- Email: [email protected]
Recent advances in the pharmacology of the calcium channel
17PS- March 1984 IIH I
ll| I
R e c e n t a d v a n c e s in t h e p h a r m a c o l o g y of the calcium channel R. Towart* and M. Schramm Immure of Phannacolog}.. Ba.ver AG. D.¢~tl/ B'~qvwmd I. FR( i
The calcium entr), blockers, or calcmm antagonim, hare become both ('hntcol!r mefid drugs and fascinating research too& Recent re.~earch with the.~ecmnpound~ t~ leading to new knott,ledge of the workb~gs of the calcium channel.~ ,,f excitahle t~.m,'. Novel compounds are appearing which stimulate, rather than hh~'k the chmmei. .4 picture is now emerging of a calcium chmmd with 'receptorF for raru,tt~ (:t'ogenou.~. and pmsibly endogenous, modulators. Introduction The drugs verapamil, prenylamine and cinnarizine were introduced in the 1960s, and were characterized as a {~adrenoceptor blocker, catecholamineuptake blocker and anti-histamine respectively. However, by the end of that decade they had been redefined as calcium entry blockers, and a new pharmacological principle, that of "calcium antagonism', had emerged, mainly due to the work of the groups of Heckenstein and Godfraind t.2. The group of calcium antagonists was extended by two other
important compounds, nifedipine and diltiazem in the early 1970s and the calcium antagonists have since found an important role both as experimental tools for the investigation of calciumdependent processes, and as effective therapeutic agents in a variety of cardiovascular diseases, such as angina and
Dihydmp)~line bindingdt~ No chemical structure has prm, ed more potent than the 1.4-dihydropyridines. of which nifedipine (Nitro ph(FlEnyl Dlhydro..~yridlNE) is the most ~'ell known example (see Fig. !). Recent years have seen the introduction of
many nifedipine analogues..some of which are more potent, and some of
which have greater tissue specificity. The use of high specific activity tntiated dihydropyridines (e:,. [~Hlnitrendipine and [3H]nimodipine)~has now indicated that a pharmacolo~cally relevant dihydropyTidine binding site or receptor closely associated with calcium channels exists in many tissues, including smooth. cardiac and skeletal muscle, brain tissue and a clonal phaeochrom(m.~.'toma cell line, PI2. This !ffjo~c has recently been reviewed in TIPS"-.
hypertension ~, A plethora of dissimilar chemical structures have been found to have roughly similar calcium antagonistic actions. Although some investigators have felt that some of these agents acted by purely physicochemical interactions with the cell membrane, their Potency, stereospecificity, and subtle structure-activity relations suggest pharmacologically relevant binding to recognition sites ('receptors') at or near the calcium channel for some of them at least. This short review will focus on recent advances in the pharmacology of agents which modulate calcium fluxes by interaction with the binding sites associated with the calcium channel.
]'hcsc delaik-d binding slud;:.-~ ~nh Inliated dihtdrop~rid|nc~ hal,: n,m begun to m)lt~." a major mx,,tcrx that o! wh~ such a chemJcalh dJ~cr,,~, group of compounds as scrapamil, ddt,t,,cm, cmnanzinc, etc. should all act bs a simd,u mechanism, that ,)! blocking calc,um channels. The dlhsdro~rMmc,, brad to a "receptor ~te" of tool. ~1 200 (11111(see e.g. Ref 6l in the membranes ol nlant tts,~.'s. Its prt~Jcrlics hate bccn descTll~.d in dclm] in the excellent rt.slcw ;~ Glossmann's group ". and arc consistent ~Ith its being a gl.woprotcm mac.~-
molecule closch ~ts~iatcd ~nh the shin cak'mm channel. The binding alfinities" and properties art.., perhaps surpnsin#y. in vie~ of the tissue %'Icctit,t~ .sht~-n b.~ dihydnq~.Tidint..s, korh ~imdar ~'hether the membrane factoP, hat,." been l~)lated f w m brain, heart or smooth m u s i c = "".
The receptor =s st'k:cific for dih',rdropyr;dines; other calcmm antagomsts do not directl,, displace [~ttlnitrendipinc. Ho~-eser. the non-dihTdrop~ndme calcium antagomst~ haxe been sho~n to interact allt~,tcncalh ~ith the dthsdropyndine binding site l see Ref. 5 ). and a recent study" has taken this one step further to examine the effect,, of combinations of non-dlhydrop}ndine eMctum anta.oonists.. The findln,,s• pro-]de e~tdence that the non-dihydrop)ndtne calcium antagomsts, such as xerapamd. diltiazem, cinnarizinc, etc. tbem.~hes interact ~ith a ~mgle rccognitkm site cl('~ to. but separate from the DHP binding site. Hms. Sn,~dcr's group ,,egoists a unitarx mtxiel for drug mt~lulatton of calcium ch~mnels, in ~htch a site specific 1\~r dihydrop.,,ndines is m citric
X2
--CFa
Xl
R100C H3C
02
OOR2 H
H3C
CH3
COOCH3 ~
CH3
R;
R~
X,
X?
Nifedipine SKF 26240 Nicardipine
-CH3 -CH3 -CH3
-CH3 -C2Hs -CH2CHzI~CH2G
- NO2 -CF3 -H
-H -H -NO2
Nitrendipine Felodipine
-CH3 -CH3
-C2Hs -C2Hs
-H -Cl
-NO2 -Cl
CH3
"Presentaddress:Miles LaboratoriesLid, Stoke Court, Stoke Poges,Slough SL2 4LY, Bucks, Fig, I, The chenutxd strucmres of some repre_sentunvedih.vdmpyru~ne calcmm an~Jgon~s~ ¢lefiJand B.4 Y EBb44 fin bo.O, a calciw~ agonL~r UK.
771'S- March 1984
112 apposition to a specific multivalent cation site, and a site for verapamil, diltiazem and diphenalkylamines, such as cinnarizine is nearby". Thus any of the compounds by itself may interfere with calciam movements through the channel. A maior problem has, however, arian with ~he advent of dihydropyridine binding studies, namely that, although in general a fair correlation e~ists between ligand studies and pharmacological effects (see e.g. Ref. ~), there are many tissues (e.g. brain, heart) where a discrepancy between binding and pharmacological effect exists (see Refs a and 9). Lee and Tsien, for example, find a ltX)fold difference ix.tween [ Hlnitrendipine bw,ding and its pharmacological effect on isolated heart ,.'ellst". Possible reasons for these discrepancies have been discumed elsewhere, but the alteration of the macromolecule properties or l-~ssof essential cofactors during cell disruption procedures are obvious ~:andidates~. Yamamura's group has begun performing binding studies i~= vivo, and this technique could start to resolve these problems' . The consequences of the "unitary, m~','el" for calcium antagonists based on a ~flcium channel with two binding sites in close apposition to each other" are that the effects of calcium antagonists, at least in isolated systems, should be fundamentally similar. Although the basic similarities of the different drugs led Reckenstein to group them together as "calcium antagonists', much work in recent ~¢ars. devoted to emphasizing the differences between the agonists, has been done. However, a recent investigation has shown that, despite the considerable differences in pharmacokinetics and tissue specificity (see e.g. Ref. 3), the major properties of dihydropyridines, verapamii and diltiazem are, in fact (at least in isolated heart cells), fundamentally similar m. The effezts of nitrendipine (a dihydropyridine), D600 (the methoxy derivative of verapaniil) and diitiazem were examined on isolated single cardiac cells under conditions allowing close control of calcium currents. The drugs were all found to block both inward and outward fluxes of Ca 2+ or Ba z+ through the calcium channels (sodium was absent, and sodium channels were blocked with tetrodotoxin) and increasing the external CaZ+-ion concentrations was found to increase the number of unblocked channels uj. The authors thus endorse the use of the term "calcium antagonist'. The major difference between the compounds was found in the degree of 'use depen-
dence', i.e. the degree with which the channel block is dependent on the frequency of activation of the channels. (The differences in use dependency go some way to explaining the different pharmacological profiles of the compounds.) The authors, however, feel that the differences amount more to a quantitative spectrum of activity rather than fundamental qualitative differences I". Control and modulation of calcium channels The availability of labelled dihydropyridincs is also leading to attempts to solubilize, purify and isolate the calcium channel. First reports of progress in this field are appearing from the groups of GIossmaan and Triggle, and this work shouht eventually lead to progress in another area of comparative mystery,, the ~ntrol and modulation of the calcium chanp.el which plays an important role in the function of calcium ions as second messengers. Other approaches to calcium channels are also bearing fruit. Electrophysiological studies on single cells have been mentioned above TM, and other studies are now being carried out with single calcium channels using the 'patch clamp" method (see Ref. 12 for review). Two types of calcium channels may be distinguished, the "voltage sensitive channel' and the 'receptor operated channel'. Little is known of the receptor operated channel, although in different tissues in the same species it may be sensitive or insensitive to blockade by calcium channel blockers. It may also represent a 'special case' of the voltage sensitive channel, in which the activation mechanisms have been transferred from potential sensitive control to control by occupation or otherwise of specific receptor sites for catecholamines, histamine or other neurotransmitters and autocoids. The voltage.sensitive channels are widely distributed in neuronal, muscle and ,secretory tissues, and although they share many common properties, among them a sensitivity to calcium channel blockers, obviously differ from tissue to tissue (see Ref. 12 for review). Thus, the calcium channels in cardiac tissue are h~gll~lysensitive to the [3-effects of catecholamines, whereas those in smooth muscle are much less sensitive, if at all. The interested reader is warned that the general concepts discussed here may not necessarily apply to particular calcium channels. Voltage sensitive calcium channels appear ~to differ substantially from the
classical I h~dgkin-ltuxley theory which so elegantly describes the bchaviour of sodium channels. A three-state model, with two closed states, and one open state, has been prol~)sed '~. As each individual channel can only be either closed or open, modulation takes place by changing the probability of opening. In general it appears that. for single channels which are in the state that allows activation, the probability thal the channel will open increa.~s markedly with depolarization Thus, depola~ation within certain limits increases the net calcium flux through the calcium channels in a particular cell'2 Various physiological and exogenous substances are able to modulate the ion flux through the calcium channel in various ways also, presumably, by altering the probability of opening 12. A potent activation mechanism for voltage sensitive calcium channels, at least in cardiac tissue, involves phosphorylation, by means of cAMP-dependent protein kina~, Thus catecholamines, phosphodiesterasc inhibitors or cAMP analogues increase calcium influx by increasing the probability of channel activation (for review see Ref. 12). Howe:vet, there is as yet no evidence that such mechanisms interact with the calcium antagonist receptor sites in the channel. As discu~ed above, the organic calcium antagonists appear to block calcium channels by binding to one of two sites close to, and presumably essential for, the function of a calcium binding site. Inorganic calcium antagonists, such as Cd "+, appear to act by binding to a (different) io calcium binding site. Calcium agonists Drugs which stimulate calcium influx by interaction with the receptor sites normally available for calcium antagonists could be termed, by analogy,'calcium agonists'. Several years ago both praziquantel and l-methyl adenosine were shown to activate calcium influx by some diteo mechanism, and their effects were inhibited by D600, the methoxy derivative of verapamil '4. However, these compounds have not been studied in detail. Recently, more conclusive evidence for calcium agonism has emerged with the description of two totally different compounds, the marine toxin maiotoxin, and the nifedipine derivative BAY K8644. Maiotoxin stimulates calcium influx at concentrations of 1 0 - ' " - 3 x 1 0 -~ g ml -I. Its effects are competitively inhibited by verapamil, suggesting that it has an agonistic action on the verapamil-binding
ITI'S- March lq84
] 13
site at the calcium channel t~. A 3-nilm analogue of nifedipine, BAY K1,~44 (see Fig. !). is also able to stimulate calcium influx in low (111 ' ~ - 3 x 10 ~ g ml- i) concentrations, and its effects are competitively inhibited by nifedipinet". Ligand binding studies with BAY K8644 show no interaction with neurotransmitter receptor sites, such as a- or fladrenoceptors, muscarinic or nicotinic receptors, but strodg interaction with dihydropyridine (e.g I~H]nitrendipine) binding sites. The effects of BAY K8644 are particularly interesting, as w|th nifedipine it provides the first example for the DHP binding site of a chemically similar agonist/antagonist pair with mutually opposing effects. Thus, candidates are emerging which activate the voltage dependent calcium channel by direct action on the sites occupied by the presently k n o ~ calcium antagonists. The pharmacological profile of these new compounds is not yet complete. BAY K8644 possesses positive inotropic actions, and directly stimulates smooth muscle u', an action which is not mediated by a- or 13-adrenooeptor stimulation. The known tissue selectivity, of the nifedipine-like calcium antagonists makes it probable that the dihydropyridine calcium agonists also exhibit a degree of tissu~ selectivity. Maiotoxin stimulates smooth muscle, but in contrast to BAY K8644 is known to increase catecholamine release from adrenergic neurones IT. A topic currently being investigated is whether an endogenous ligand (agonistic or antagonistic) exists which can interact with calcium antagonist sites and could be involved in the physiological regulation of calcium channels. We are now entering a new age of calcium channel pharmacology. In addition to the binding techniques and the prospect of purified calcium channel preparations, we now have potent and specific drugs which stimulate, rather than block, the voltage sensitive channel. These drugs promise to become important pharmacological tools, and their novel mechanism of action promises new therapeutic actions in diseases where the calcium-dependent contraction or secretion is pathologically depressed.
Acting through ~peczilic surh.'e recet,tor~ ,.I t',,r(,Ihlr~ m~,t)tift'~. ~l,,~t~l~,~:Ptt" t~ki'~ regulate hwal myocardial pofttsion m rev,t,n.w t,, tlhttl~t',~ ill tdrdtdt t t t i , r t [lh' structure--activity rehttionshq~.~ of d~h'tto.~llte anah~g~ .~l¢~gt'~t thal tilt" p|lrt,'h" ,IH,i
Acknowledgement The authors are indebted to Mrs K. B. Crocker for typing the manuscript,
specialized sires which e]'ti'ct l(~and tundmg andor fix'ephor attJta:ton. 5,oh correlations htenti~, the steric.hwtors tt'htth iqlluent'e binding and hint at the thtma'al forces which gorem a[finiO'.
.~ I l c n n . | ' I) I I ~ l l i ) *lm ] I , , , , / , d J~ 11147 I1~,~ 4 l n g g l c . D Iitr,~2) 1-rend~ Pbar, n,u,d ~,~ 1
5 G ~ a n n . II. l--crrr~.II Iz . lucb~-t-k¢. | . M¢~¢',. R and |lo:tmann. I- ~!'~2~ I'r~md~ Pharmawo/ ~ t ~. 4~1-4.~7 6 N ~ a n . R I . Ik~r~tt,~. M . h~,~¢t M and I~lzdumkl. M (It~3l B,~hcm Bmph~ ble, ( "ommun I I I. ~7~-~] 7 Bdkmann. P. i-'cm l). I ucbtw~kc F ~nd (;k~mmann. It 11'~12l Arzm'tm t . r ~ h ~'.. .~1 -.~.~ N Jant~. R A . Maurct. % (" . %armu:nto. I (i Bolgcr. ( i I and l n g g l c , l ) J I ! ~ 2 ) I:ur 1 Pharm~rol ~;2. ltH- It)4 9 Muq~h.',. K M M . ( b r a i d . R J . I J r g ~ n L B. L and S m d e r . S It (198311 l'r,~ "g'all
Robbie Towan rece~,.ed hU Ph.D..from Gla~g*,~ U nive~av in 1974. Alter a brwf ~pell teachm~ pharmacology at Trinity ('ollege. DM~n. hi" joined Bayer A. G. in Wuppertal. t~here he hi'came interested in cardiac metabolum and cahTum entrt blockers Since 1081 he has been ttorl~m¢ .~g~ Mil~ Laboratone~ on vanom a s p e ~ ,,f rcsp~rat~,rt pharmacology.
I Fl¢ckenstein, A., Tritthardt, H. a~d Fleckenst©in, B. (1969) P fldgen An.'h Gesamre
Physiol. Memchen Tiere 307, R25
2 Godfraind, T. and Kaba, A. (1969) Br. J.
IO ~hlramm. %1 l h o m . i .
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.MaRhta~ .¢~hram~. ~radl~ated m m , zlherrd,~,, :,: lO*l and obtained l,.t~ Ph I) tr. p t ~ , , , rr,,,r. Cologne t ~ t t ert_-,~ ,n l~ "~ HI ~hc,..'atre, .:d :~,,.'fl,,le~ at the lr.~n,',.~" ~,, P;,.;,:,,/,,:~ .,~,: ;,,,rid Ba~t'r .4 (; m ttt,pp..'~a; :~./V'~ ,tit. :, -~,,','t',:,':'d cilh'lta'vt mq~hfiddlor~
Structure of the coronary artery adenosine receptor R. A. Olsson SIOI~'(~iM ( "ardh)t~t~t'l~hlr R¢.~¢',t,xh I dbo~tlh ~rt. i)t'l,~trt,W~tt ~,' I,:tt',,n~: Ih,:,, :,'.c I "~.~,,,~.', Florida ('olh'ge ~l Medwmc. 12qOi .'~ .lOfh .~f . B,,I I0 lamr,,. ~1 ¢3~12 I ~ t
r/~o$(' do?/'/aifls o f
Re~h~ llst
ST.I
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,' ~ ~.,',?
arh'rv ddett()Mtlt" rt't'tT~h~r e~lth t'()tthllH ~t'tt'r,i"
A 1970 report that adenosine stimulates brain adenylate cyclase and that theophylline antagonizes this effect established the existence of adenosine recep-
tots t. lnve~igations since then have d~.'umented the ubiquity of adenosine receptors and are now defining their imlx~rtance in cardiac, neural, endcK:rine.
ll| I
R e c e n t a d v a n c e s in t h e p h a r m a c o l o g y of the calcium channel R. Towart* and M. Schramm Immure of Phannacolog}.. Ba.ver AG. D.¢~tl/ B'~qvwmd I. FR( i
The calcium entr), blockers, or calcmm antagonim, hare become both ('hntcol!r mefid drugs and fascinating research too& Recent re.~earch with the.~ecmnpound~ t~ leading to new knott,ledge of the workb~gs of the calcium channel.~ ,,f excitahle t~.m,'. Novel compounds are appearing which stimulate, rather than hh~'k the chmmei. .4 picture is now emerging of a calcium chmmd with 'receptorF for raru,tt~ (:t'ogenou.~. and pmsibly endogenous, modulators. Introduction The drugs verapamil, prenylamine and cinnarizine were introduced in the 1960s, and were characterized as a {~adrenoceptor blocker, catecholamineuptake blocker and anti-histamine respectively. However, by the end of that decade they had been redefined as calcium entry blockers, and a new pharmacological principle, that of "calcium antagonism', had emerged, mainly due to the work of the groups of Heckenstein and Godfraind t.2. The group of calcium antagonists was extended by two other
important compounds, nifedipine and diltiazem in the early 1970s and the calcium antagonists have since found an important role both as experimental tools for the investigation of calciumdependent processes, and as effective therapeutic agents in a variety of cardiovascular diseases, such as angina and
Dihydmp)~line bindingdt~ No chemical structure has prm, ed more potent than the 1.4-dihydropyridines. of which nifedipine (Nitro ph(FlEnyl Dlhydro..~yridlNE) is the most ~'ell known example (see Fig. !). Recent years have seen the introduction of
many nifedipine analogues..some of which are more potent, and some of
which have greater tissue specificity. The use of high specific activity tntiated dihydropyridines (e:,. [~Hlnitrendipine and [3H]nimodipine)~has now indicated that a pharmacolo~cally relevant dihydropyTidine binding site or receptor closely associated with calcium channels exists in many tissues, including smooth. cardiac and skeletal muscle, brain tissue and a clonal phaeochrom(m.~.'toma cell line, PI2. This !ffjo~c has recently been reviewed in TIPS"-.
hypertension ~, A plethora of dissimilar chemical structures have been found to have roughly similar calcium antagonistic actions. Although some investigators have felt that some of these agents acted by purely physicochemical interactions with the cell membrane, their Potency, stereospecificity, and subtle structure-activity relations suggest pharmacologically relevant binding to recognition sites ('receptors') at or near the calcium channel for some of them at least. This short review will focus on recent advances in the pharmacology of agents which modulate calcium fluxes by interaction with the binding sites associated with the calcium channel.
]'hcsc delaik-d binding slud;:.-~ ~nh Inliated dihtdrop~rid|nc~ hal,: n,m begun to m)lt~." a major mx,,tcrx that o! wh~ such a chemJcalh dJ~cr,,~, group of compounds as scrapamil, ddt,t,,cm, cmnanzinc, etc. should all act bs a simd,u mechanism, that ,)! blocking calc,um channels. The dlhsdro~rMmc,, brad to a "receptor ~te" of tool. ~1 200 (11111(see e.g. Ref 6l in the membranes ol nlant tts,~.'s. Its prt~Jcrlics hate bccn descTll~.d in dclm] in the excellent rt.slcw ;~ Glossmann's group ". and arc consistent ~Ith its being a gl.woprotcm mac.~-
molecule closch ~ts~iatcd ~nh the shin cak'mm channel. The binding alfinities" and properties art.., perhaps surpnsin#y. in vie~ of the tissue %'Icctit,t~ .sht~-n b.~ dihydnq~.Tidint..s, korh ~imdar ~'hether the membrane factoP, hat,." been l~)lated f w m brain, heart or smooth m u s i c = "".
The receptor =s st'k:cific for dih',rdropyr;dines; other calcmm antagomsts do not directl,, displace [~ttlnitrendipinc. Ho~-eser. the non-dihTdrop~ndme calcium antagomst~ haxe been sho~n to interact allt~,tcncalh ~ith the dthsdropyndine binding site l see Ref. 5 ). and a recent study" has taken this one step further to examine the effect,, of combinations of non-dlhydrop}ndine eMctum anta.oonists.. The findln,,s• pro-]de e~tdence that the non-dihydrop)ndtne calcium antagomsts, such as xerapamd. diltiazem, cinnarizinc, etc. tbem.~hes interact ~ith a ~mgle rccognitkm site cl('~ to. but separate from the DHP binding site. Hms. Sn,~dcr's group ,,egoists a unitarx mtxiel for drug mt~lulatton of calcium ch~mnels, in ~htch a site specific 1\~r dihydrop.,,ndines is m citric
X2
--CFa
Xl
R100C H3C
02
OOR2 H
H3C
CH3
COOCH3 ~
CH3
R;
R~
X,
X?
Nifedipine SKF 26240 Nicardipine
-CH3 -CH3 -CH3
-CH3 -C2Hs -CH2CHzI~CH2G
- NO2 -CF3 -H
-H -H -NO2
Nitrendipine Felodipine
-CH3 -CH3
-C2Hs -C2Hs
-H -Cl
-NO2 -Cl
CH3
"Presentaddress:Miles LaboratoriesLid, Stoke Court, Stoke Poges,Slough SL2 4LY, Bucks, Fig, I, The chenutxd strucmres of some repre_sentunvedih.vdmpyru~ne calcmm an~Jgon~s~ ¢lefiJand B.4 Y EBb44 fin bo.O, a calciw~ agonL~r UK.
771'S- March 1984
112 apposition to a specific multivalent cation site, and a site for verapamil, diltiazem and diphenalkylamines, such as cinnarizine is nearby". Thus any of the compounds by itself may interfere with calciam movements through the channel. A maior problem has, however, arian with ~he advent of dihydropyridine binding studies, namely that, although in general a fair correlation e~ists between ligand studies and pharmacological effects (see e.g. Ref. ~), there are many tissues (e.g. brain, heart) where a discrepancy between binding and pharmacological effect exists (see Refs a and 9). Lee and Tsien, for example, find a ltX)fold difference ix.tween [ Hlnitrendipine bw,ding and its pharmacological effect on isolated heart ,.'ellst". Possible reasons for these discrepancies have been discumed elsewhere, but the alteration of the macromolecule properties or l-~ssof essential cofactors during cell disruption procedures are obvious ~:andidates~. Yamamura's group has begun performing binding studies i~= vivo, and this technique could start to resolve these problems' . The consequences of the "unitary, m~','el" for calcium antagonists based on a ~flcium channel with two binding sites in close apposition to each other" are that the effects of calcium antagonists, at least in isolated systems, should be fundamentally similar. Although the basic similarities of the different drugs led Reckenstein to group them together as "calcium antagonists', much work in recent ~¢ars. devoted to emphasizing the differences between the agonists, has been done. However, a recent investigation has shown that, despite the considerable differences in pharmacokinetics and tissue specificity (see e.g. Ref. 3), the major properties of dihydropyridines, verapamii and diltiazem are, in fact (at least in isolated heart cells), fundamentally similar m. The effezts of nitrendipine (a dihydropyridine), D600 (the methoxy derivative of verapaniil) and diitiazem were examined on isolated single cardiac cells under conditions allowing close control of calcium currents. The drugs were all found to block both inward and outward fluxes of Ca 2+ or Ba z+ through the calcium channels (sodium was absent, and sodium channels were blocked with tetrodotoxin) and increasing the external CaZ+-ion concentrations was found to increase the number of unblocked channels uj. The authors thus endorse the use of the term "calcium antagonist'. The major difference between the compounds was found in the degree of 'use depen-
dence', i.e. the degree with which the channel block is dependent on the frequency of activation of the channels. (The differences in use dependency go some way to explaining the different pharmacological profiles of the compounds.) The authors, however, feel that the differences amount more to a quantitative spectrum of activity rather than fundamental qualitative differences I". Control and modulation of calcium channels The availability of labelled dihydropyridincs is also leading to attempts to solubilize, purify and isolate the calcium channel. First reports of progress in this field are appearing from the groups of GIossmaan and Triggle, and this work shouht eventually lead to progress in another area of comparative mystery,, the ~ntrol and modulation of the calcium chanp.el which plays an important role in the function of calcium ions as second messengers. Other approaches to calcium channels are also bearing fruit. Electrophysiological studies on single cells have been mentioned above TM, and other studies are now being carried out with single calcium channels using the 'patch clamp" method (see Ref. 12 for review). Two types of calcium channels may be distinguished, the "voltage sensitive channel' and the 'receptor operated channel'. Little is known of the receptor operated channel, although in different tissues in the same species it may be sensitive or insensitive to blockade by calcium channel blockers. It may also represent a 'special case' of the voltage sensitive channel, in which the activation mechanisms have been transferred from potential sensitive control to control by occupation or otherwise of specific receptor sites for catecholamines, histamine or other neurotransmitters and autocoids. The voltage.sensitive channels are widely distributed in neuronal, muscle and ,secretory tissues, and although they share many common properties, among them a sensitivity to calcium channel blockers, obviously differ from tissue to tissue (see Ref. 12 for review). Thus, the calcium channels in cardiac tissue are h~gll~lysensitive to the [3-effects of catecholamines, whereas those in smooth muscle are much less sensitive, if at all. The interested reader is warned that the general concepts discussed here may not necessarily apply to particular calcium channels. Voltage sensitive calcium channels appear ~to differ substantially from the
classical I h~dgkin-ltuxley theory which so elegantly describes the bchaviour of sodium channels. A three-state model, with two closed states, and one open state, has been prol~)sed '~. As each individual channel can only be either closed or open, modulation takes place by changing the probability of opening. In general it appears that. for single channels which are in the state that allows activation, the probability thal the channel will open increa.~s markedly with depolarization Thus, depola~ation within certain limits increases the net calcium flux through the calcium channels in a particular cell'2 Various physiological and exogenous substances are able to modulate the ion flux through the calcium channel in various ways also, presumably, by altering the probability of opening 12. A potent activation mechanism for voltage sensitive calcium channels, at least in cardiac tissue, involves phosphorylation, by means of cAMP-dependent protein kina~, Thus catecholamines, phosphodiesterasc inhibitors or cAMP analogues increase calcium influx by increasing the probability of channel activation (for review see Ref. 12). Howe:vet, there is as yet no evidence that such mechanisms interact with the calcium antagonist receptor sites in the channel. As discu~ed above, the organic calcium antagonists appear to block calcium channels by binding to one of two sites close to, and presumably essential for, the function of a calcium binding site. Inorganic calcium antagonists, such as Cd "+, appear to act by binding to a (different) io calcium binding site. Calcium agonists Drugs which stimulate calcium influx by interaction with the receptor sites normally available for calcium antagonists could be termed, by analogy,'calcium agonists'. Several years ago both praziquantel and l-methyl adenosine were shown to activate calcium influx by some diteo mechanism, and their effects were inhibited by D600, the methoxy derivative of verapamil '4. However, these compounds have not been studied in detail. Recently, more conclusive evidence for calcium agonism has emerged with the description of two totally different compounds, the marine toxin maiotoxin, and the nifedipine derivative BAY K8644. Maiotoxin stimulates calcium influx at concentrations of 1 0 - ' " - 3 x 1 0 -~ g ml -I. Its effects are competitively inhibited by verapamil, suggesting that it has an agonistic action on the verapamil-binding
ITI'S- March lq84
] 13
site at the calcium channel t~. A 3-nilm analogue of nifedipine, BAY K1,~44 (see Fig. !). is also able to stimulate calcium influx in low (111 ' ~ - 3 x 10 ~ g ml- i) concentrations, and its effects are competitively inhibited by nifedipinet". Ligand binding studies with BAY K8644 show no interaction with neurotransmitter receptor sites, such as a- or fladrenoceptors, muscarinic or nicotinic receptors, but strodg interaction with dihydropyridine (e.g I~H]nitrendipine) binding sites. The effects of BAY K8644 are particularly interesting, as w|th nifedipine it provides the first example for the DHP binding site of a chemically similar agonist/antagonist pair with mutually opposing effects. Thus, candidates are emerging which activate the voltage dependent calcium channel by direct action on the sites occupied by the presently k n o ~ calcium antagonists. The pharmacological profile of these new compounds is not yet complete. BAY K8644 possesses positive inotropic actions, and directly stimulates smooth muscle u', an action which is not mediated by a- or 13-adrenooeptor stimulation. The known tissue selectivity, of the nifedipine-like calcium antagonists makes it probable that the dihydropyridine calcium agonists also exhibit a degree of tissu~ selectivity. Maiotoxin stimulates smooth muscle, but in contrast to BAY K8644 is known to increase catecholamine release from adrenergic neurones IT. A topic currently being investigated is whether an endogenous ligand (agonistic or antagonistic) exists which can interact with calcium antagonist sites and could be involved in the physiological regulation of calcium channels. We are now entering a new age of calcium channel pharmacology. In addition to the binding techniques and the prospect of purified calcium channel preparations, we now have potent and specific drugs which stimulate, rather than block, the voltage sensitive channel. These drugs promise to become important pharmacological tools, and their novel mechanism of action promises new therapeutic actions in diseases where the calcium-dependent contraction or secretion is pathologically depressed.
Acting through ~peczilic surh.'e recet,tor~ ,.I t',,r(,Ihlr~ m~,t)tift'~. ~l,,~t~l~,~:Ptt" t~ki'~ regulate hwal myocardial pofttsion m rev,t,n.w t,, tlhttl~t',~ ill tdrdtdt t t t i , r t [lh' structure--activity rehttionshq~.~ of d~h'tto.~llte anah~g~ .~l¢~gt'~t thal tilt" p|lrt,'h" ,IH,i
Acknowledgement The authors are indebted to Mrs K. B. Crocker for typing the manuscript,
specialized sires which e]'ti'ct l(~and tundmg andor fix'ephor attJta:ton. 5,oh correlations htenti~, the steric.hwtors tt'htth iqlluent'e binding and hint at the thtma'al forces which gorem a[finiO'.
.~ I l c n n . | ' I) I I ~ l l i ) *lm ] I , , , , / , d J~ 11147 I1~,~ 4 l n g g l c . D Iitr,~2) 1-rend~ Pbar, n,u,d ~,~ 1
5 G ~ a n n . II. l--crrr~.II Iz . lucb~-t-k¢. | . M¢~¢',. R and |lo:tmann. I- ~!'~2~ I'r~md~ Pharmawo/ ~ t ~. 4~1-4.~7 6 N ~ a n . R I . Ik~r~tt,~. M . h~,~¢t M and I~lzdumkl. M (It~3l B,~hcm Bmph~ ble, ( "ommun I I I. ~7~-~] 7 Bdkmann. P. i-'cm l). I ucbtw~kc F ~nd (;k~mmann. It 11'~12l Arzm'tm t . r ~ h ~'.. .~1 -.~.~ N Jant~. R A . Maurct. % (" . %armu:nto. I (i Bolgcr. ( i I and l n g g l c , l ) J I ! ~ 2 ) I:ur 1 Pharm~rol ~;2. ltH- It)4 9 Muq~h.',. K M M . ( b r a i d . R J . I J r g ~ n L B. L and S m d e r . S It (198311 l'r,~ "g'all
Robbie Towan rece~,.ed hU Ph.D..from Gla~g*,~ U nive~av in 1974. Alter a brwf ~pell teachm~ pharmacology at Trinity ('ollege. DM~n. hi" joined Bayer A. G. in Wuppertal. t~here he hi'came interested in cardiac metabolum and cahTum entrt blockers Since 1081 he has been ttorl~m¢ .~g~ Mil~ Laboratone~ on vanom a s p e ~ ,,f rcsp~rat~,rt pharmacology.
I Fl¢ckenstein, A., Tritthardt, H. a~d Fleckenst©in, B. (1969) P fldgen An.'h Gesamre
Physiol. Memchen Tiere 307, R25
2 Godfraind, T. and Kaba, A. (1969) Br. J.
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.MaRhta~ .¢~hram~. ~radl~ated m m , zlherrd,~,, :,: lO*l and obtained l,.t~ Ph I) tr. p t ~ , , , rr,,,r. Cologne t ~ t t ert_-,~ ,n l~ "~ HI ~hc,..'atre, .:d :~,,.'fl,,le~ at the lr.~n,',.~" ~,, P;,.;,:,,/,,:~ .,~,: ;,,,rid Ba~t'r .4 (; m ttt,pp..'~a; :~./V'~ ,tit. :, -~,,','t',:,':'d cilh'lta'vt mq~hfiddlor~
Structure of the coronary artery adenosine receptor R. A. Olsson SIOI~'(~iM ( "ardh)t~t~t'l~hlr R¢.~¢',t,xh I dbo~tlh ~rt. i)t'l,~trt,W~tt ~,' I,:tt',,n~: Ih,:,, :,'.c I "~.~,,,~.', Florida ('olh'ge ~l Medwmc. 12qOi .'~ .lOfh .~f . B,,I I0 lamr,,. ~1 ¢3~12 I ~ t
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arh'rv ddett()Mtlt" rt't'tT~h~r e~lth t'()tthllH ~t'tt'r,i"
A 1970 report that adenosine stimulates brain adenylate cyclase and that theophylline antagonizes this effect established the existence of adenosine recep-
tots t. lnve~igations since then have d~.'umented the ubiquity of adenosine receptors and are now defining their imlx~rtance in cardiac, neural, endcK:rine.