functioning of the ceIl, and in certain cases, the abnornaf or pathologicaJ- behaviour of ceLl-s, Free calcium is not only involved in the direct regulation of certain ce1lu1ar functions (E-C coupl_ing) but al-so acts in rnaintaíning the cell-rs structural integrity. The following díscussion will examine in further detail the role of ca2+ in the ceÌl. Thê preceding treatise on excitation-contraction coupling in cardiac, skeletal and smooth muscle described the crucial role of ca2+ in the contraction process. ca2+ts regulatory role is, however, not limited to the contraction process. The opening and closing of cellul-ar gap junctions is also dependent upon the intracel_lutar Ica2*]. cardiac gap junctions are connections formed betvreen tv¡o opposing sarcoÌe¡nrnaI nenbranes, These connectj-ons are hydrophillic protein channels (connexons) Ínsulated from the extracell-ular space spanning between the cells.102 EtectrícaJ- uncoupling between normal cardiac cells occurs upon intracellular injection of Ca2+ which is completety reversed when the intracellular ¡Ca2+¡ is 1owered.1o3 The increased ICa2*] eLectricall-y uncoupJ-es ceII-to-cetI interactions by increasing the junctional resistance and abolishing the cel1-to-cell novenent of molecules. The rnechanism by which this happens is not conpletely understood but two hypotheses have

26 been proposed: (i) Ca2+ ions bind to the gap junction phosphotipids resulting in a conformationaf change and closure of the gate betrveen the celIs104, and (ii) ca2+ triggers an enzynatic reaction that afters the confornation of gap junctional protein and. causes blockage of the channel.1o5 The refease of neurotransrnitters during stimulus-secret ion coupling in neurons is also controlled by changing l-eve1s of intracelluLar Ca2+. Douglas and poisner first denonstrated the necessary role that ca2+ plays in the neurosêcretory process.106 Since then, the ¡nechanisrn by which Ca2+ regulates this process has been further elucidated. Upon excitation, activation of voftage- dependent Ca2+ channels e¡nbedded in the nerve terminal menbrane results in transmernbrane inf l-ux of Ca2+ frorn the extracell_ul_ar =pace.107 The resulting increase in intracellular Ica2+] initiates exocytosis, where neurosecretory vesicl-es fuse v¡ith the plasma menbrane and rel-ease their contents (neuropeptides) into the synaptic junction.108 In addÍtion, ca2+ not only appears to controf the neurosecretory process but rnay also regulate the devefopnent of the neurons thenseÌves. During chick embryonic development, there is a period of naturally occurring notoneuron cefl death. It is evident that ca2+ leve1s preceding this event are crit.ical in the regulation of this process.1og Therefore, there rnay be a causal association between the onset of cerL death and the expression of 1Lo 11L the ca2+ channel-s necessary for ca2+ novement. ' In a structural capacity, ca2+ has been recognized as necessary for proper cell-to-ce]l adhesion, as well as for naintaining cêIl- nenbrane structural integrity,112 The exact neans by which ca2+ associates with the components of the rnembrane to estabfish its structuraJ. integríty is not yet known. Ho\^¡ever, removal- of Ca2+ from the surrounding nedium appears to disrupt ca2+- dependent connections between the surface coat and the externaf larnína, resuJ-ting in a ffuid-fifled separation.ll3 These connections may be ca2+-carbohydrate bridges which require ca2+ for stability. 114 In certain pathologicat conditions, aJ.terations in Ca2+ levels and êf f l-ux/inf l-ux rnechanisms results in irreversibJ_e damage and cel-I death. One such condition is that of the rCa2+ paradoxr, observed when ca2+ is removed from the interstitial fluid and then restored. There is a resul_ting dranatic increase in the intracellular ¡Ca2+1 causing celJ- damage and. eventual cell death. Several routes of ca2+ influx have been examined including Na/ca exchange, ca2+ channels, K/ca exchange, and even the passive novement of Ca2+ through the danaged cell nembrane.115r116,116a,117 Irregularities in normal beating of the heart are referred to as cardiac arrhythmias, Cardiac arrhythnias can be separated into two large classes; (i) those due to abnorrnal initiation of írnpulses for contraction, and (ii) those caused. by abnormal irnpulse conduction.llS ca2+ plays an irnportant role in the generation of both types of arrhythnias. Ca2+ can affect both the autornati.c celJ-s required for the initiation of contraction and the conducting fibres required for the spread of the contraction signa1.119 ln addition, ca2+ can induce arrhythmias indirectly by inftuencing the

2A cardiac oxygen suppty and demand. These indirect effects invofve ca2+ altering the coronary or peripheral circul_ation, and changes in the heart rate or myocardial contracti Ì ity. 120 calciun channels: Types andl L,ocation

There are currentl-y four types of ca2+ channefs that have beên discovered in both excitable and non-excitabLe ceLls. These include: (i) T-channefs, (íi) N-channels, (iii) p-channels, and (iv) L-channels. The separation of these ca2+ channeLs into their respective groupíngs has comê fron biophysical, pharmacologica l, and structural data . 121 T-type Calcium Channels. The T-type Ca2+ channel was first described in vertebrate sensory neurones by carbone and Lux.122 They described thís new channef as a fully inactivat.ing, low voltage activated Ca2+ channel .122 since their discovery, the T-type ca2+ channel has bêen described in a wide variety of excitabl-e and. non-excitable cer_r_s including cardiac tissue, skeletal muscLe, smooth muscl-e and neurons. 123 T-type channel-s have a Iow activation voltage (-50 nV to _60 nV), peak at -30 nV and are inactivated quickty (5-30 msec).s Thi= quick inactivation resufts in a s¡nal1, unitary cond.uctance of ca2+.r24 In cardiac tissue, the conductance of ca2+ is too brief to be a najor contributor to the E-C coupÌing process.s However, this bríef conductance may be sufficient to pray an important rore in the activity of the pacenaking cer.Ls in the sinoatriaf node where

29 the majority of the Ca2+ channels are of the T-type. s,126 Pharrnacological- data has sho\^¡n the T-type channels to usually be considered as insensitive to dihydropyridines, and, ín sensory neurons, to veraparnil (a phenytal.kylamine ) .r23,r24 Sensitivity of the T-channels to feJ-odipine (a dihydropyridine ) has, however, been de¡nonstrated.l2s The dihydropyridines, phenytalkylamines and benzothiazepínes are three groups of a class of organic compounds referred to as calciurn channel blockers (antagonists). These conpounds will be discussed in detail subsequently. Drugs Like

6, 7 tetramethrinl2 diphenylhydantoín12 , and a¡niIoridel28 have al_L shovJn to act on the T-type ca2+ channel, although none appear to be sel-ective for the channel-. Nickel and cadmium are also capable of blocking the T-channel, with nickef usually ¡nore effective.L29 N-type calcir¡m channels The N-type Ca2+ channef was first described by NovJycky in the chick dorsal root gang1ion.130 N-type ca2+ channels appear to be present only in neuronal tissue and do not seem to exist in ¡nuscl_e tissue. N-channel-s have also been found in certaín endocrine ceLrs such as the pancreas, anterior pituit.ary and the adrenal gIand.130 N-channers are activated by rnernbrane potentials si¡nifar to those that activate L-type ca2+ channels (-30 mV to -40 mV) and inactivate within tens to hundreds of ¡nifriseconds at potentiars si¡nílar to L-type channers.t3l The actuar- differences between N- and L-type Ca2+ channels appear to be rninimaL. They activate and inactivate at sirnilar nenbrane potentials and their ca2+ conductance arso appear to be very simirar.132 The distinguishing

30 factor between N- and rJ-type ca2+ channers ís their pharrnaco logica 1 properties. N-type Ca2+ channefs are inhibited by a group of snail toxins, cl-conotoxins, whiJ.e being insensitive to modulation by 130, L33 dihydropyridines . The prirnary means of modufation of the L- type ca2+ channels is via dihydropyridines (calcium channel blockers) for which they are sefective. In neuronal mêmbranes, both N- and L-type Ca2+ channels have been identl¡ia¿.132 Ho!¡ever, there, appears to be a specific distribution of the two channel types, Radioactive antibody binding studies have shown that N-channels are particularJ-y associated with the neuron terninus, probably functioníng in neurotransnitter release.134,135 It appears that the N-type Ca2+ channels carry the bulk of the inward current during activation of the neuron, and the L-type channels play a much srnaller rol_e in this process.136 P-type calcium channets

Certain neurons exhibít a Ca2+ channel that is actÍvated at. high menbrane potentiars and insensitive to both dihydropyridines and o-conoto*itr. J'38 r 139 This particular channer- has characteristics which are dissirnilar to other ca2+ channels. These new ca2+ channer-s are abundant in cerebellar purkinje cells and virtuarry absent in other neurons.139,L40 Due to their predoninance in nurkinje cêLLs, they were denoted as p-type channels, though it is not known whether it is one type of channer or a group of si¡niIar channels. These P-type channers are not blocked by either the o-conotoxin or DHP, but are bl-ocked by another toxin derived frorn the veno¡n of the spider Agelenopsis aperta.139 Littr-e work has been carried out into the biophysicaÌ and structuraL make-up of the p-type channel, but it is suggested from prelirninary work cornpleted that the p-channers have a similar subunÍt structure to that of L-channe1".1.41 L-tyþe calcium Channets The L-type Ca2+ channel has been the most extensively studied of aLf the ca2+ channer.s. The L-type ca2+ channer- is often referred to as the dihydropyridine-sensitive Ca2+ channel, because much of the characterization of the channeL has come from the use of dihydropyridine ca2+ channel ligands which specifícaI1y bind to this type of Ca2+ channel. The L-typê Ca2+ channel has been identifÍed in all types of cetls within the body, including skeletal, cardiac and srnooth nuscle, and nervous tissue. within these different tissues, there are certain unifying characteristi-cs arnong the L-type ca2+ channels found there. These include: (Í) sensitivity to inorganic and organic blockers, (ii) stereotypical response to dihydropyridine ca2+ agonists, (iii) activation range (-40 mv to -30 nV)s, (iv) slow inactivation when ca2* is not the prirnary charge carrier, and (v) steady-state inactivation at posítive hording potentíars.124 variations between the various tissue type ca2+ channel-s exist as weII, such as ca2+-dependent inactivatíonl42 and the increase ín channeÌ activity by cycric-A-!{p- dependent kinase activity (seen in cardiac tissue) 143. The most variable paraneter beti,reen the tissue L-type Ca2+ channefs is voltage-dependent inactivation, The most rapidly inactivating channels are frorn cardiac tissue, whife neuronar or secretory L-

32 type Ca2+ channels appear not to have any vol-tage-dependent inactivation. Variability in the rate of inactivation has also been observed i-n channeLs from the same tíssue.144 To expLore the L-type Ca2+ channel in further detail, a tissue specific examination of skeletal- muscle, cardiac tissue, and smooth muscLe foI]ows. SkeletaT l"IuscTe L-type CaTcium ChanneTs. Skefetal- muscfe has a very high concentration of dihydropyridine-binding sites, and therefore, much of \^rhat Ís known about the dihydropyridine receptor and the L-type ca2+ channel has cone from study of skeÌetal nuscle tissue. Howêver, the dihydropyridine receptors found in skeletal muscl-e tissue are not all functional L-type Ca2+ channels. Schwartz determined that the density of dihydropyridine receptors was 35-50 tirnes the nurnber of functional L-channel-s.160 Sol-ublization studies of the dihydropyridine-binding site and reconstitution into phospholipid vesicl-es resul-ted in an active L-channel. The solublization process yieJ-ds a mul-tisubunit complex.r{st146 The skelet,al L- channel- has been shown to be a pentameric for¡nation composed of 4 distinct subunits, a1 (170 koa), a2/6 (175 kDa) | ß (92 kDa) and the y subunit (32 kDa). The 6 subunit can be released frorn the a2l6 configuration by reduction of â disul-f ide bond.147 The dl subunit sequence shor+s considerable homology t,o those of other menbers of a voltage-dependent ion channel superfarnily which includes K+ and Na+ channe1s.145 For example, there is approxirnately 30å hornology in the ske]etal nuscl-e c, subunit and

33 the voJ-tage dependent Na+ channel. The d1 subunit in skeletal nuscle contains four internaÌ repeating units which contain six nenbrane-spanning helices. The vottage-sensing l-ocation of the channel is thought to reside on the fourth of êach these heLices where there is a positively charged anino acid every three to four residues.121 The o, subunit is al-so where the organic compounds, the ca.Icium channel antagonists, are thought to bind. The dihydropyridine-binding site is believed to be located on the extracel-lu1ar surface of the c, subunit.l4s Expression of the a1 subunit in mouse cells devoid of ß,a2/6t and y subunits dernonstrated the appearance of dihydropyridine- sensitive L-type ca2+ channeLs.149 This observation woul-d suggest that the o, subunit itself can forn a functional Ca2+ channel, and. that the additional subunits rnay provide functional and structurat support. The a, subunit is also thought to play a distínctive role in skeletaL muscle by acting as a voltage sensor in the excitation- contraction coupling process.146 The previously described E-c coupling process in skeletal nuscl-e indícated that the rel_ease of Ca2+ from the SR stores rnay be rnediated by a linking of the dihydropyridíne voltage sensor to the junctionaJ- SR ryanodine- sensitive Ca2+ release channel . Depolarizing stirnulus is tsensedr by the dihydropyridine-voltage sensor resulting in a conformational changè in the o, subunit. This conformatíonal- shift is transrnitted to the ca2+ release channel causing it to open and. rel-ease the ca2+ stores,59 AdditionaL support for this hypothesis co¡nes fro¡n the observation that not. all the dihydropyridine receptors are actually

34 Iinked to functional L-type ca2+ channels and nay act as voftage sensors.16o Therefore, the c, subunit of skel-etal- nuscl-e appears to not onl-y act as the functional- unit for the Ca2+ channel but nay aLso act as a sensoring mechanism necessary for triggering release of ca2+ from the sR during E-C coupling,

fhe a2/6 subunit (175 kDa) can be reduced to a l_bo kDa d2 subunit and 3 peptides of 25,22, and I7 kDa forming the 6 subunit.146 The separation of the cornplex occurs \^rhen disulfide bonds which hofd the subunits together are reduced. ,!he e2/ 6 conpl-ex is the product of a single gene with the d2 sequence forning the N-terninal and the 6 sequênce forrning the c-ternit,.1.151 T}:e d2/ 6 compl-ex has a high leve1 of glycosylation with both subunits being glycosylated. 150 The structural arrangenent of the a2l6 conptex rvithín the Ca2+ channel has not been full-y elucidated, but it is thought that the 6 portÍon may act as an anchor for the d2 portion.l4s'l'so Functional-ly, the 02/6 conpl-ex appears to be abfe to modify the activity of the Ca2+ channel. Coexpression studies of a, and o2ló showed altered ca2+ current, in both nagnitude and 152 153 kinetics, in skeletal muscLe preparations, ' rn addítion, the rate of ca2+ influx through L-channel-s r^ras enhanced in liposornes reconstitut.ed v¡ith c1 and c2/6 subuni¡".154 The ß subunj-t was originally identified as a protein that consistently copurified with the a, subunit,l4s The ß subunit contains several phosphorylation sites. ca2+ channef function ís known to be nodulated by phosphorylat.ion event.s, and the ß subunit may be the area upon which the enzyrnes act to alter Ca2+ channeL kinetics. The ß subunit is believed to be associated with the cytoplasrnic conponent of the c1 subunit.155 The consistent copurifícation of the ß subunit with the o1 subunit suggested that the ß subunit interacted with the a, in a functional manner.

Coexpression studies of û1 transfected L-ceLIs aLong with ß subunits shov¡ed a dramatic i.ncrease in the number of dihydropyridine bindíng sites but no effect on current densíty. There was, however, an increase in the rate of Ca2+ current activation. l-49, 156 coexpression studies in which tne a2/6 subunit was added (dt/d26 /ß conbination) yielded a peak current above that seên with the d!/ß conplex, indicating a close rêgul_atory interaction between the various subuni_ts. !4r,r57 The y subunit is the l-east characterized of the skeletal muscle L-channel_ subunits. The y subunit consistently copurifies with the c1 subunit, appears to be very hydrophobic and is extensively glycosylated.145'158'1s9 Coexpression studies usíng the skeletar nuscle y subunit with the other subunits has shown l-ittle functional significancel4l and requires further research,

Cardiac L-type caLciun channeLs . The cardiac L-type Ca2+ channel plays a signif icant,l-y different functionar rore than does the skeLetaf muscLe L-channeL. In cardiac tissue, the release of SR Ca2+ stores is induced by transsarcolemmal influx of Ca2+ duríng the plateau phase of the cardiac actÍon potential. This transsarcolemmal influx of Ca2+ is rnediated by L-type g¿2+ s¡¿¡¡.1=,161 This ca2+-induced ca2+ rel-ease

36 mechani.sm in cardiac tissue, as opposed to the possib]e voLtage gated rerease in skeLetar muscle, may account for the observation that the najority of dihydropyridine receptors found in cardiac tissue are functional L-type ca2+ channeLs.162 The L-type Ca2+ channeL has been characterized, and has been found to consist of four subunits; a1 (170-190 kDa), a2 (l-70 kDa), ß (52 kDa), and 6 (28 kDa) 163¿164, The T subunit found in the skel-etaL rnuscle L-channel has not been found in cardiac tissue.165 The cardiac dl subunit shows structural similarities to the skel-etar muscre ct subunit. The cardiac c, subunit contains the four repeating rnotifs, each consisting of 6 transnembrane d.omains simíl-ar to that of skeletal rnuscle. !66t167 HoÌ^rever, there are several (5) protein kinase A phosphorytatíon sites identified in the skel-eta1 rnuscr-e c:. subunit r,¡hich are rnissing in the cardiac for¡n and are repr.aced by four new sites.166 structural differences have also been noted with respect to the extracellular protein regions, which nay account for the lack of cross-reactivity of antibodies raised against the skel_etaf muscle û1 subunit..168

Differences have also been observed in the Lengths of the 5r and. 3 l ends of the .DNA crones that arê responsible for encodÌnq the skeretal and cardíac muscle a, isoforms.167 The cardiac isoforn of the a1 subunit appears to have thê same functional characteristics as does the skeletaL muscle forn. The o, subunít is capable of functioning as a L-channer and is the site of binding of the calciurn channeL antagonists, 168 The ß subunit has been found and cloned in cardiac tissue, Coexpression of this ß subunit with a cardiac cl- subunit resufted in altered ca2+ channel kinetics including increased peak currents, accel-erated activation kinetics and shifts in the voltage-current

relationship to more hyperpolarized potentials. The cardíac ß subuni-t is si¡nilar in structure to the skeretal muscl-e isofor¡n and is thought to act in a modulatory rofe.2oB The additional subunits that have been reported likety function in structuraf support (anchoring) and in regulation of channel kinetics.168 Snooth MuscLe L-type CaTcium Channets The smooth rnuscle L-type ca2+ channel is not as welf characterized as the cardiac or skel-etat nuscle channel. There are fewer dihydropyridine binding sites in s¡nooth nuscfê tissue, as conpared to the nunerous avaitability found in skeLetar muscl-e, which has made characterization of the channel difficult. The snooth nuscLe L-type Ca2+ channel is likely very sinilar to the Ca2+ channel found in cardiac nuscre. rt is proposed to be a quaternary structure, composed of o1r c2r ß and 6 subunits. The y subunit r'¡hich was obsêrved in skeretal nuscre but not in cardiac muscl-e, also appears to be absent in smooth nuscle. Using a reverse_ transcribed polyrnerase chain reaction technique, the presence of the 1 transcript in RNA isoÌated fron nouse brain, card.íac muscle, spleen, kidney, liver, and stonach as well as frorn human brain and cardiac muscl-e was undetectabre. The y subunÍt was detected in hunan and rnouse skeletaL nuscle preparations , 16s

A cDNA library isolated from rat aorta has shown the aortic o1 .DNA to be very si¡nilar to 01 .DNA isoLated from cardiac tissue.

38 The cfose identity of the two 01 cDNÀ libraries has suggested that the û1 subunit fron these t\,/o tissues arise frorn the sanê gene.207 Differences that exist in the smooth nuscle isofor¡n are thought to arise from alternative splícing that occurs in both the cardiac and snooth muscle a, subunits.2oT Ion Move¡nent Through calcíum channels There are currently two popular hypotheses about the rnethod of ca2+ ion movenent through ca2+ channel-s: (i) the allosteric or one- point rnode1169, and (ii) two-site rnodel.r7o,!7r The strengths and weaknesses of these two modefs wí11 be discussed. The al-l-osteric rnodel (one-site nodel) prêdicts that, there is a high-affiníty Ca2+ binding site located on the external surface of the ca2+ channel and has a net charg¿ o¡ -2 This ca2+ "r72. binding site controls the selectivity of the channel-. When the extracellul-ar ¡ca2+¡ is greater than l- ¡.1M, this ca2+ binding site is primaríJ-y occupì-ed by ca2+ and there is a resulting conforrnationaL change in the channel- resulting ín only ca2+ ions passing. The ions that are noving through the channel, transiently bind to a site ín the pore itself, but only weakly. When the Ca2+ binding site on the external surface is not occupied by Ca2+, the channel pore becones nonselective, allowi-ng passage of monovalent ions . 124 The weakness of this nodel is the confornational_ change that occurs upon bindíng of ca2+ to the regulatory site, resulting in selective ca2+ íon perrneation, The character of thís confor¡national change is difficurt to assess and therefore the precise mechanism by which the channel sel_ects between bj-ocking and permeant ions re¡nains uncLear. r24,113 The two-site ¡nodel- proposes that there are two hígh-affinity ca2+ binding sites, Iocated at either end of the channel. An ion moving fron the outside to inside of the celÌ has to first bind to the outer síte, then inner site, and then fÍnatly passíng into the cytoplasm.124 when both sites are occupied, the affinity of each of the binding sites for the Ca2+ Íon is reduced due to electrostatic repulsions of the tv¡o sane charged ions, This electrqstatic repulsion speeds up the departure of the other ion.

The nodel makes the assurnption that an ion can onl-y move into a site that is vacant. Thereforê, at high ICa2*], both binding sites are likely to be occupied. This destabilizes the inner bínding site ¡,¡hich íncreases thê probability that the binding site will lose it.s ca2+ ion to the cytoplasrn. r74tr75 The outer site ca2+ ion can then move to the free inner site. Binding of another ca2+ ion to the outer site, again destabifizes the inner site and that ion then moves into the cytopJ-asm.r12,!74tr15 The selectivity of the channel is deter¡nined by selectivity sequences contained within the high- affinity binding sites.124 The tv¡o-site modeÌ is able to expJ.ain many of the experirnental resuLts obtained using dihydropyridine-sens itive ca2+ channels. For exarnple' the tv¡o site model can explain nonovalent conducti_on through the channel in the absence of divalent cations, saturation of current as a function of lCa2*), the ability of very Iow divalent concentrations to inhibit nonovaLent conduction, and the

40 ability of fow Ica2*] to inhibit Ba2+ conduction through the channel.112 The rnain weakness in this theory is that it assu¡nes syrnmetry of the channel energy profile, so that the forces at work at the outer binding site are equivalent to those at the inner site" In addition, the model assunes the channef to be a rel-atively inert structure that does not interact r,¡ith the ions passing through the channel, except for binding and unbinding reactions" There is, therefore, no confornation change associated v¡ith the channel- upon ion permeation.l24 Regulation of Catcium channel Activity The Ca2+ channel- can exist in one of three conformational states at any one time. Thosê states are: (i) activated. or open, (ií) inactivated, or (iii) resting. The activated or open state is evídent upon depolarization of the nenbrane resulting in opening of the Ca2+ channèl from the resting state. In the actívat,ed state, there is conductance of i-ons across the sarcorern¡na1 menbrane into the cytoplasn. InactivatÍon is the procêss by which the Ca2+ channel enters into; (a) a non-conducting state once it has been activated, or (b) a state where it is not availabl_e for activation. The inactivated state is an interrnediate stage in q¡hich the channel- is not conducting any ion current (closed) but it not presently available to be reopened with another depolarizing stimulus. The recovery process noves the ca2+ channel frorn the inactivated state to the resting state. At this stage, the channel- is closed (non- conducting) but is availabl-e to be reopened upon being presented with a depolarizing stinulus.5 The nornal progression of the ca2+

4L channel upon being presented with a depolarizing stirnulus is to nove into the open conducting state from the resting state. Following the open state, the channel ¡noves into the inactivated state r¡¡here there ís no further ion rnovement and the channel- ís not available to be reopened if another stimul_us were to be presented. When the mernbrane potential reaches a certain point, the channel moves frorn the inactivated state back to the original resting state. For the channêf to be opened it has to first be in the resting statê, and for the channeL to get back to the resting state it has to go through the inactivated state. The current flow through the ca2+ channel nay be nodulated by several enzyme and organic ligand cornpounds. The resulting current fLow nay be increased or decreased depending upon the compounds antagonistic or agonistic tendency, One wefl known modul_ator of Ca2+ current is the stírnulatory effect of ß-adrenergic agonists on heart cells. ß-adrenergic st.irnulation of ca2+ current in heart ceLls may occur via two means: (i) indirectly via the phosphorylation of the ca2+ chânnel-, and (ii) directly by the stinulation of the Ca2+ channel by bínding of activated c protein. 176 The application of a ß-adrenergic agonist (Iike isoprenaline) to heart cel-ls resuLts in a cascad.e of events culrninating in a stimulation of Ica (calciunì current). ß-adrenergic binding to ß- receptors located on the sarcore¡nmal menbrane stirnulates adenyrate cyclase activity. Àdenyr.ate cycrase activation i-ncreases the levels of cycJ-íc adenosine rnonophosphate (cAMp), an intracel- l-ul-ar second messenger. f ncreased level-s of cA.l"fp activate the enzyrne protein kinase A which phosphorylates the a1 subunit of the Ca2+ channel protein compl-ex. phosphorylation of the d1 subunit al-ters the ca2+ channel properties causing an increase of f"".176 The resulting increase in I". by cÄMp-induced phosphorylation of the ca2+ does not change the rate or the amount of ca2+ that enters via an individual channel, but it does increase the probabíIity that the channel will open. Therefore, there v¡il1 be an increased number of channeLs open at any gÍven moment increasing the inf l-ux o¡ gu2+.180 Experinental evidence supporting this cascade and the resulting increase in I". by phosphorylation of the Ca2+ channel cones , from the use of protein kinase inhibitorslTT and phosphatases. 178 eddition of a heat stabre protein kinase inhibitor via internal ceII dialysis to inhibit c¡\Mp production resulted in the suppression of the ß-adrenergic agonist enhanced ¿au.rn In addition, the intracel-l-u]ar appì.ication of a protein phosphatase (calcineurín) !¡hich wourd dephosphoryrate the ca2+ channel \^'as arso ablê to reverse the incre¡nent in I"u by ß-adrenergic stimulation.lTS ß-adrenergic stimulation of Ïca ïnay aLso cone from the direct coupling of the activat,ed G" subunit with the Ca2+ channef. When the cÄMP-dependent phosphoryration pathway was blocked, addition of a ß-adrenergic agonist (isoprenaJ.ine) stiJ-r evoked an increase in the Ig3r176 The resulting increase in f"" by Gs was relatively small conpared to that invoked by the cAMp-mediated pathway. It is thought that Gs nay act to prirne the ca2+ channer.s for up-regulation by the cAlUP-dependent phosphorylation. 176

43 Phosphoryl-ation of the ca2+ channel inay not be a means of increasing lca alone but nay also be necessary for the channel- to respond to membrane depolarization. Studies in which the a,

subunit of the Ca2+ channef has been used to reconstítutê a

voLtage-activated channel has shown phosphoryl-ation by the cÄ_I{p- dependent protein kinase is necessary and sufficient to restore activity of these preparations, Addítion of a protein kinase inhibitor to the preparation blocked activity" Ä,n endogenous protein kinase A is believed to be associated with the membrane in close proximity to the channel to regulate its gating activity.179 ß-adrenergic stirnulation al_so increases the rate of resequestering of ca2+ back into the SR, via the SR ca2+ punp. Phosphoryl-ation of the protein phospholamban by cAMp-induced

protein kinases results in increased activity of the SR Ca2* pump.183 This increased activity of the pump al_1ows for a faster re-accumulation of Ca2+, allowing for the relaxatJ-on process to occur at an accelerated rate.181 edditional proteíns Like Troponin I, C, and nyosin light chain kinase are al_so phosphorylated by cAMP-dependent kinases upon ß-adrenergic st,irnulat,ion, The phosphorylation of these protein complexes aIl-ovrs for quicker cycJ-ing of the nyofifanents during the contraction process.182 The overal-1 consequence of ß-adrenergic stirnuration is an increase in the chronotropic and inotropic aspects of the cardiac cyc1e. L8o,184 . A group of conpounds that decrease the conductance of ca2+ via the Ca2+ channels are the calcium channeL bl_ockers (antagonists). This particuLar group of compounds wil1 be discussed in detail later, and it will be sufficient at this tirne to sirnply outline their effects on cal-ciun channeL kinetics. The calciun channel bfockers bind to the a1 subunj-t of the L-type ca2+ channel. There are currently, three rnain cfasses of cal-ciun channêl blockêrs, the dihydropyridine ( dihydropyridines ) , phenytalkylarnines and benzothiazepines . 185 All three classes have their receptor-sites on distinct but closely retated sítes on the a1 subunit of the L- type ca2+ channel ,186 It is thought that the calciun channeL antagonists preferenti-alIy bind to the deporarized or ínactivated state of the calciurn channeJ-, and in sone way decrease the probability of the channel reopening.lss The process by !,rhich the calcium channel antagonists prevent or slow the reopening of the channel is thought to be by slowing channel rephosphorylation. 179 Arnst.ronq proposed that for the Ca2+ channel to be opened upon mernbrane depolarization, it first must be phosphorylated by an associated protein kinase A.179 rf the calcíurn channel brockers sl-ov¿ the rephosphorylation process, the channels $¿ould not. be avaiLable for opening upon further membrane depolari zatíons . l-?9 The resul_t of binding of the calcium channel blockers to the L-type Ca2+ channel- is to red.uce the influx of Ca2+ ions via the channels. This reduction in rtriggerr ca2+ causes a d.ecrease in contractility in cardiac tissue, and reLaxation in vascurar smooth muscLe. 185 ReguLatÍon of catcium channel Density one of the prirnary means of controlling the ca2+ available for cel-lul-ar functions is via the activity of the ca2+ channeLs, The

{5 regulation of the number of ca2+ channels present ín a tissue at a particular ti¡ne is al-so controlled. It is v/elf known that the number of Ca2+ channels present in a specific tissue is influenced by circutating drug (hormone) levels and particular disease states. The regul-ation of ion channel-s and their densities appears to follow a similar pattern to that observed in the reguJ-ation of nembranê receptors. Receptor regulation can be separated into two main categoríes: (i) homofogous regulation, in r^¡hich a ligand regulates its own receptor, and (ii) heterologous regulation, v¡here the receptor is regulated by a ligand/process occuring at a discrete receptor system.187 The exact rnechanism by which a Iígand wilJ- regulate its receptor is different and specific for that particular receptor systen. Receptors can be regulated after either long- or short-tern exposure to a ligand. It is usually foLlowing a prolonged exposure that changes in receptor regulation are noticed. Short term exposurês usually resul-t ín tenporary nodifications to ¡nembrane potential, coupling systerns, receptor distribution, phosphoryl_atÍon state and menbranê lipid environ¡nent causing theír change in receptor metabolis¡n267'268 (i.e. synthesis, membrane insertion, internal i zation, recycling and degradation26s ) . Chronic or 1ong- term ligand exposure usually results ín a decrease (down- regulation) of receptors v¡ith a agonist ligand and. an increase (up- regulation) with an antagoníst.26s,269 The regulation of ion channels (e.g. Ca2+ channels) is likety similar to those procêsses that rêgulate cefl surface receptors as

46 both are menbrane proteins and therefore have siniÌar processing mechanisms. The following discussion wiLL examíne the influence on ca2+ channel density of certain dísease states and after exposure to chronic circutating drugs (hormones).

C ircu 7 at ing Drug s / Hormone s There are several- $¡elL characterized responses of ceÌl-surface receptors to circulating horrnones. These include down-regul-ation of ß-adrenergic receptors266,270 | ínsulin receptors2?2 and low- density lipoprotein receptors2T4 to high circutating hormone level-s. The down-regulation seen in ß-adrenergíc receptors occurs after exposure of the ß-receptors to high circulating l-evels of epinephrine. This exposure of the ß-receptors to high circulatÍng epinephrine results in a nonfunctional binding of ligand to receptor, and effective down-regulation of the ß-receptors. The ß- receptors are still abl-e to bind epinephrine, but the bound epinephrine Ís not abl-e to activate the adenylat,e cyclase systen necessary for cel-Lular action and this inactivation is thought to bè nêdiated by phosphorylation of the ß-receptor. 266 t27 o t27 7 The down-regulation seen in insuLin and low-density tipoprotein receptors is ¡nediated by a different process. High circulating levels of ínsulin hornone or lov¡-densíty lipoproteins resul-ts in cel-I-receptor int.ernalization by endocytosis.272,273,274 This effectively reduces the nurnber of receptors present on the surface at any one tirne, resulting in a down-regulation of the receptors. Exposure to circul-ating compounds such as ethanol, 1ead, insulin, thyroid hornone and calciu¡n channel antagonists all have been implicated in altering the number of ca2+ channefs present in certain tissues. 187 For exampfe r anj-mals or celfs chronicalfy exposed to ethanol or l-ead resulted in an increase in the numbêr of

1, 4 -dihydropyrídine-binding sites present in certain brain regions. L.,ead treatnênt resul-ted in an increase (482) in the B^"* (nitrendipine binding) in rat striaturn and cortex, but was found not to change in the hippocampal region.188 The developmênt of ethanol dependence is proposed to involve an increase in the number of l-, 4 -dihydropyridine-binding sites as a result of chronic ethanol adninistration. EthanoL is known to inhibít ca2+ influx in cel-ls following chronic treatnent, resuJ.ting in an increase in the density of ca2+ channels. L89' i'9o Thê bodyts own circulating hormones may al-so regulate thè density of channels. Treatnent with the hornone insulin over a 21- day period resulted in an increase in the density of 7,4- dihydropyridine binding sites in cuLtured human muscl-e cel-ls.191 However, an increase in the number of caz+ channels was also found. in cardiac muscl-e ¡nembranes isol-ated fron streptozocin induced diabetic rats.337 Additional functional alterations may also result fro¡n al-tered hornone levels as r^ras observed i-n rat ventricular rnuscle after chronic diabetes ne11itus.192 Chronic thyroid hor¡none treatment in chick ventricufar cells resulted in an increase in the nunber of 1, 4 -díhydropyridine binding sj-tes and a concurrent increase in the nu¡nber of ß receptors present.193 However, in heart mernbranes obtained fron hyperthyroid rats, a decrease in the number of ca2+ channel-s present with a concurrent increase in ß-receptor density was reported. uypothyroid rats fron the sane study shovred the opposite effect, with an increase ín Ca2+ ctrannel-s and. a decrease in the number of ß-receptors present.194 The discrepancy rnay be .due to a species specific dífference, but additionaf work into thyroid hormone reguJ-ation is required" The coÍrpounds that would be expected to regul-ate the density of the Ca2+ channels to the greatest. degree and with the most specificity vrould be the calciun channel antagonj-sts. Several studies have been undertaken looking at different types of calciurn channel- antagonists, modes of administration, species and tissue reactions.187 In me¡nbranes prepared fron mouse brain tissue, a 4OB reduction ín 1, 4 -dihydropyridine binding sites was noted after oral treatment with either nifedipíne or verapaníI for 28 days and no change $¡as noted. hrith oral diltiaze¡n over the same period.2'15 A 232 decrease in 1, 4 -dihydropyridine sites in brain and 49e" in heart was noted in a rat rnodel after 20 days of intravenous nifedipine.2T6 No change nas noted in heart tissue prepared fron râts treated with a lower dose of oral nifedipine for 14 d.ays217 , indicating that alterations may be tirne, node of application and dose dependent.

1, 4 -dihydropyridíne binding sites in Pci-z cel-l-s were also noted to increase (292) after 5 days of nifedipine treatrnent, whife a d.ecrease (24å) was noted after 5 days of treatment with Bay K 8644 (Ca2+ channel agonist) .279 It is difficult to obtain reliable infor¡nation on the action of the cafcium channel antagonists on ca2+ channel density due to the variabílity in experirnental protocol-s .

49 Ðisease States Several pathological conditions are associated with an alteration in the nunber of ca2+ channels present in certain tissues. Studies conductèd on hypertensive rats have shown alterations in 1 , 4 -dihydropyridine-binding sites and has 1êd researchers to suggest that these al-terations may be a result of some functionaL change in these animals, playing a rol-e in their hypertensive condition. Several studíes were conducted on a strain of rats referred to as sHR (spontaneously hypertensive rats) in which, due to a genetic condition, al-1 develop hypertension. Radioactive nitrendipine binding on heart membranes prepared frorn 24-vreêk-old sHRs and g-week ol-d SHRS showed an increase in Ko and B*.* in the 24-\,reek but not the 9-week group, as reLated to nornotensive control-s.195 This was confir¡ned later with elevated nitrendipine sites in cardiac tissue isolated fron 16-weèk-old sHRs but not in l-o-!¿eek-old sHRs.196 An up-regulation in the number of

1, 4 -dihydropyridine sites was noted in heart tissue isol-ated from spontaneousl-y hypertensÍve rats (33? increase) and salt-sensitive rats (55å increase) afber 7-27 day nitrendipine treatrnent.2TS It has, therefore, been suggested that alterations in the density of Ca2+ channels rnay be related to hypertension and the possible devefopment of the disease. Àn increase in the number of ca2+ channels has been reported L97 ,79a r99 ,2oo in hypertrophied cardiac ¡nuscle. ' The Syrian cardiomyopathic hamster serves as a ¡nodel for human hypertrophy. Radioactive labelJ-ing studies have shown increased dihydropyridine

50 bindíng sites in heart, brain, skeletal nuscle and srnooth rnuscle in the cardiomyopathic hanster.2o0 Additional support for the increase in cardiac binding sites has cone fron hypertrophy induced via aortic stenosis. .A.n increase in the number of dihydropyridine binding sites was noted after 5 days and 3 weeks post surgery.2ol controversy has been added, however, by the fíndings of Howlett, et a-l , who have shown that there is no significant aLteration in the number of Ca2+ channels obtained fron cardiomyopathic ha¡nster cardiac tissue.202 No sígnificant changes in dihydropyridine binding sites were observed fron cardiac tissue obtained from 3s- to 41-day old rnyopathic harnsters. 2o2 | 2o3 Further study is required in order to resolve this controversy, and factors Ìike cardiornyopathic strain, age and membrane preparation differences wíl-Ì have to be addrer".¿.18? Dihydropyridine-binding sites were also increased in human hypertrophied cardíac ¡i"=n".204,205

Patients suffering frorn parkinsonrs disease have shown a decrease in the nu¡nber of dihydropyridine-binding sites in varj_ous areas of the brain.206 Nitrendipine binding showed a d.ecrease in Bmax from the areas of the caudate nucleus, substantia nigra and putanen with no change noted in the affinity of the channels. Parkinsonrs disease is characterized by a degeneration of substantia nigra doparnine neurons which is suggested to be the cause of the loss of I,4 dihydropyridine-binding sites. CÀIJCIU¡'Í CHANNEI., ANTAGONISIS Hi story The cal-ciun channel antagonists are a heterogeneous group of

51 related conpounds, with one com¡non factor linking them together, their action on the voLtagè-gated ca2+ channef. Research into their structurer mode of action, cLassification and clinical use has been undertaken for 30 years since their discovery. Recognition for nuch of the early \,rork reLated to cal-ciurn channel antagonists is attributed to Albrecht Fleckenstein, who Ín 1-963 was approached by two pharmaceutical cornpanies to look into th¡o ne\,/ conpounds that had vasodilatory and unexpl-ained cardiodepressant r5i1i¡i.r.209,21s Dr. Fleckenstein and his colleagues' original work, al-ong with work carried out by Winifred Nayler, utitised one of the originally discovered cafcium channel- antagonists: preny1anine.210 This compound was shown to produce coronary vasodilation and had a strong effect on electromechanical_ uncoupling in cardiac tissue and this effect was similar to that seen by the removal of extracelLular ca2+. The inhibitory effects on contractility could be overcone by approaches that íncreased cellul-ar ca2+ mobilization. 2r7,272 Prenylamine's inhibitory action on rnyocardial contractility was questioned as to whether it was a resuft of adrenergic ß- receptor blockage, catecholamine depletion or some other unidentified interventíon. At a conference in Capri, Dr. Fleckenstein and Dr. Nayler presented their work and proposed that prenylanine action was ¡nedíated by inhibition of calcium perrneation across cardiac ceflular me¡nbranes.210 This rvas the starting point for a varied and extensive research history that has spanned severaL decades.

52 The rrCalciurn Antagonistsrt nomencl-ature vJas coined by Fl-eckenstein in L966 | and they r¿ere described as a group of conpounds that restricted calcium-dependent ÀTP utílization, contractile energy expenditure and oxygen requirement via an interference with activator cal-ciun in active cardiac ¡i""l.r".2J-0'213 Prenylanine v¡as a nember of this new group of compounds, but additionat compounds with even rnore specific and more potent antagonistlc characteristics were discovered incl-uding verapamil (one of the two original coÍrpounds)21s, rnethoxyverapami I (D 600) and nífedipine.214 EarJ-y experimental studies shor,¡ed that these conpounds (verapamil, D 600 and nifedípíne) were able to sel-ectively block thê ca2+ inffux via the slow-rnediated channel in depolarized cardiac nembranes without affecting the fast transmembrane inward Na+ current that initiates the action potential.21o It is this ability to interfere with the voltage-gated Ca2+ channets in the cardiovascular system that has been the major focus of research i¿ith these compounds. Additional research lines have focused on vascular smooth muscle, nonvascular s¡nooth muscLe, and neuronal syst.erns.2o9

clas s if icat ion Fleckenstein's originaJ- qrork allowed for the classification of cal-cium channel antagonists into tv¡o groups: (i) Group A: Highty specific ca2+ antagonists, and (ii) croup B: Less specific ca2+ antagonists. 216 Group Af or the highly specific ca2+ channel ant.agonists,

53 incLuded one of the oríginaJ.ly discovered antagonists, veraparnil (phenyl-aLkylanines). Al-so incl-uded in this highly specífic group krere nifedipine (dihydropyridines) and dil-tiazem

( benzothiazepines ) .216 These conpounds were able to interfere v¡ith the transsarcolem¡naL influx of Ca2+ via the slow-entry channel wíthout major inhibition of the fast inr,¡ard Na+ current. They were also able to depress ca2+-dependent excitation-contraction coupling

in marnmalian ventricular rnyocardiun by 9OZ-l-OOå before the fast Na+ current was also .¡¡""¡.¿.216r217 The Ì{HO (Wor1d HeaLth Organization) has subdívided thê croup A calciun antagonÍsts into classes I, II, and III: veraparnil-Iike, nífedipine-1ike and diltiazem-like cornpounds. 218

Verapanil was the prototype Ca2+ channel- antagonist and a me¡nber of the phênyl-alkyLarnine group, introduced Ín Europe in 1963. Verapanil has been one of the ¡nost extensively studied experimentally, as v¿e1l- as cIinically, of at1 the antagonists. Verapamil was originally used clinically as an antianginaÌ and antihypertensive agent.219 However, it soon becane evident that verapamil had a rnuch morê drarnatic effect in treatnent of supraventricular arrhythmias.220 AdditíonaI members of the veraparnil-1ike antagonists include galloparnil (D 600), anapamil and . _ t1ô crapamrl- . --- tlifedipine was the prototype of the dihydropyridine group of Ca2+ channel antagonists. Nifedipine was d.iscovered by professor Kroneberg, who in 1-969 asked Dr. Fleckenstein to investigate this new substance, denoted. as Bay aLo4o,2r5,22o Nifedipine r,ras found to act at a different site on the sto\,r' ca2+ channel- than veraparnil, and had powerful arterial vasodílatory ability but had littIe effect at the AV node. Nifedipine was found to be useful in the treatnent of all grades of hypertensj.on but had littfe or no direct effect on supraventricufar arrhythrnias . 219 The dihydropyridine

( nifedipine- l ike ) cl-ass of ca2+ channel antagonists has undergone an explosion with respect to new additions to the group. There are currently dozens of ¡nembers to this group including nitrendipine, felodipine, isradipine, PN-2OO-i-l-O and amLodipine to ¡nention a f er.¡. Díl-tiaze¡n is the prototype nenber of the benzothiazepine group and was initiaJ-ly developed in Japan and is nor¡/ available worldwide. Diltiazem acts and j-s used ctinically for the same spectrum of disorders that verapanil is used for. This 1ed researchers to think that dil-tiazern interacted wíth the same site on the Ca2+ channel that veraparnil did. It is now knovrn that êach of the groups of Ca2+ antagonists binds to its own receptor site on the c, subunit of the L-type Ca2+ channel. Diftiaze¡n is clinically usêd. for conditions such as angina pectoris, hypertension and 219 supraventr icul-ar arrhyth¡nias .

The croup B ca2+ channel- antagonists are less specific and less potênt than those menbers of croup A. These compounds are stil-1 capable of interferÍng v¡ith the excitation-contraction coupling process, resulting in a decreased rnyocardial contractíIity. However, due to their fess specific nature, they also affect the fast Na+ influx during the initial phase of the actìon potentiaJ. as well as ínterfering with Mg (Magnesium) -

55 dependent pheno¡nena.215,222 Members of thís group include prenyLamine, flunarizine, bepridil, caroverine, perhexiline, and cinnariz ine.215 Flunarizine has been sho\,¡n to have prorninent cerebral- vasodil-atory effects and is consid.ered a mixed antihístaminic. ClinicaL uses include rnigraine, vertigo and transj-ent ischerníc

attacks. Bepridil- is considered a rnixed sodium btocker that may have clinical- use ín angína and arrhythnias, but has been rernoved. fron the Àmerican market due to prolongation of the eT interval in the cardiac cycle.219 Mechanís¡r of Action

The ca2+ channel antagonists are known to bind to the a1

subunit of the l-,-type ca2+ channel in order to mediate their Ca2+ blocking influences . 164,234 The a1 subunit of the L-type ca2+ channel ís cornposed of four internal repeatíng sequences, with each sequence consisting of six ¡nembrane spanning hel-íces.121 Molecular characterization studies have been carried out in order to determine the precise location on the c, subunit where each of the ca2+ channel blocking drugs bind. Modelling studies carried out by Langs, et al . proposed that the binding síte of the dihydropyridines in skeletal_ rnuscle nay be Located on the 54 helix of the L-channeL c, subunit.223 However, studies carried out by Rugella, et a7.224, proposed another site of dihydropyridine molecufar binding. Employing irradiated 3H [ ] nitrendipine to covalently bind to the purified skeletal nuscle o1 conplex, followed by digestion and. sequencing of the peptides,

56 indicated that the nitrendipine bound. to the cytosol-ic taif in the region of the 56 helix of the IVth repeating subunit.224 Analysís of L-type Ca2+ channels frorn other tissues (cardiac and smooth muscLe) showed this particular region to be highly conserved

between tissues, suggesting a possíble reason as to $rhy dihydropyridines bind with high affinity to ca2+ channels frorn al-l tissues.22s The sequence inrnediately following this proposed dihydropyridine binding siÈe is thought to be the ca2+ binding donain for the channel, which has al-so been found to be highly conserved betv¡een tissues.226 Association between these two sites nay pfay an important functional rol_e in regulating channeL activity. The phenylalkylarnine binding region also appears to be located on helix 6 of the IVth repeating subunit on the a,! subunit, Photoaf f inity Iabe11ing rr'ith a phenylatkylarnine-receptor-selective verapanil derivative (arylazide) locafized the receptor binding site to the intracel-Iu1ar end of the M6 helix and adjacent intracellul-ar arnino acid residues on the intracellul-ar side of the ca2+ channel .227 The anino acid sequence of the IVs6 and surrounding C-ter¡ninal tait is híghly conserved among 01 subunits isofated from other tissue and specíes types (rabbit and carp skel-etal- rnuscle and rabbit cardiac rnuscle) ,228,23o,23r In addition, phenylal-kylamine binding properties and modulation of their binding by cations from these different tissues and species are also very =i*11.r232'233 suggesting that this highly conserved. area is important in the forrnation of the phenylalkylanine receptor. Localization of the receptor binding site to an intraceflular dornain is consistent with functional studies v¡hích have shown that phenylalkylarnine Ca2+ blocking drugs nust be apptied to the intracellular surface of thê channel before they becone u"¡¡u".22a r229 In identifying the channel drug binding sites, the prinary focus has bèên on the three rnajor ca2+ channel antaqTonists (dihydropyridines, phenylalkylamines and benzothiazepines), Hovrever, evidence has bèen gathered to suggest that there are ¡¡r"225 possibly six234 discrete binding sites associated with the ca2+ channel . On the basis of functional and binding experirnents there is evidence for! (i) a dihydropyridine site, (ii) a phenylalkylamine (veraparnil) site, (iii) a benzothiazepine (diltiazen) site, (iv) a site for fluspirilene and pinozide 235, ( díphenylbutylpiperidines or diphenytalkytarnines ) and (v) a site for the indol-izine SR33557, a novel potent non-dihydropyridine236. As new synthetic drugs becone avaílable, additional ínforrnation about the ¡nol-ecul-ar binding sites present on the ca2+ channel v¡iII become evident. Drug interactions with their binding sites on the channeLs are often dependent upon the state of the channel. As previously discussed., the ca2+ channef rnay exist in one of three states at any one tirne; open, closed or Ínactivated. The interaction of Ca2+ channel antagonists with the Ca2+ channel are voltage-dependent, with affinity increasing with increasing rne¡nbrane depolarization. Therefore, the Ca2+ channel- antagonists preferentially bind to the

58 inactivated state of the ca2+ channel, r¿hich is the state that is favoured by depolari zaluion.2o9 t23'1 '23a There is conversely, a 1ow affinity for the other two states, closed and. open.22s ca2+ channel- activators, such as Bay K 8644, have been shown to preferentiarry bind to the open state, reducing the deactivation of this state and resurting in increased channel opening tines in the presence of this díhydropyridine activator.225 conformatÍon of these electrophys iologic observations has come frorn radiol-igand binding studies carried out in polarized and depolarized cell-s.239 t24o

Additional_ reactions with the chânnel rnay be related on a frequency dependent basis, vrhereby affinity increases with j-ncreasing frequency of the depolarizing stirnulus. This is apparent for the chârged ca2+ channer antagonists, verapamil and. dil-tiazen.241 verapamir. and dirtiazern, being charged and more por-ar species, have to access the inactivated state of the channel through the open state. Increasing t,he frequency of the depolarizing stinulus increases the probability that the Ca2+ channel will be in an open state. Àccess to the inactívated state by veraparnil and dir.tíazern through the open configuration is then afso increased. The L, 4 -dihydropyridines, being nonpoLar and noncharged' are abl-e to access their preferred binding site directJ.y through the membrane.242

once the ca2+ channel antagonist has bound to its nof ecur-ar si-te on the inactivated state of the channel , the brocker, in sone yet unknown fashion, is able to stabirize the inactivated confor¡nation. stabilization of the inactivated state prevents or

59 sl-ows the channers transition into the restíng or cl-osed state upon repol-arization of the nembrane to its resting membrane potential. For the channel to be reopened upon further depolarizing stimuti, the channel rnust first be in the resting or cl-osed state. By maíntaining the channel in the inactivated state, the Ca2+ channel antagonists red.uce the nunber of Ca2+ channels availabfe to be opened upon further depoJ-arizing stirnuJ-i. This effectively reduces the fca (inward Ca2+ current) upon nenbrane depolarization. In cardiac tissue, this reductíon in inr¿ard ca2+ movenent wirr reduce the rel-ease of ca2+ from the sR stores via the ca2+ induced ca2+ reLease mechanisrn, resulting in decreased. contractility. fn vascuLar srnooth nuscle, Ca2+ channel blockage results in vasodilation due to reduced inward Ca2+ movement. One theory that has been proposed for how the ca2+ channel antagonísts stabilize the inactivated state of the ca2+ channef has come fron Arnstrong, et at,!72 Arnstrong and his colleagues suggest that for the Ca2+ channel to be opened upon presentation of a depolarÍzing stinulus, the channer- ¡nust first be phosphoryrated by an endogenously linked protein kinase. The ca2* channel antagonists bind to the inactÍvated state of the channer- and inhibit or sr.ow the repofarization phase of the channel. This effectivery reduces its ability and availability to be reopened upon further stimulation. 172 Different isorners for each of the ca2+ channel antagonÍsts exist, and these isoners differ in their ability to modur-ate ca2+ channel kinetics.245 verapamir exÍsts in two isoners, the d and r.

60 forn. cIinically, verapanil is administered in a racernic mixture of d- and -Z-verapamil, producing a 1o-fo1d greater irnpairment of 243 atrioventricular conduction than the d-isorner . t244 Four diLtiazern stereoiso¡ners exÍst incruding; d-cis, 7-cis, d-trans and l.-

trans, Al-l four isomers were found to bind to the same benzothiazepine receptor site but had differences with respect to their activity. At1 four isoners r,¡ere abre to inhibit bínding of radioactivery labelled diltiazem but the potency of inhibítion $/as different (d-cis>7-cis>d-trans=r-trans ) . Arl iso¡ners were ar-so found to modulate ¡3H1eNzoo-:_ro (a dihydropyridine ) binding with the d-cis isomer stirnulating binding and the others showing inhibition.246 Enantiorners of certain dihydropyridines have differing potencies and arso act at different binding sites, with antagonístic or agonistic properties. The dihydropyridine pN 202- 791 exists in two enantiorneric formations tS(+) and R(_)1. The S(+)-PN 2O2-79L isoforn is an agonist but its optícaI isomer R(_)_ PN 202-79! is an antagonist.24T rt is generarly berieved that the antagonistic and agonistic dihydropyridines mediate their action by binding to the sane receptor sit,e248. The dihydropyridine pN 202_ 791 appears to bind to tr"¡o separate sites as determi-ned frorn conpetitive binding studies, which assurnes if the isomers bind to the sa¡ne site they should compete for binding to the site. rt was found that the concent.ration-response relationship between the enantiomers showed no cornpetition, suggesting that the action of these tr+o forns invol-ve two separate binding sites.247 The three rnajor ca2+ channer antagonist binding sites on the

61 o1 subunit are alfosterically linked to one another.209 These al-l-osteric linkages have been deterrnined by the use of radioligand bindlng studies carried out in nenbrane preparatíons from excitable tissues.249 These aLlosteric Linkages aLl-ow for positive and negative heterotropic interactions between the various Ca2+ channel- antagonists bínding sites. Binding of any one of the ca2+ channel antagonist groups to their respective binding site on the a, subunit will result in a negative interaction with the Ca2+ pore, decreasing rca2og,242. Bindíng of one of the antagonist groups to their respective site wilL also have eíther a positive or negative effêct on the binding of the other antagonists to their sites. For exanple, binding of veraparnil (phenylalkylarnine ) to its receptor site will have a negative alLosteric affect on the bindinq of dihydropyridines to its receptor site. Thêrefore , phenylalkylanines bound to their receptor site wiLL inhibit or reduce the binding of dihydropyridines . phenylal-kyl_amines also have a negative inftuence on the benzothiazepine receptor site, Binding of a dihydropyridine antagonist to its receptor site negatively infruences the phenylalkytarníne and benzothiazepine sites. However, binding of a benzothiazepine antagonist to its site wí11 posítívely influence binding at the dihydropyridine receptor site whiÌe negatively infLuencing bindíng at the phenylalkylarnine site.242 The posítive binding inffuence on dihydropyridine binding showed by dirtiazern (a benzothiaz epine ) is specific to one stereoisomer of the diltiazen antagonist, the d_cis isomer. The other thrêe stereoisomers of the dittiaze¡n nolecure

62 shor^red inhibítion of binding to the dihydropyridine receptor síi-e,246t253 The positive influence that dil-tiazem and other positíve reguLators [ (+) -tetrandine] have on dihydropyridine binding is postulated by Staudinger, et al . , to be an interdependence of the dihydropyridine binding site and the ca2+ binding site on the c, subunit.2s2 The positive heterotrophic arlosteric regulators affect ca2+ rate constants and optimize coordination of ca2+ in the channel pore which in turn increases the affinity of the channel for the dihydropyridines. 2 s2 The binding of the ca2+ channel antagonists to their receptor sites has been knov¡n for sone ti¡ne to be rnodulated by the presence of divalent cations. Studies have shor,¿n that high affinity 1,4_ dihydropyridíne interactions with the cl. subunit critically depend on the avail-abifity of divalent cations such as Ca2+ 25o ln the presence of ¡nicromolar Ica2+], the ca2+ channel bl0cking receptor site was stabífízed in a conforrnation that alrowed high-affinity binding of phenylarkylarnines. Àt mil-limorar ca2+ concentrations, a l-ow-affinity state of binding on the stabfe ca2+ channef brocking receptor was observed..251 The binding of ca2+ antagonists to their tissue receptor sites is al-so modulated by temperature2s4, ¡nernbrane lipid peroxidation25s, and rnembrane cholesterol_ content.2s6 Àn increase in Ko with increasing tenperature has been observed and this is thought to be a resul-t of an increase in the dissociation rate of the ligand- receptor complex, rrith little change with respect to the association rate.258 The increase in the dissociation rate with

63 increasing tenperature is suggested to be mediated by a configurational change in the dihydropyrÍdine receptor induced by the increased tenperature,25T Lipid peroxidation (an irnportant mechanism in tissue ischernic damage)259,26o has al-so been shown to induce a confornational change in the ca2+ channef264 such that thêre ís an altered binding of radioactiveJ-y label]ed dihydropyridines ( t3HlpN-200-11-o) resulting in altered ca2+ ion fluxes.255 Alterations in membrane cholesterol content (as seen with aqe261, diabetes262, and atheroscLerosis263 ¡ has been observed. to alter binding of ca2+ channel antagonists.256 An increase in menbrane cholesterof content resul_ted in a d.ecrease in Ca2+ channel antagonist binding, whÍch is thought to be a consequence of the increased freê cholesterol aJ_tering drug partitioning coefficients. 256 Al-terations in cardiomyocyte nenbrane composition by addítion of l-ow density lipoproteins2so and oxidized 1ow densíty lipoproteins265 has beên shor^rn to increase ca2+ transient,s by rnodifying ca2+ transport through L-type channels. cells treat,ed with oxidized 1ow density J_ipoprotein were found to be more sensitive to the blocking acLion of nicardipine (a dihydropyridine) than controt cardiornyocytes. 26s cLinical Applícation

The capacity of Ca2+ channel antagonist to block the sLoh/ inward ca2+ current has rnade these cornpounds an irnportant addition to the group of drugs used in the treatment of conditions rel-ated to the cardiovascular system. Much of the original clinical us of ca2+ channel antagonists vJere as antianginar and antihypertensive agents, though their scopê of use has now becone nuch more extensive. 219

The Ca2+ channel- antagonists tend be funped together into one large group of conpounds because of their unifying blocking action of the L-type Ca2+ channel. However, the Ca2+ channeL antagonists arê commonly broken down into three subgroups which have different physioJ.ogical actions upon adninistration in an experirnentat or

clinical- situation.281 Veraparnil ( phenylalkytamine ) shows the ¡nost sígnificant affect on cardiac ( atrioventr icul-ar ) conduction but has ninimaf vasodiLatory properties. llifedipine (dihydropyridine) has the nost potent vasodilatory effects but has rnini¡nal_ affect on the cardiac conduction system. Dittiazen ( benzothiazepine) has intermediate actíon on cardiac conduction and vasodí Lation. 281 ca2+ channel antagonists are currently used to treat a variety of complications q¡ithin the cardiovascul-ar systen. The abil-ity of compounds like veraparnil and diltiazern to slov¡ conduction velocity through the atrioventricular node has made these drugs useful in the treatnent, of cardiac arrhythnia s2ar,2g2 such as supraventricular tachycardia. 283 The ca2+ channef antagonistst peripheral vasodíl-atory and negative inotropic properties have made then useful in the treatrnent of ischemic heart conditions such as angina pectoris (Prinzrnetalts or variant angina and chroníc stable angina) 2ar | 244-2a9

ca2+ channel antagonists ability to btock the slow inward ca2+ current, made then attractive prospects in protecting the heart fron danage during ischernic events. post-ische¡nic infarcts are

65 generally thought to be a direct resuft of Ca2+ overload, possibly occurring vja the ca2+ channefs.29o Therefore, the capacíty of the

ca2+ channeJ. antagonists to block the channel and prevent ca2+ overload coupled with the favourable hemodynarnic benefits of vasodilatíon were believed to be benef i-ciaI in heart fail-ure

conditions. This has not turned out to be the case. In fact, Ca2+ channel. antagonist treatnent in heart failure has an adverse effect on patient recovery. 28r | 244 '292-29't The negative inotropic properties of Ca2+ channef antagonists293 | 296 (possibly increased effect in faiJ.ing heart292¡ and the activation of neurohor¡nonal systens (e,g. renin-angiotens in systen)294 have been proposed to undertie the adverse effects in heart faiture. Bersohn and Shine (l-993) have shown so¡ne protectj-on by veraparníI in ischemic rabbit heart.291 The protectíon was dependent upon pretrêatnent of hearts with verapanil prior to the ischernic episode.291 The second generation ca2+ channel antagonists (verapamil, dil-tiazem and nifedipine being first generation) like isradipine, appear to havê more specific actions on vascuLar srnooth nuscle with negligible cardiopressive effects. Isradipine nay provide favourabÌe hernodynamic benefits without the detrirnental cardiodepre==iotr.295,298,299 The ca2+ channel antagonist verapamil has been shown to beneficial in preventing cardiornyopathy in diabetically induced rats.3o0 Hypertension Ís a major risk factor for the developrnent of cardiovascular diseases, Íncluding coronary artery disease, stroke, Left ventricular hypertrophy, congestive heart failure, renal failure and aortic aneurysrns.3o1,306 Therefore, the control- of

66 efevated blood pressure is an irnportant consideratlon in ord.er to prevent the above conditions from deveJ.oping. ca2+ channel antagonists have been used in the treatment of hypertens.ion, alnost fron their concêption some 30 years ago. A1I three groups of antagonists have some effect on the hypertensive condition, but it is the dihydropyridine (e.g. nÍfedipine and isradipine) group that has been shown to have the most favourable resuLts.302-305 one of the characteristics of hypertension is an elevated systemic vascular resistance, ca2+ antagonists reduce the ca2+ influx- dependent cornponent of vascular contraction, therefore, inducing vascul-ar refaxation and a concurrent d.ecrease in blood 3o7 308 pressurer. ' The ca2+ channel antagonists have been shown to be potent arterial vasoditators that result in reduced blood pressure without actj-vation of sympathetic reflexes that resul-t in sodiurn and vol-ume retention, 307,308 Treatment with ca2+ antagonists has been shown to prevent and reverse left ventricular hypertrophy induced by the hypertensive condition. 3o9, 310 They have aLso been shov¡n to slow the progressíon of renat darnage in chronic renaL disease and following chronic hypertension. 311' 316 ca2+ has, for a long tine, been recognized as an integral part of an atherosclerotic plaque. The process of for¡nation of atherosclerotic plaques includes the accunulation of Iipids in the arterial waII, myocyte rnigration to and proliferation within the

intimal layer, and the accurnulation of ca2+.312,313 The ca2+ antagonÌsts have been observed to retard plaque for¡nation and may affect aIt levels of the plaque developrnent process.313 Ca2+

67 channel antagonists have been shorrrn to enhance cholesterol- ester hydrolysis and decrease total cholesterol- accunuLation in aortic tissue, thereby retarding plaque devefoprnent. 314 In addition to having anti-atheroscLerotic action, ca2+ antagonists are able to provide beneficial hemodynamic effects in animal-s already having atheroscleros is. 315 Excess ca2+ accunulatíon in plaque cefls is inhibited by ca2+ antagonist blockade of the ca2+ channels.312 ca2+ channel antagonists are now being observed. to have beneficial actions in cerebrovascular disease.3rT'322 Because of their potent dilatory ability, Ca2+ antagonists are abl-e to provide systemic arterial dilation that nay ameliorate cerebral ischernia by providing increased cerebral- blood fl-ow.317 I srad ipine

( dihydropyrídine ) appears to bind preferentially to cerebral ca2+ channel-s. Applicatíon of isradipine to rats experirnentally induced to undergo a stroke, shor+ed a 50å reduction ín infarct size as conpared. to rcontrolt stroke induced anima1s.319 Thís reduction in infarct size rnay be a resuÌt of increased cerebral- blood flohr (vasodilatíon) and prevention of neuronal death by reducing the ca2+ infLux into the neurons.319'322 This rnay red.uce the amount of brain darnage íncurred by stroke victims.319 ca2+ antagonists also appear to have beneficía1 resufts in treating subarachnoid haernorrhages 318

As research into ca2+ antagonists continues, new cLinical uses are being discovered for these compounds. ca2+ channel antagonÍsts have now been linked to inhibition of cancer celt growth323 and treatment of conditions including nigraine324, vertigo324 and rnood

68 disorders325. As research continues and new derivatives are discovered, additional applications of this diverse group of conpounds wiff become realized. Method s Drug treatnent protocol. Female Sprague-Da$/l-ey rats (CentraI Aninal University of Manitoba) r,reighing l-50-2oO grans .Care, were given twice daiJ-y subcutaneous (s.c,) injections of verapamil in 100 pl of water at varying concentrations: 2.5, 10, 20, 25,30, 50, 60, and 75 ng/kg/day. The treatment was carried out once in the morning and once in the l-ate afternoon for varying periods of time. control animaÌs received thê vehicle injection. Aninal- weight was monj-tored over time in order to ensure that the drug concentration administered was constant. VerapamiL concentrations of 2,5, lO, 20 and 30 mg/kg/day vrere injected for a period of 8 weeks in order to determine if the channel was sensitive to increasj-ng verapamil concentrations. A standard 1-0 ng/kg/day verapamil injection was carried out over a span of 2, 4,8, and 16 weeks in order to assess whether any changês occurred as a function of time with a fixed concentration. In order to ascertain whether changes occurred over a shorter tirne interval with nuch higher (near toxic) drug concentrations, doses of.25,50, 60 and 75 ng/kq/day were injected over a span of 24 hours . A noveL rneans of drug application, the irnplantabl_e slow- refease pe1Iet was tested. The pèllets were designed to rel-ease a constant dosage of drug over a 3-week tirne span. An initial study v¡as.undertaken in which a 5O mg verapamil pell-et (Innovative Research of Anerica, Toledo, oH) r^ras carefully implanted s.c. in the naþe of the neck of separate rats. Care was taken to ensure

70 that the pel-l-et was not danaged at atf and the snafl incision carefully sutured. The 50 mg peflet size was desígned to release a verapanil- dose of If .9 ¡¡lq/lKg/day over a three-v/eek period. Control- anirnal-s were implanted with a placebo peltet, Additional verapanif implantable pelfet concentrations were tested with varying times of study duration. Veraparnil pellets of 0.25 ng (0.06 ng/kglday) , 1-.5 mg (0.36 mg/kglday) , 5 ng (r_.t9 ng/kq/day), 10 ng (2,38 if.q/kq/day) and 25 mg (5,95 mg/kglday) were irnpJ-anted for a period of 24 hours and 7 days. Twenty-four hour studies were also carried out with 35 ng (8.33 ng/kg/day) and 50 nì9 (]-]-.9 n.g/kg/day) verapanil peLlets. ControL anirnals were irnplanted v¡i.th pl-acêbos of the same size as the corresponding experirnental anirnal pel1ets.

In order to compare the various means of drug appJ-ication, a short term (24 hour) study was carried out using SO ng (j-l_.9 :ng/kglday) implantable verapamil pell-etsf LL.9 rng/kgl¿lay s.c. injection and 11.9 ng/kglday oral dosage. The s.c. injections and. oral- dosages were given twice daily. The oral- dosages were adrniniqtered with a p.o. intubator. Radioligand bindíng. Rats were sacrificed after a predeterÍrined tíme of treatment. In the initíal studies rvhich assessed changes occurring after l-ong-term exposure with a constant drug dose and varying the drug concentrations after a fixed time, three different tissue types \^/ere exarnined. These included ventricular tissue, brain (cebrurn) and a segment of skeletal nuscle (quadriceps) . L.,ater studies assessing short terrn changes v¡ere carried out using

7r ventricular tissue on1y. Membranes were prepared from each tissue fraction by the nethod of wagner and colleagues.339 Tissue v¡as scissor minced and then honogenized for z X Zo seconds v¡ith a polytron pT-20 on a settj-ng of 5 in a solution containing bO nM Tris-Hcl (pH 7.4) at 4 degrees C. This homogenate r,/as centrifugêd at 1OO0 X g for 10 minutes and then the supernatant v¡as recentrifuged at 48,OOO X gr for 25 minutes at 4 degrees C. The resultant pe11et was vrashed tv¡ice at this speed in the 50 nM tris-Hct (pH 7.4) and was finally resuspended in the 50 nM Tris-HCl (pH 2.4) mediu¡n for protein ana1ysis340 and radioligand binding. Calciun channel density was assessed via specific binding at the dihydropyridine receptor r¡ith the radioligand t3HlpN 2OO-11-0.338

Approximately 50 ¡-¿g of skeletal ¡nuscle menbrane protein, 150-300 pg cardiac menìbrane proteín and 250 ¡rg of brain membrane protein were incubated for t hour at 25 degrees c in 0.5 rnl- of a medium containing 0.025 to 4.0 nM ¡3H1rW 200-110 and 50 mI"t Tris-HCl- (pH

7.4). Nonspecific binding was assessed in the presence of 2.5 pM nifedipine. The reaction was terminated by filtration through Gelnan Type A/E glass fíber filters (which had been presoaked in 0.3å polyethylenirnine to reduce background activity) . The filters ¡,¡ere washed twice with 2 ml of ice-cold 50 nM Tris-Hcl (ph 7.4) buffêr, dried and then the radioactivity neasured by standard liquid scíntil-fation spectrophoto¡netric techniques. In some cases, the reaction nediun contained O. j- to l-o0 ¡JM veraparnil + l-. O ¡nM Ca2* or l-o¡JM veraparnil to determine the afrosteric interactions between

72 the veraparnil binding site and the [3H]pN zoo-11-o binding. AII reactions were carried out under dinly lit conditions. saturation binding data were anaJ-yzed using the nonl-inear J-east-squares curve- fitting program LIcAND329.

Verapanil quantítation. plasna was prepared from blood sarnpJ.es coLl-ected fro¡n the taÍl severaL tí¡nes during the treatment regirnes. The plasrna was stored at -85 degrees C untiL the time of extraction. VerapamiJ_ was extracted into a heptane organic phase as described by Kapur et aI .333, v¡ith back extraction into o.l- M H2SO4. A 10p1 extraction sarnple vras run on a waters High

Perfornance Liquid Chronatography (HPLC) system with a Waters C1s /-¿Bondapak (10-pm particle size) reversed-phase column (30 cn X 3.9 mm) and eluted with acetonitri fe-KH2po4 (o.l_ M; pH 3.0) (34:66) at a flow rate of 1 n]/min. Detection was carried out on a waters 47o FLuorescence detector with 203 and 320 nn \,ravelengths for the excitation and ernissíon bands, respectively. Standards were preparqd by spiking control plasna samples with known quantities of veraparnil and its netabol_ites ( D-6f7 | D-620 and norverapamil). 6tatísticar analysis. statistical significance was d.eterrnined by one-way analysis of variance test and Duncanrs rnultiple range test.341 significance was arbitrarily set at a o.05 level-. uateriars. Al1 chemicars were of standard reagênt quarity except for the chromatographic sorutions which $rere HpLc grade. verapanil was obtained frorn Signa ChernicaL co. (St. Loiusf MO). Nor_nethyl veraparnil hydrochloríde (norveraparnil) was purchased frorn Resêarch Bioche¡nicals Tnc. (Natick, MÀ). The veraparnit ¡netabolites 5-

73 nèthylanino-2- (3 ,4 -dimethyloxyphenyl ) -2 - isopropylva leronitri Ie (D-

6t7) and 5-amín o-2- (3, 4-dimethyophenyl ) -2 -isopropyl-valeronitr i l-e (D-620) were kindly donated to us by KnoÌl- pharmaceutica l-s Canada (Markhan, Ontario, Canada). Results The first series of experirnents in this investigation addressed the issue of optimizing the node of verapamil delivery to the rats. fn our initial trials utilizing the bo mg (calculated daily release of 11.9 ng/kq/day) implantabl-e slow-release verapamiJ- peflets, a 63å morta].ity rate was observed in the first 18 hours post-irnplantation. There were no deaths in animafs irnpJ_anted with the placebo pellet or those given injections of an identical d.osage (1L.9 ng/kglday) of veraparnil. Later studies utilizing different sizes of inpLantable pellets dênonstratèd a dose dêpendent nortality rate (Figure 1). A very different dose dependent nortafity rate was observed at increasing doses of verapamil injected subcutaneously (Figure 1), The dose at h'hich rnortality

first occurred with the slow-reLease irnplantabJ-e pe11et $ras considerably lower than for the injected group. The verapamil pellets were designed to release the drug continuousfy over a 3- week period. This v¡as obviously not the case and the striking differences in rnortality between the initiaL two rnodes of drug deJ-ivery (s,c. irnplant and s.c. injection) suggested that the circutating veraparnil concentration nay not be similar in the two rnodels .

Another set of animal-s r^¡a s irnplanted with the verapamil pel-let.s (50 ng) and bloÕd samples wêre colLected regularly in order to carefuLly nonitor the refease of the drug from the pe1let into the bloodstream. The concèntration of veraparnil- rnetabolites was aLso rnonitored to deterrnine if the ¡node of adninistration may have

75 100

90

Verapamil Lnpl BO

Ëoo Verapamil injections cd !)ti Å€50 P ()B40 f-{ 0) Êr 30

20

10

0 0102430405060708090 Verapamil Concentration Administered (mg/kg/ day) f igure 1. Percent mortality as a firnction of verapamil concentration administered (mglrg/day) vta slow-release implant pellets or subcutaneous iqjection. The theoretical values for daily release of verapamil presented above were calculated for the 5,10,25,50 and 75 mgpellets. aftered the ¡netabolism of the drug,

A representative tracing of a typicaf chrornatogran produced by the HPLC after injection of a plasna sarnple spiked ¡vith a 1oo ng standard of veraparnil and its rnetaboLites is seen in Figure 2À. The primary verapamiL ¡netabol-ites D-620 and D-617 are the first to appear. These tr1¡o metabolites appear around. the 5-7 ninute mark after injectíon of the sarnple into the HPLC colunn. D-620 appears first fol-l-owed very closeLy by D-617. Àt the !7 to 2! ¡ninute interval, the netabolite norverapanil and the parent drug verapamil peaks appear. The norverapanil peak inrnediateJ.y precedes the appearance of the veraparnil peak. Using the absorbance peaks produced by this spiked pLasma sampfe, a standard curve r^ras produced. Conparing our unknov,¡n plasna sanples absorbance to the standard curve, the concentrations of verapamil and its ¡netabol-ites was abre to be deterrnined, A representative tracing of an unknown pl-asma sample obtained fron a verapanil implanted rat is presented in Figure 28. The net,abolites D-617 and D-620 were usually not detectable or detected in very small_ quantities. For comparative purposes, veraparnít \,¡as ad¡ninistered to separate rats via s.c. injection or p.o. intubator and blood was col-lected in an identical fashion. As sho!,¡n in Figure 3, pfasna veraparnil concentration was significantry higher in the pertet inpLanted rats than in the two other groups. The verapamil concentration rose over tirne and reached its peak within I hours after inplantation. Thereafter, it renained at a fever - r.o-for_d higher than the other treatnent regirnes. veraparnil $ras not

77 0.10

0.08

D-6 T7 N ,/ c) 0.06 .lC) .ocü

(Ao .o 0.04 .-t oo

0.02

0.00 36912 ls18 Time (Minutes)

Figure 2A. Representative tracing of a High Performance Liquid Chromatography (IIPLC) chromatogram showing verapamil and its metabolites (norverapamil,D-617 and D-620) as a fi¡nction of time. The tracing is from a control plasma sample spiked with i00 ng each of verapamil, norverapamil, D-617 and D-620 standards. 0.5

0.4

0¡45 ()C) 0.3 d -o oL< (A o.2 -o Norverapamil

0.1

0.0

15 18 Time (Minutes) Figure 2.4. Representative tracing of a High Performance Liquid Chromatography (FIPLC) chromatogram showing verapamil and its metabolites (norverapamil ,D-617 and D-620) as a function of time. The tracing is from a blood sample obtained from a verapamil implanted rat (50 mg) 8 hours post-implantation. J 2.5 E

cI) ì 2.O

o E TN 1.5 o o- mplonted Pellet 1.0 n jected =E o o_ o t_ 0.5 o t___J 0.0 0 2 4 8 i0 i214161820222426 Time (hrs)

Figure 3. Plosmo veropomil concentrotions during the first doy of treotment with different modes of veropomil odministrotion (11.9 mg/kg/doy). Volues represent the meon * S.E.M. for four to eight seporote qnimols. *P<.05 vs. other treotment modo lities. detectable in pJ.asma fro¡n control animals. The concentration of the primary veraparnil netabolites norverapanit (Figure 4) | D-6L7 (Figure S), and D_620 (Flgure 6) were afr increased in the plasina fron veraparnil pel ret- implanted rats. rn genêral, the metaborites appèared later in the treatrnent regirne than when verapamil was detected. The quantity of each of the netabolites was an order of rnagnitude higher l day aftêr implantation in plasma sanples frorn the pel 1et_irnpLanted rats as conpared to the other rnodes of treatment. The metabofites D-6r_7 and D-620 were èither not detectable or mêasured in extrener-y 10w quantities in the rats given veraparnil orafly or by injection. The drug was not released in a continuous, even fashion over the two-v¡eek period that it was monit,ored. At both r- and 2 weeks after inpJ-antation, the drug levels were lower or sinilar to the concentrations observed with the injection protocoL (Figure 7, upper graph) . The norveraparnil concentration also dropped precipitously frorn 1 to 7 days after inplantation (Figure 7, lower graph) . The other metabolites I D-6I7 and D_62O, vrere not detectable at 1 to 2 weeks (data not sho!rn). The analysis of the binding characteristics of [3H]pN 200_ 110 in these experiments r+as carried out using the LïGAND progran. This progran anaLyzes the specific and non_specific binding charact'eristics of the radioactive ligand (pN 2oo-11-o) and al10ws for a scatchard plot analysis of the Clata. A representative tracing of a typicar scatchard plot of binding data from a controL heart presented is in Figure B. scatchard pJ-ot analysis allows for

81 Li tiãr I ,î,i l_tr 1.0 $i Wi ;.¡ cD l,;: ì lmplonted Pellet ,^ 0.8 )l;' ln jected iir o i1,, E Orol 'i'r' (n :,; :i o ,t¡ 0.6 ,Ìi' o_

=E o o.4 o_ co a\) o \_ a) o.2 t_ o Z. 0.0 0 2 + 6 B 10 12 14161820222+26 Tîme (hrs)

Figure 4. Plosmo norveropomil concentrotions during the first 24 hr ofter treotment with different types of veropomil odministrotion (11.9 mg/kg/doy). Volues represent the meon f S.E.M. for four to eight rots. *P(.05 vs. other treotment modolities. o.4 I c lmplonted Pellet E tr lnjected ,À Io) 0.J Orol

o * E a, o o.2 Õ_ l\ l_ (o 0.1 I ô L-t 0.0 0 4 1012 14161820222+26 Time (hrs)

Figure 5. D-617 concentrations over time in plasma from rats treated with different types of verapamil administration (11.9 m,g/kg/day). Values represent the mean + S-E.M. for four to eight rats. *P<.05 vs. other treatment modalities. o.20 I a E lmplonted Pellet 0.1 6 t lnjected oì I ^ Orol o o.12 Ë (n o ã 0.08 O N (o I 0.0 4 Õ t___J 0.00 0 8 1012 14161820222+26 Time (hrs)

Figure 6. Concentratione of the verapamil metabolite D-620 over the course of I day in plasna from rats treated rith different modeg of verapamil administration (11.9 m.g/kg/day). Yalues represent the mean * S.E.M. for four to eight rata. *P<.05 vs. other treatment modalities. 3.0 o E ø 2.5 o lm p lo nted o- 2.0 '=r ln jected t* (- t. 1.5 o-\ ool 1.0 O->r- -ì t---J 0.5 0.0

o 1.0 E Ø o 0.8 o- lm plo nted = t-l 0.6 ln jected o o_ r- Lg)o\ o.+ O-l ( \-. 0.2 o z. t_t 0.0 0 2 4 6 8 10 12 14 Time (doys)

Figure 7. Plosmo concentrotions of verqpomil (upper groph) ond norveropomil (lower groph) ofter 1, 7 ond 14 doys of treotment with veropcmil in jections ( 11 .9 mg/kg/ doy) or the implonted pellet (colculoted to releose 1l .9 mg/kg/doy). Volues represent the meon + s.E.M. for three to six rots. xP(.05 vs injected group. 85 0.16

0.74

(l) q) 0.12 1-r !-r è 0. i0 o m O 0.08 oo I o\ o 0.06 c\ì z oi 0.04 rJ.'( c.)

0.02

0.00 0.00E+000 1.008-011 2.00E-011 3.00E-011 4.00E-011 5.00E-011 6.00E-011 l3rilpu 2oo-r1o Bound (M) F igure 8. Represent ative datademonsfrating Scatchard plot analysis of ¡3H1eN 200-110 binding to a crude membrane fraction of control ventricular tissue. B,o*: 6.067 x 10-11 M,

Kr: 3.941x 10-10 M andprotein concentration: 0.184 mgml. the deter¡nination of B^.* and KD. In order Lo ensure that there h¡as saturation of binding of the radÍoactivety Labelfed tigand (pN 2OO_ l-10) to the nenbrane bound ca2+ channel-s, a saturation curve r,¡as produced (Figure 9). The saturation curve d.emonstrates that saturation of [3H]pN 2Oo-110 binding occurred at the higher pN 2oO_ 1l-0 concentrations (2-4 ntf) which is in agreement with others338. The rats that survived the initial verapamíI irnplant (50 mg) drug treatnent were exanined for specific binding of [3H]pN 2oo_1j-o binding to cardiac and brain nenbranes, tr,¡o weeks post_ irnplantation. Thê results are presented in Tabfe 1. Both the Bmax and KD for pN 200-1i-o binding to cardiac nembranes vrere significantly depressed in rats ad¡ninistered the verapa¡nil via the pellets. Rats injected with an equivalent dose of verapamil (11-.9 ng/kg/day) did not demonstrate any significant alterations in these parameters, Bindíng to the brain mernbranes was unaffected by either .of the drug treatnent protocols. The unreliability of the irnplantable slow_release verapamil pellets to release a constant non_toxic dose of the drug persuaded us to discontinue their use at this stage of the study. Instead., we decided to continue our initiar- experirnents on the ef fect.s of dose and ti¡ne on the ca2+ channel biochernistry via s.c. injection as a node of drug administration. rnplantabfe slow_rer.ease verapamil per-lets were used in later experirnents to exanine the effects of short-ter¡n exposure. Results from Table L denonstrated that there r¡/ere no significant effects on Ca2+ iharacteristics after 2 v/eeks of s.c. injection at l-1.9 ng/kglday. It is possible

a7 180

160 ôo t40 o H ql 120 ôo 100

m O oo r-l oo r-{ O¡ O (-ì z Êi )f< cît_¡

0 1000 2000 3000 4000 5000 [3rilpN 2oo-r1o (pM) Figure 9. Representative data demonshaling saturation of binding of ¡3H¡fN 200-100 to a crude membrane fraction of control ventricular tissue. The specific binding is a function of,different concentrations of ¡3H1eN 200-110. B,o*: 1.648 x 10-13 (ûnoVmg), Ko: 3.941 x 10-10 M andprotein concenfration: 0.184 mglml. Table 1. Specific bindìng characteristics of [3H] PN 200-110 to crude membrane

fractions of heart and brain tissue in control, verapam'i 1 injected and verapamiì ìmplanted (2 weeks) rats.

K¿ (nM) Bru* ( fmo l/rng )

Tissue Control Injection Implant Control Injection Implant

Ventricle 0.25 r 0.05 0.i5 r 0.02 0.i0 r 0.01* 263 t 55 191 * 16 120 * 9*

Brain 0.14 * 0.02 0.12 t 0.01 0,12 t 0.02 192 t 25 211 r 18 173 r 16

Data are expressed as mean * S.E,M. of 5 - B. K6l dissociation constant; B,n¿¡: maximal density. *P<0.05.

89 that longer treatment periods are necessary to induce changes in receptor characteristics. Therefore, rats were injected with 10 ng verapanil /kglday for 2 to L6 veeks and pN 2oo-110 binding was assessed in mernbrane fractions fron three tissues: ventricul-ar, brain and skeretal nuscr-e. The resufts from these experiments are shown in Tabl_e 2. There were no significant differences in B^u* or KD vaLues for ¡3H¡ell 2oo-L10 binding in any of the tissues examined .

The concentration of veraparnil injected nay be an important factor in inducing changes in ca2+ channel receptor characteristics . Therefore, the verapaxnil concentration injected daily over an 8-v¡eek period was varied fron 2.5 to 30 nS/kS. Plasma samples were taken at the end of the I week adrnínistration period and neasured for verapanit and norverapanif content. These resuLts are shov/n in Table 3. There $ras a concentration dependent rise in the circulating leve1s of both the parent drug (verapaniL) and its prirnary metabolite, norverapamil, Consistent with previous results, the other veraparnil metabolites I D_6L7 and D_620, were not detectable . Specific binding of pN 200-l-10 $¡as neasured ín cardiac, brain and sker-etar nuscle mernbranes isofated fron controf aninar-s and those treated with varying veraparnil concentrations, There were no changes detected in cardiac or brain fract.ions (Table 4), but there was a trend for increasing B*.* and KD in skeletal ¡nuscle rnembranes with increasing verapamil concentratÍons. This attained statistical_ significance at the highest dose (30 ng/kglday) used Table 2- Specific bindÍng characteristics of [3H]-PN200-110 to cardiac, brain and skeietal muscìe nembranes of rats treated for varying times with verapamil injections.

2 weeks 4 weeks I weeks 16 weeks

VI VI VI

L. Cardiac

K¿ (nM) 0.25 r 0.05 0.15 t 0.02 0. 11 r 0.01 0.11 r 0.01 0.17 * 0.04 0.20 * 0.03 0.22 * 0.04 0.22 t 0.04 Bmax (fmol/ng) 263 r 55 191 r 16 222 x 20 260 * 26 315 r 53 296 ¡ 48 341 t 59 480 t 90 2. Brain

K¿ (nM) 0.14 r 0.02 0.12 a 0.01 0.10 t 0.01 0.11 t 0.01 0.L2 * 0.02 0.13 t 0.02 0.13 r 0.04 0.21 10.06 Bmax (fmol/nq) 192 r 25 2L1, x 1,8 2Bl * 25 2BB * 26 337 È 48 317 * 38 520 ¡ 120 500 + 110

3. Skeletaï Musc ìe

K¿ (nM) ND ND 0.35 r 0.02 0.40 j 0.05 0.35 r 0.07 0.44 t 0.05 0.73 + 0.72 0.40 r 0.03 Bnax (pmol/mg) ND ND 4.02 t 0.28 4.20 ¡ 0.52 4.19 r 0.53 4.80 r 0.48 4.00 r 1.10 2.80 t 0.40

Values represent the nean a S.E. (n=8-10).

ND: not determined; C: controì group; VI: veraparniI ìnjected (10 mg veraparni I /kglday ) . Table 3. Pìasna verapamil and norverapami1 concentratìons jn rats injected for B weeks with varying concentrations of verapamiì.

Expenimental (ng/mì Group Verapami I ) Norverapamì ì (nglml )

Control ND ND 2.5 mg veraparnìl/kg 23.8 + t5.B 6.9 È 6.4

10 mg verapamiì/kg 51.5 r 16.5 17.3 t 7.7 20 ng verapamii/kg 95.4 * 34.1 2S.Z x 8.5 30 mg verapamil/kg IIt.l r 24.6 57.4 r l3.z

Values represent nean r S.E. of 5 - 9 separate determinations. ND: not detectab I e.

92 Tab'ne 4. Specific bindìng characteristics of l3¡1i-p¡200-110 to cardiac, brain and skeletal muscìe nenbranes of rats treated for B weeks with varying concentrations of verapanil.

0 mg verapamil/kg 2.5 mg veraparnil/kg 10 mg veraparnil/kg 20 mg verapamil/kg 30 mg verapamil/kg

1. Card i ac

K¿ (nM) 0.i7 * 0.04 0.15 * 0.03 0.20 * 0.03 0.24 r 0.03 0.18 * 0.03 Bmax (fnoì/mg) 315 * 53 249 t 39 296 + 48 324 x 42 357 * 38 2. Brain

K¿ (nM) 0.72 ¡ 0.02 0.16 i 0.02 0.13 t 0.02 0.11 r 0.02 0.14 t 0.02 Bmax (fmoì/mg) 337 r 48 302 x 40 317 t 38 264 x 38 305 r 38

3. Skeletal Musc le

K¿ (nM) 0.35 r 0.07 0.41 r 0.06 0.44 r 0.05 0.48 * 0.06 0.54 t 0.05* Bmax (pmol/mg) 4.19 * 0.53 4.39 È 0.39 4.80 r 0.48 4.82 x 0.45 5.94 t 0.37*

Values represent the mean r S.E. of B-10 independent observations.

* P < 0.05 vs. control values (0 mg verapamil/kg), (Table 4). The above studies denonstrated that there v¡ere no significant arterations ín the cardiac ca2+ channer receptor characteristics v¡ith either long-term drug dosage or varying drug dosage vja s.c. injection, though a change v¡as noticed Ín skeretal nuscr-e (Table 4). The sêrum plasma veraparnil concentratíons (- 2.5 to 250 nglnl) for our injected doses (2.5 to 30 ng/kg/day respectively) were in the range of the reported therapeutic concentrations of verapamil in the serun (BO to 4oo ng7rnl¡ 219. It is apparent, therefore, that in the therapeutic drug dosage range of veraparnil, there was no significant arteration in the receptor characteristics of the cardiac ca2+ channers, A depression in cardÍac binding sites was noted, however, in the 50 ng veraparnir impranted animars (Tabre 1), p]âsna The veraparnir l-evels attained in these ani¡nal_s reached a -2.2 LeveL of ng/ml by the 8th hour post-irnplantation (Figure 3). This prasna verapamil concentration is higher than the therapeutic -]-o-for-d. lever by rt is possible that thêre nay be an arteration in the ca2+ channel binding characteristics at near t.oxíc doses of veraparnil. In order to test this hypothesis, a group of rats were inplanted with a range of concentrations of sIor,¡-release veraparnil pell-et,s. The decision to use the implantable pellets again was taken because they had shown an arteration in the cardiac ca2+ channê1 binding characteristics previously, and they refeased nassive, potentially toxíc concentrations of veraparnil. contrary to their design, the sr-oru-release pelrets rereased these toxíc lor near toxic) doses of veraparnil in a short tirne period post_

94 irnplantation, and. were able to maintain that levef for at least 24 hours (Figure 3), A range of pellet sizes were tested (e.ZSt L.5l 5, 10, 25t 35t 50, and 75 ng) for varying periods of tíne (24 hours and 7 days) . The 75 mg pelfet inplant group had a t OOZ nortalíty rate, and the 50 mg implant group had only 2 out of 5 anj-nals survive, too snalL a nurnber in order to get an accurate result. The Bmak results for the renaining groups are prêsented in Figure LO (24 hours) and Figure J.l (7 days). No significant differences were noticed bêtween the various irnplant groups t/ith respect to t3HlpN 2oo-1r-o bínding to cardiac menbranes. The KD values for the 24 hour study ranged fro¡n 0.9 + 0.1 x L0-10 nM (control) to 1.1 + 0.2 x 10-10 nM (25 ng). The 7 day treatnent group had a KD range frorn + 0.9 O.1x 10-10 nM (control-) to 1.L t 0.2 x 1O-1o ntf (25 ng). No significant differences h'ere noted in KD amongst any of thê groups.

Br'ood sanples taken from the above verapamil pellet irnptanted groups dernonstrated a siniLar variabiLity in the reLease of verapamil that the original 50 mg verapanil petlet impLant study denonstrated (Figure 12). There was a drarnatic rise in pl-asna verapaniJ- concentration in the first 24 hours post-irnplantation which dropped siqnificantly by 3 days and re¡nained at a relativery constant and fow level for the rernainder of sarnpling. The concentration of veraparnil present in the plasna was increased with increasing perlet size. rt is evident that the unreLiabiì.ity of these sl0w-rerease veraparniJ- pelrets is not restricted to only the 50 ng pel_let size,

95 Þ0

o d= bo

\Ô É o\ ri I O c-{ z Êr

cot-J Control 0.25 1.5 5 10 25 35 Implanted Verapamil Pellet Size (mg)

Figure 10. Specific binding characteristics of ¡3H1PN 200-110 to crude membrane fractions of,heart tissue from control and verapamil implanted (24 hours) rats. Verapamil pellet size is in mg and implanted into 200 gram female Sprague-Dawley rats. Data are expressed as mean * S.E.M of five to nine animals bo

o È Òo

\o -J Êa

l O (\ z È

c.) Control 0.25 1.5 5 10 25 Implanted Verapamil Pellet Size (mg)

Figure L1. Specific binding characteristics of [3H]PN 200-110 to crude membrane fractions of heart tissue from control and verapamil implanted (7 days) rats. Verapamil pellet size is in mg ¿1d implanted into 200 gram female Sprague-Dawley rats. Data are expressed as mean * S.E.M. of five animals. 550

500 a 550 {. soo ff +so 450 - 4oo Ë ã sso Ð É soo É 400 É. zso Fl I zoo É # 150 at ¡-¡ Ê 550 1oo rt ¡{ o Ëuo Þ Ë0 300 r{ A 0 4 B 12 1620242A ó Tlme (Houre) É D ft 250 A E c o 0.26 200 o 1.õ al v6 ã (, vto È 150 ¡2õ Þ

100

50

0 4 Tlme (Hours)

Flgurc 13. Plasma vera¡lamll eoneeatrattonr (ng/ml) over tlmo from ratr lmplanted rlth d,lflereat verapamll peltet ttzea (mg). Data repreaent Dlea¡. + S.E.II. of flve to etght anlmal¡. A final t3HlpN 2oo-110 binding study was carried out on cardiac tissue isolated from rats that had received near toxic doses of verapamil via s.c, injection over a 48 hour time span. The injection groups consisted of 25,50, 60, and 75 ng/kg/day. The resufts for the B^"* data obtained frorn these experirnents are presented in Figure 13. Due to the high rnortality rate in the 75 ng/kglday group (823), no rel-iable binding data v¿ere obtained. No significant difference was noted betr¿een the other groups of injected anirnals with respect to contrors. The KD values ranged from 1.7 + 0.3 x 10-10 nM (60 nq/kg/day) to l_.9 + 0.3 x 1O-1o nM

(controL) . No signíficant differences were detected Ín these Ko val-ues amongst the groups . Blood samples were obtained duríng the drug treatment regirnen at 4 hours and 4g hours post first injection. The anarysis of the veraparnil concentrations obtained frorn these sarnpJ.es are presented in Figure 14. The pr.asna veraparnil concentration increases v¡ith the increased drug treatnent group and Ís hígher at 4 hours than at 48 hours in aLl- respective groups. rt is significant to note that the plasna veraparnil concentrations observed in the 48 hours group were obtained approxirnatery tv¡elve hours after the rast injection. The potential for allosteric interactions between the dihydropyridíne (PN 200-110) binding site and the phenyrarkyJ-arnine (verapanil) binding site were rnonitored throughout aL1 the t3HlpN 200-110 binding experiments. rt is known that ar-rosteric interactions exist between the various binding sites l-ocated on the ca2+ channer and these interactions are ¡nodi.f ied by the presence of

99 180

ä0 160

o t40 di¿-i 120 ä0

100

Êq

ri o I O c.ì z Ê.i cî

Verapamil trnj ection C oncentration (mglkg/ day)

Figure 13. Specific binding chmacteristics of [3H1eN 200-110 to crude membrane fractions of heart tissue from control and verapamil i4jected (48 hours) rats. Verapamil iqjections were in mglkglday and injected into 200 gram female Sprague- Dawley rats. Data are expressed as mean + S.E.M. of six to twelve anim¿15. 1200 bô 1000 o

CÛ ¡-r 800 () C) o O 600

c6 400 o

(B 200 (t) (d

0 25 50 60 75 25 50 60 75 4 Hours 48 Hours Verap¿rmil lqjection Concentration (mglkgiday)

Figure 14. Analysis of blood s¿tmples obtained from verapamil iqjected animals at4 and 48 hours after the initial injection. Plasma verapamil concentration is in nglml. Data are expressed as mean+ S.E.M. of two to twelve animals. cations such as ca2+.338 fn order to ensure that there v/as no alteration in the nornaf interaction between the dihydropyridine and the phenyJ.aJ-kylanine binding sites due to the verapamÍl treatment regime, al-l-osteric studies were perforrned.. To test if verapanif had altered this allosteric interaction, cardiac membranes were isolated from control and verapani 1-treated rats and PN 200-110 binding r¡tas determined in the presence or absence of verapamil and al-so in the presence or absence of Ca2+. Verapamil inhibíted t3HlpN 2oo-110 binding and this effect was enhanced. if caLcium was ornitted from the reaction mediurn (Figures l-5 and 16). However, there was no change in the percentage of inhibition by verapamil Ín vitro as a function of verapamil treatrnent of the rats (see Figures 15 and 16 and Tab]e 5). 140 f- lo.lo uM ffi t.oo uu tzo VZto.o u.u 1¡{ {J É Fffitoo. uu ()o Ès 100 d É Bao trl C)

or6o o 6ll î, 40 t-,

Control Verapamil Verapamil Injeotion Implant

Figure 15. Inhfbttion ot [3n]ftt 200-110 binding to crude membrane preparations of ventricular tissue by verapamil. V¡:ntricular membranes were incubated with 0,25 nM ["H]PN 200-110 with various concentrgfiong of unlabeled verapamil in the presenee of 1 mM Ca--, Values represent mean + S,E,U. of eight erperiments,

103 f ]o.1o uM ffit.oo uu o V7)to.o au ¡.. +¡ Ê ffiroo. uu ()o N d É É o trt o

I o cu z È

git t-¡

Control Verapamil VerapamÍI Injection Implant

Figure 16. Inhibition of feff]fW 20O-110 binding to crude membrane preparations of ventricular tissue by verapamil. VBntrÍcular membranes rere lncubated rith 0.â5 nM ["H]PN 200-110 with various co¿centrations of unlabeled verapamil Ín the absene e of e a-' . Values represen! mean + S.E.M. of eight experiments.

104 Table 5. Inhibition of 0.25 nM t3Hl PN 200-110 binding by 10 pM verapamil. Inhibition is a percent of conhol, where 100% is binding of [3H]PN 200- I 10 in the absence of verapamil.

Verapamil Implarts

Conhol 0.25 ms 1.5 ms 5 ms l0 ms 25 ms. 35 ms

24 Hours 68.9t3.3 68.7 x2.9 71.9tL7 69.9+3.9 65.1Xz.l 70.5t2.9 67.8X2.3

TDays 67.4t1.1 69.3 x2.8 '12.2 t.3.9 75.3r5.4 71.3t4.7 74.9 *.4.I

Verapamil Injections

CorLlrol 25 me/r.glday 50 mgkglday 60 mgkglday

48 Hours 71.0 t 2.'l 65.8 t 2.4 66.8 t 2.7 67.2 f{.3

Values represent mean t S.E.M. of 5 to 10 separate experiments.

t05 Di scus E ion In order to determine the biochemicaf status of the Ca2+ channel in the present study, a radioactive ligand binding assay was enpfoyed. t3HlPN 2oo-110, a dihydropyridine, was used as the radioactively labelled Iigand because of its high binding specificity for the L-type Ca2+ channel. A possible functional problem arises in using a dihydropyridine ligand when the drug r¡¡e are testing is fro¡n another group, a phenylalkylamine. Howêver' the choice to use a highly specific dihydropyridine in assessing chanqes in the ca2+ channel after phenylalkylarnine treatment is justified in two ways. First, although radioactively labeIled 3H verapamil cornpounds are available (e.S. ¡ l verapa:niI) , their specificity for the ca2+ channel is low and, therefore, it would have been impossible to accurately deterninè any subtle changès occurring in the channels, Second, it is highly irnprobable that there would be an alteration in the binding site of one of the compounds without the concurrent al-teration in the other. The binding sites for the ca2+ channel antagonists are al-1 located on 234, the e! subunit of the ca2+ Mo l-ecular "¡.tttt.1164r characteri zation studies have isol-ated the proposed bínding sites for the dihydropyridines and the phenylalkylarnines to helix 6 of the rvth repeating subunit of the c1 subunit,224,227 rt is, therefore, tikely that if any change v¡ere to occur at the verapamil binding site, a concurrent change would also occur at the PN 200- 1L0 binding site. CurrentLy, it is not conpletefy understood hrhat effects

106 chronic Ca2+ channel antagoníst treatment has on the biochenicaf

characteristics of the ca2+ channeL. panza, et aL,215, showed. a 3H d.own regulation in the number of [ ] nitrendipine binding sites in menbranes prepared fron mouse brain tissue after tong-tern treatment (28 days) v¡ith nifedipine (2BO mq/kglday) and verapamil (27o ;mg/lKg/day) but not with diltiazem (380 ng/kg/day).27s The drugs were administered to the experirnental rnice by feeding them powdered food containing the drugs. À down-regulation in the nunber of neuronaL (brain) and cardiac Ca2+ channels was sho\,rn by cengo, et u7.216, in rats receiving chronic intravenous

adninistration of nifedipine (0.864 and 8.640 ng/kg/day) for 20 days.2?6 A concurrent do\,rn-regulat j-on in the nunber of ß- adrenoreceptors vras al-so noted. Le Grand, et a7., observed that there was a depressÍon in ca2+ current in hunan atrial nyocytes after chronic oral treatment with Ca2+ antagonists nifedipine (BO- 120 ng/day) , nicardipine (60-80 mg/day) and diltiazen (120-180 rng/day) .326 They attributed this Ca2+ current depression to a dokrn- regulation in the ca2+ channels, In opposition to these studies, Nishiyarna, et a7.277, showed that there qras no change in the ca2+ channel density (or ß-adrenergic receptor density) after chronic treatment with oraL nifedipine in rats, They administered 1OO mg/kg/day of nifedipine vja oral stornach tube for a period of tr,¡o v¡eeks. Much of the controversy, with respect to these drugsr act.ions on ca2+ channel biochernistry following chronic adninÍstration co¡nes from a variety of fact.ors: anirnals, duration, type of drug and

to7 concentration, tissues and nodes of drug administration. For exampLe, in order for the Ca2+ antagonist to mediate its blocking effects, it has to reach the specific tissue vja the circulation. Differential plasma antagonist concentrations rnight be attained using the various nodes of drug administration available and, thereforê, could account for the variability in results noted above. In order to correlate any differences r"re nay have observed in ca2+ channel- biochernistry in our experirnents, we felt it irnportant to ¡nonitor the circulating plasma veraparnil concentrations at varíous points in the treatment regirnens. Three modes of drug adrninistration were tested with respect to their ability to raise the circul-ating verapamil concentrations. These included an irnplantabLe slow-release verapanil peJ.let, subcutaneous (s.c.) injection and oral ad¡ninistration (via p.o. intubator), Slow-release pellets used to ad¡ninister drugs have become nore prevalent in both experirnental and cl-inical situations. The use of an implantable sl-ov¡-release pellet is a very convenient means of administering a constant drug dosage over a period of tine, thus elíninating twice or thrice daíly injections or oral dosages. In certain sítuations these sl-ow-release pellets have proven to be an effective neans of adninistrat íon.327 For exanple, a slow-release calcitriol pelIet has been used in avian ernbryos and showed. no difference in rnortality (with respect to controfs) at lower concentrations, while there was an elevation above cont.rol rnortalities with higher d.osages.327 Therefore, the availability of an implantable slow-reLease veraparnil petlêt presented a very

l-08 attractive neans of administering the drug as it el-iminated the need for a twice dai-Iy injecfion, 7 days a week. In our initial" studies utilizing a 50 mg ímplantable pellet, the pel-l-et was to deliver a constant veraparnil dose of I]-.9 mg/kg/day for a period of three weeks. In the first 18 hours post- inplantation, we observed a high mortality rate (63å) in the aninals. As there were no deaths in our pLacebo irnplanted group, and care was taken not to darnage the pelIêt during inplantation¿ we hypothesized that the pellets \,rere not releasing even doses of the drug, but instead \^¡ere releasing toxic dosages of veraparnil. In order to test our hypothesís, we directly exarnined the plasna concentrations of verapamil and its prinary metabolites (norverapamil, D-617 and D- 620) from impJ-anted animals, and anirnaLs that had received a sí¡nilar dose of verapanil (l.7,9 nLq/lr,g/ day) via s.c. injection and ora11y (via p.o. intubator). The pl-asna verapamil concentration was sígnificantly higher in the pelIet inplanted rats than in the other tv¡o treatment groups (Figure 3). Verapamil concentration rose over ti¡ne and peaked at approximately I hours, v¡here it stayed at a l-evel - 1O-fold higher than the other treatment nodes. There was then a dra¡natic drop in the pi-asma veraparnil concentration between 1- to 7 days (Figure 7, upper graph) , hrhere it rernained at this foïer level for the remainder of the rnonitoríng period. The primary veraparniJ- metabolites norverapamil (Figure 4, Figure 7 llower graph]) | D-6L7 (Figure 5), and D-620 (Figure 6) alt showed elevated levels in the plasna from veraparni 1- implanted animals compared to the other two treatnent modes. D-617 and D-620 were,

109 Ín fact, very difficult to neasure (not detectable) .in rats adrninistered veraparnil by injection or oral means. Additional verapamil pellet irnplant experiments using a variety of pellet sizes (0.25 ng to 75 nS) confirmed our observations that drug rel-ease was indeed unpredictabl-e and uneven. Furthermore, there was a dra¡natic increase in the nunbêr of deaths in the impJ-anted groups with increasing pellet size (> 10 ng). Standardization of the theoretical daily drug dose between the verapamil imptanted and s.c. injected animals, índicated that the dose at which death was occurrj.ng was nuch lower for the imptanted group than for the injected group. Therefore, the veraparnil implanted rats must have been receiving a toxic dose of the drug within a short time after irnplantation. Death always occurred within 18 hours post- implantation (data not shown). It is evident from thäse data that the irnplantable slovr-release verapamíJ- pelÌets were not delivering a 1ov¡, even dose of verapamil over the prescribed 3-weêk period. They were, in fact, releasing large quantities (often toxic dosages) of thê drug withín a short ti¡ne after irnpJ.antation, peaking within 24 hours then fall-ing to 1o!¡er level-s for the remainder of tine. Therefore, although the use of irnplantable slow-release pelfets could have irnportant advantages as a means of drug administratíon, these experirnents do not support its use as a reliabfe method of verapamil adninistration. In those animals that did survive the verapanil implantation, t3HlPN 200-110 binding v¡as carried out in nembranes isofated from a variety of tissues, Analysis of pN zoo-110 bindinq data was

]-t 0 carried out using a Scatchard plot fron r¡/hich receptor characteristics such âs Bm.* and KD can be determined. Bmax refers to the rnaximu¡n binding capacity of the ligand to the rnenbrane

fraction. Ko i-s referred to as the dissociation constant and is a neasure of the affínity of the particular recept.or for the Iigand. PN 200-11-0 binding denonstrated saturation at the higher pN 2OO-1j-O concentrations (2-4 nM). This vaÌue is in agreernent with other reportêd studies of PN 2oo-1tO binding338, À d.ecrease in the 8..* and KD values for PN 200-110 binding to rnernbranes obtained from

aninal-s v¡hich survived the 50 rng verapanil pell-et irnplant r¡ra s noted after thro weeks duration with respect to control_s. No significant al-terations in Ca2+ channel characteristics were observed. in the s.c. injected or orally adrninistered groups (equivalent 11.9 ng/kglday for 2 weeks) . The unreliability of the ímplantabl-e slow- release pelIet forced us to contÍnue our experinents utilizing s.c. injection as the means for drug adrninistration instead of the peLLet impJ.ants. Since there $rere no changes noticed after 2 weeks at 1l-.9 nq/kg/day, it was hypothesized that a hígher drug dosage or possibly l-onger duration of treatment was required in order to observe a sj-gnif icant change in channeL characteristics . There is a precedent that increased leve1s of the Ca2+ channel antagonist nay alter channel characteristics . cengo and his colleagues

3H demonstrated a reduction in the number of ¡ l nitrendipine bindÍng sites after 2O days of intravenous treatment with 0.864 mg/kg/day nifedipine. There v¿as a further reduction in binding sites in anj-mals treated r^rith a higher dose of nifedipine (g.640 mg/kg/day

111 i.v.).276 Therefore, a verapamiL concentration range of 2.5 to 30 ng/kg/day was chosen to be ad¡ninistered by s.c. injection into rats over an 8-week period. This particular range was al-so chosen because of other studies demonstrating this drug concentration to be effective in the treatment of conditions such as diabetic cardiomyopathy in t.¿=300'33o, genetic cardionyopathy in hamsters33l and hypertrophic cardiomyopathy in humans332. cardiac, brain and skefetal nuscle tissue were examined and no sígnificant changes in receptor characteristics were noted at any of the concentrations for eíther brain or cardiac t,issue (Tabfe 4), However, a trend. v¡as observed for íncreasing Bmax and KD with increasing veraparnil concentration for skeletal- muscle v¡hich reached significance at the hÍghest concentratíon (30 ng/kglday) (see Table 4), The increase in B*u" and KD observed in skeletal ¡nuscle nay have been a consequence of the high local veraparniJ. concentrations resulting from the s.c. injections. The s.c. injections !¡ere adrninistered in the hind region which night have resulted in a high local veraparní1 concentration in the area fron ¡vhich we obtained our muscle sanples

(quadriceps ) . In order to deter¡nine whether the duration of the drug treatnent had any effect on channel characteristícs, a constant dose of 10 mg/kglday was adrninistered via s,c, injectíon for up to 16 weeks. Cardiac, brain and skeletaÌ nuscle were again excised and PN 200-110 binding was carried out on the crude menbrane fractions. No significant changes wêre noted in the Ca2+ channel characteristics (Table 2). The circulating verapamil concentrations observed in the present study have cfinical relevance. Therapeutic concentrations of verapamif in the serum range fron 80 to 4oo ng/ml .219 The verapanil injections in this study resuttêd in plasna veraparnil concentrations of ' 25 to 250 ng/nl_, Verapamil is approximately 9Oå bound to serun protein 219, therefore, the effective circulating verapamiJ- concentration range was 2.5 to 25 ng/nl . This range of circulating verapamil concentrations may dispJ-ay some pharmacological action on contractile functj-on of the hêart, Verapanil at 2,5, 25 and 250 ng/ml can depress developed tension by 16 + 2t 49 t 6 and 76 ! 4 Zt respectively, in cardiac ¡nuscLe.116 Therefore, the cj-rculating veraparril concentrations deterrnined in the present study would be expected to have had sone pharmacofogical effects on cardiac performance. Hov¡ever, no change was noticed in cardiac ca2+ channel characteristics i-n the therapeutically relevant veraparnil range. A change i,¿as noticed in the toxic dosage range after 50 ng verapamil pellets inplantation. At this point in our work, therefore, we consid.ered it possible that in order to see alterations ín channeL characteristics, a higher (near toxic) dosage of veraparnil was required. To deternine what the threshold veraparnil dosage (and the threshold circulating veraparnil concentration) was which is required to el-icit a change in the biochernical characteristics of the channel, experiments were performed utilizing the implantable slow-release verapamil pellets and s.c. injections. The verapamiJ- pell-ets (pell-et size range from O,25 mg to 75 ng) r,¡ere utiÌized in this case to take advantage of their capacity to release high levels of verapamil- and maintain thern for at least 24 hours. Fron our previous results it roas known that these peltets del-ivered a high (near toxic) dose of verapamil' rn addition, S.c. verapamil injections at a concentratíon of 25 to 75 mg/kg/day r,¡ere a1sÕ administered. Mortality data (Figure 1) indicated that the levels of verapamil being adrninistered by these treatment protocoLs were potentialJ-y toxic (up to 8o to l-00å nortafity) . Plasma verapamil concentrations were determined at various points ín the drug to be eLevated. Hovtever no treatnent reginen and found ' signíficant changes were noted in B*.* or Ko in cardiac nembranes preparêd frorn either the veraparnil ínplanted rats (Figures 1-0 and 11) or the verapanil injected rats (FÍgure 13). It is apparent that even at high doses of the drug, the ca2+ channels appear to be highly resistant to alteration. There are currentl-y ¡1u'"225 (possibly .i*226¡ identified molecul-ar binding sites on the d1 subunit for the various ca2+ channel- antagonists and noveL derivativesr alf af losterical-l-y linked to one another.209.243,245 These aLlosteric interactions aflo\^r for increased or decreased binding of one ca2+ antagonist in the presence of another bound antagonist. For example, the binding of dihydropyridines is inhibited in the presence of verapami12o9, while stiÍrulated in the presence of the d-cis Ísomer of diltÍaze¡n246. el-terations in the all-osteric interaction between the two receptor subcLasses as a function of veraparnil treatnènt would be expected to be a sensitive index of change in one or the other

lL4 receptor. nadioligand binding studies v¡ere carríed out in v¡hich all-osteric changes between these two receptor subclasses r¿Ìere exarnined as a function of verapanil treatnent. veraparnil inhibited the binding of PN 2oo-l-l-o as expect e,d,242 t25oa . However, addition of o.l- to 1oo pM verapamil into the binding reaction ¡nedium in vitro showed a si¡nilar inhibition of t3HlPN 200-l-10 binding in both verapamil treatèd animal-s and controf anímaIs (Tabl-e 5). This woul-d strongly suggest that verapamil treatrnent of the aninals díd not after allosteric interactions between the two binding sites for phenylal-kylamines and dihydropyridines, This concLusion was further supported by data obtained with varying the [Ca2+] in the binding reaction nìedia. The cation ca2+ is an ínportant nodulator of ca2+ antagonist bíndi¡g.250,2s0a In the presence of 1 nM ca2+, the ability of veraparnil to inhibit PN 200-1-l-0 binding was reduced (Figure 15), as expected338. The effects of veraparnil on PN 2oo-l-l-o binding were not different betv/een the trvo groups in the absence or presence of ca2+. The above interventions would be expected to detect changes in a1l-osteric interactions betr¿een the receptor sites had they exísted. No differences hrere noted with respect to all-osteric interaction or rnodulation by cations, therefore, ít is unlikely that verapamil treatrnent caused even subtl-e changes in the biochemical characteristics of the ca2+ channel in the heart under the experirnental conditions used in the present investigation.

115 CONCIJUSIONS 1. The biochenical characteristícs of the cardiac L-type ca2+ channel appears to be very resistant to afteration during chronic administration of the ca2+ channel antagonJ-st, veraparnil to rats, No alterations in channel biochemical characteristics were observed in either a therapeutically rel-evant veraparnil range or in toxic dosages. Tn addition, there was no change observed after varying durations of treatment. The one exception to this was noted when a decrease in Bmax and KD was observed in the 50 ng inplanted ânimafs. 2. Brain and skeletal muscl-e L-type channel-s also appear to be resistant to alteration by verapanil. No changes in channel- biochernical characteristics in brain were observed. The trend of increasing Bma" and Ko noted in skeletal- rnuscle rnay be a consequence of high localized veraparnil concentrations. 3. As no changes in the cardj-ac ca2+ channel bioche¡nical- characteristics were noted after long-term usage of therapeutic dosages of verapanil, it is unl-ikely that verapamil treatment would significantly alter these characteristícs when used clinicaJ.Iy in the treatnent of certain disease states like diabetes and cardiomyopathy,

Howêver, it should be noted that the disease states may respond to the drug with a different sensitivity and, therefore, this v¡ould have to experimentatly tested before definitive concfusions rnay be attained.

1l_ 6 4. Theoreticall-y, j-mplantable sLow-rel-ease peJ-1ets are a very attractíve means of drug administratíon. Hov¡ever, our studies denonstrate that verapamil pellets are unreliabLe in their release of the drugr and often release toxic doses v¡ithin a short tirne post- impJ-antation. Quantitation of circulating pl-asna verapamil concentrations from the larger pe1let sizes

resulted in Levels (-2,2 u,g /mL) sinilar to clinical cases of verapanil toxicity where serun concentrations of 1.5 to 5.3 ¡.¿g/nl have beên reported.336 These implantabLe pellets are not a practical alternative for veraparnil drug administration.

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