Tissue specificity of dihydropyridine-type calcium antagonists in human isolated tissues

T I P S - January 1988 [Vol. 9]

37

Tissue specificityof dihydropyridine-typecalcium

Membranes isolated from various tissues possess specific binding sites for at least three chemical classes of Ca 2+ entry blockers: dihydropyridines, phenylalkylamines and benzothiazepines. These sites are distincL but interact with each other (see Refs 1 and 2). Overall, the properties of the binding sites are remarkably similar in isolated membranes from different tissues, including nervous and endocrine tissues. Furthermore, the size and immunochemical features of the polypeptide bearing the three binding sites are very similar in skeletal, cardiac and smooth muscle 2. Thus, at the molecular level little selectivity may be detected in the interaction of Ca 2+ entry blockers with various tissues (see Refs I and 4).

antagonists in human isolatO,d

tissues

I Huma=

,',,,,,,, ;' 1

T. GodfrainJ, N. Morel and M, Wibo

S

Tissue specificity is essential for the multiple indications of the large group of drugs listed under the heading "calcium antagonists'. Theo Godfraind and colleagues analyse this specificity in the cardiovascular system. The species differences evident in specificity emphasize the importance of studies with human preparations. In the case of dihydropyridines, tissue specificity may be related to factors that modulate the state of Ca 2+ channels and to the r?,lative importance of stimulus-dependent Ca z+ entry versus stimulus-dependent mobilization of Ca 2+ stores.

There is an obvious need for quantitative information on the action of drugs in human beings and the use of human isolated tissues, when not limited by ethical considerations, may in part answer this need. It may also provide an insight into the mechanism of the therapeutic effect of drugs in humans which may differ from that in animals. Analysis of the actions of Ca 2+ antagonists in h u m a n isolated coronary arteries and cardiac preparations may serve as an illustration of such studies. An important question that has been raised several times is the tissue selectivity of Ca 2+ antagonists: does this selectivity exist, and if so, to what properties of the antagonists and tissues is it due?. This question has not only an academic interest, but it may also justify the use of different agents for selective therapeutic purposes as well as the search for novel agents. In this paper we report observations on human preparations and attempt a rational explanation of tissue selectivity based on studies with both human and animal tissues. The pharmacological actions of any drug may be studied at various levels (the molecule, whole cell, Theo Godfraind is Director of the Laboratoire de PharmacodynamieGdndraleet de Pharmacologie Univer$itd Catholique de Louvain, UCL 7350,Avenue EmmanuelMounier, 73, 8. 1200 Brussels, 8elsium. N. Moreland M. Wibo are Research Associates in the same laboratory. Theo Godfraind is Secretary General of IUPHAR.

tissue in vitro, organ in w!vo, clinical disorder), as shown in Table I, which illustrates in f~ve steps a pharmacotherapeutic ca~cade for Ca 2+ antagonists. 1\~ assume a link between the variou: steps, some consistency shoulo exist in the order of magnitude of the quantitative parameters estimated for the various levels of action. An analysis of the pharmacotherapeutic cascade is possible for the action of nifedipine in angina pectoris. Nifedipine-evoked relaxation of de= polarized human coronary arteries in vitro is observed at concentrations close to therapeutic plasma levels. Comparison of IC5o values of nifedipine in rat aortas and in human coronary arteries shows that they are of the same order of magnitude. In rat aorta, IC5o values are of the same order as Kd values measured on isolated membranes, indicating that the effect observed at the tissue level is related to the molecular interaction with Ca 2+ channels. However, ICso values on intact cardiac tissue preparations are usually considerably higher than the dissociation constants t.

Tissue selectivity In contrast, at the tissue level, the selectivity of Ca 2+ entry blockers is obvious. It is well known, for instance, that neurotransmitter release from brain tissue is little affected by dihydropyridines, and that cardiac contractility is much less sensitive to dihydropyridines than is smooth muscle contraction. Species differences affect this tissue selectivity and in this instance animals are not predictive of humans. For ;;xample, in isolated human myo(~rdium and coronary arteries (~;ig. '1), nisoldipine shows a vasct'~lar selectivity 100 times higher th,i,n nifedipine, (comparing conce~itrations producing 50% reductior:: of contractile activity of myi~cardium and K+-depolarized arteties with nifedipine and nisoldipineS); in rats, vascular selectivity of nisoldipine is lower than in humans (see Fig. 2). Sorr,e agents may even show a selectbrity within the arterial tree 6. For example, whereas nifedipine appear.,; equipotent on rat aorta and mesenteric artery (contracted by K + depolarization or receptor stimulation), flunarizine is

TABLE I. The pharmac otherapeutic cascade level of pharmacological :_.~!on molecule cell tissue (in vitro) organ (in vivo) clinical disorders

qualitative effect

.....

binding tO Ca =+ channel changes in Ca =+ fluxes and action potential,; cardiac negative inolropio and chronotropic .=Jffects, smooth muscle relaxation hemodynamlcmodiflt,atlons (ECG, cardiac output, blood pressure) antlanglnalend antihypertenslve effects= ~) 1988, Elsevier Pub:

.

quantitative parameter Kd, K0 ICso ICso, pA= plasma levels plasma levels

~ns~Cambridge {)165- 0147/88/$02,00

TIPS- January 1988 [Vol. 9]

38

,oo

8o

o0

o ~

60

o~... 60

•-

4O

"~

40

g o

2o

o o

20

o~

+

"--"

¢::

o

,o-',

.

.

•

,o-,O ,o-'

,o-'

0

o

,;-°

:'°

nifedipine (M)

q

,0-'

,0

,;-'

nisoldipine (M)

Fig. 1. In-vitro study of the inhibition of K+-depolarization-induced contraction of human coronary arteries (0) and of the contraction of the electrically paced human ventricular muscles (0) by nifedipine and nisoldipine. Taken from Ref. 5.

aries, the source of activator Ca 2+ is likely to be extraceUular. ~'atch clamp analysis has indicated two other fundamental causes of tissue selectivity: multiplicity of C a 2+ channel subtypes and of C a 2+ channel states. This is now being confirmed in ligand binding studies on intact cells. At least two subtypes (L and T) of voltage-dependent Ca 2+ channels have been demonstrated electrophysiologically in several cell types, incl:iding neurons, cardiac and smooth muscle cells. Only the L type is blocked by classical C a 2+ entry blockers. Neurotransmitter release may depend primarily o n C a 2+ influx via N channels, which are insensitive to dihydropyridines, but blocked by Q-conotoxin 9. Three main states of voltage-dependent Ca 2+ channels have been postulated:

distinctly more potent on the mesenteric artery 7. What is the source of this tissue selectivity? The extent of intracellular C a 2+ stores may contribute. For example, in rat aorta, some 50% of the norepinephrineinduced contraction is resistant to C a 2+ entry blockers whereas in rat mesenteric artery this proportion is only 10% (Ref. 1). This difference is related to the greater role of intraceiiular Ca 2+ mobilization in the c¢-adrenoceptor-stimulated response in the aorta. However, again, these results in rats are not predictive of the human situation. In human coronary arteries, contractions evoked by 5-HT, an agent likely to be responsible clinically for coronary vasospasm and spontaneous rhythmic contractions, are completely blocked by dihydropyridines ~. Thus in human coronrat aorta papillary

"--=-

.-o_

muscle

o-.-

_-

=

atria

human coronary artery

---o-

ventricular muscle

c.

atria

-o10"1o

10-9

=

"=l(~-e

10'--7

lO'e

concentration (U)

Fig. 2. Concentrations of nisoldipine (11)and n/fed/pine (O) producing a 50% reduction of the contractility of isolated preparations obtained from rat (from Ref. 17) and from human (from Ref. 5). Arteries (rat aorta and human coronary arteries) were stimulated by K ÷. depolarization. Cardiac preparations were electrically paced. Note that vascular selectivity of nisoldipine is more pronounced in human preparations.

resting, open and inactivated. The state of the L channel dramatically influences its affinity for dihydropyridines. Thus, in both cardiac and smooth muscle cells, dihydropyridines may show an affinity three orders of magnitude higher for the inactivated state as compared with the resting ~,~ate!°,1z. As the inactivated state is promoted by membrane depolarization, these drugs will be more effective in smooth muscle tissues, which are characterized by less negative resting potentials and long lasting depolarizing stimuli, than in myocardial tissue, which shows a more negative resting potential and is usually submitted to short repetitive depolarizing stimuli. Using [3H](+)PN 200-110, we have recently performed an analysis of dihydropyridine receptor sites in rat intact mesenteric arteries z2. As shown in Fig. 3, arterial segments incubated in depolarizing medium bind this ligand with high affinity (Kd 44 pM). The affinity is lower (Kd 200 pM) when segments are incubated in physiological medium. The Bmax is not significantly influenced by depolarization. These results are compatible with the view that dihydropyridines bind preferentially to the inactivated state of the channel. The effect of depolarization on affinity explains why the inhibitory effect of (+)PN 200-110 on arterial contraction increases with time after depolarization. Upon depolarization, the inhibition of contraction of rat aorta by (+)PN 200-110 develops with a timecourse similar to that of the specific binding to the high-affinity state of the channel in isolated mere-

TIPS - J a n u a r y 1988 [Vol. 9]

39

branes (Fig. 4) 13. Thus, binding to this high-affinity state, which is induced b y depolarization in intact arteries, appears to be the rate-limiting step of the inhibition of the contraction. Observations similar to those reported on Fig. 4 have been made with nisoldipine on isolated h u m a n m a m m a r y and coronary arteries 14. In addition to membrane potential, a variety of factors (protein kinases, G proteins) may influence the functioning of Ca 2÷ channels in some tissues and contribute to the tissue selectivity of Ca 2+ entry blockers. Another major control of vascular smooth muscle is the endothelium-derived factors which act as functional antagonists for vasoconstrictors TM. In norepinephrine-stimulated arteries Ca 2+ entry into smooth muscle cells is reduced by an endothelial factor (EDRF) acting via cGMP 16. []

[]

[]

In conclusion, the puzzling discrepancy between selectivity at the tissular level and non-selectivity at the molecular level has been clarified. Hopefully, similar studies may help to clarify the mode of action of calcium entry blocking drugs in disease states, in particular in ischemic or hypoxic conditions and in hypertension. However, when attempting to relate the therapeutic effects of drugs to their pharmacological properties, it is important to take into account the tissular conditions prevailing in pathological state. For instance, in ischemic or anoxic conditions, there is an increase of extracellular K + which will modify the membrane

I00 80 A

tO

60

Jp~-

v

o

40

10

.c: CD

20 0

I0

20

~O

40

TiME (mln)

0

10

20

30

Time (rnin) Fig. 4. Comparison between kinetics of [3H]( +)PN 200-110 association to its specific binding site [broken line, @: O.1 riM; I1:0.38 nM] and (+)PN 200-110 inhibition of K +evoked contraction of rat aorta [solid line; O: O.I riM; [7 0.3 nM (+)PN 200-110]. Immt: Mechanical responses to KCI-depolarization of rat aortic rings. Control (upper curve), rings treated with O.I nM (0) or 0.3 nM ([3) ( + )PN 200-110. Taken from Ref. 13.

potential and the state of calcium channels, and thereby their interaction with calcium channels ligands. It is also important to take into account the fact that studies on tissue selectivity in rats are not predictive of humans. Acknowledgements This work has been supported by F.R.S.M. Grant no 3.9006.87. References 1 Godfraind,T., Miller,R. C. and Wibo, M. (1986) Phannacol. Rev. 38, 321-416 2 Glossmann, H., Ferry,D. R., Striessni8, J., Goll, A. and Moosburger, K. (1987) Trends Phannacol. Sci. 8, 95-100

Fig. 3. Specific binding of [3H](+ )PN 200.1 I0 in rnessn. teric artery rings bathed in physio. Iogical solution (@)or in K+.depolarizing solution (&) (fiom Ref. 12). The highest specific uptake in depolarized artery may be related to increased binding site affinity for the dihydropyridins 00 100 200 since Bm= is not (/) significantly medl. [3H] (+)PN 200-110 (pM) fled, although there is a significant change in K~ values after prolonged clepolartzation.Ke is 200 :!: 20 and 44 ± 2 pMin physiological and KCI solution, respectively, as calculated by non.linear regreselon from the binding data.

6t

80

II.

3 Schmid, A., Barhanin, J., Coppola, T., Borsotto, M. and Lazdunski, M. (1986)

Biochemistr9 25, 3492-3495 4 Triggle, D. J. and Janis~ R. A. (1987) Annu. Rev. Pharmacol. ToxicoL 27, 347369 5 Godfraind, T., Egl~me, C., Finet, M. and Jaumin, P. (1987) Pharmacol. ToxicoL 61, 79-84

6 Cauvin, C. and van Breemen, C. (1987) ISI Atlas Sci. Pharmacol. 1, 13-19 7 Godfraind, I". (1985) in Calcium in Biolosical Systems (Rubin, R. P., Weiss, G.B., Putney, J.W., eds), pp. 411-421 Plenum Publishing Corporation 8 Godfraind,T., Finet, M., SocratesLima, J. and Miller,C. (1984)]. Pharmacol. Exp.

Ther. 230, 514-518 9 Rivier, J., Galyean, R., Gray, M.R., Azimi-Zonooz, A., Mclntosh, M., Cruz, L.J. and Olivera, B.M. (1987) ]. Biol. Chem. 262, 1194-1198 10 Bean, B., Sturek, M., Puga, A. and Hex~nnsmeyer,K. (1986)Circ. Res.59, 229235

11 Sanguinetti,M. C. and Kass,R. S. (1984) Circ. Res. 55, 336-348

12 Morel, N. and Godfraind, T. J. Pharmacol. Exp. Ther., in press 13 Wibo, M., DeRoth, L. and Godfraind,T. Circ. Res., in press 14 Godfraind, T., Egl~me, C., Finet, M., Debande, B. and Jaumin, P. in Proceedinss of First International Nisoldipine Symposium (Hugenholtz, P. G. and

Meyer,J., eds), SpringerVerla8, in press 15 Alosachie, I. and Godfraind, T. (1986) Br. i. Pharmacol. 89, 525-532 16 Godfraind, T. (1986) Eur. I. Pharmacol. 126, 341--343 17 Kazda, S., Garthoff, B., Meyer, H., Schlossmann,K.,Stoepel,K.,Towart,R., Vater, W. and Wehinser, E. (1980) A~neim..Forsch.IDvu 8 Res. 30, 21442162