Naunyn-Schmiedeberg's Arch. Pharmacol. 287, 23--32 (1975) �9 by Springer-Verlag 1975

Apparent Equilibrium Constant between ~-Adrenoceptors and a Competitive Antagonist

on a Cultured Pacemaker Cell of Mammalian Heart *

Alberto J. Kaumann** and Ruiner Witbmann

Physiologisehes Institut der Universitgt Diisseldorf, Lehrstuhl fiir Klinische Physiologic, Diisseldorf

Received September 9/Accepted November 22, 1974

Summary. Heart cells from newborn rats and kittens were cultured. In both species the rate of pacemaker activity of single, cultured cells was increased by low concentrations of catecholamines. The fl-adrenoceptor blocker (-)-KL255 anta- gonized competitively the effects of both, (-)-noradrenaline and (-)-isoprenaline. A mean equilibrium constant of 3.6 • 10 -1~ M between (-)-KL255 and fl-adrenoeeptors of single cells was estimated.

Key words: Drug-fl-Adrenoceptor Complex -- Single Heart Cell.

Apparent equilibrium constants for drugs are a measure of their affinity for the receptors which mediate their physiological effects. These constants may serve as u reference point to investigate and chemically identify drug receptors. Apparent affinities for fi-adrenoceptors of an extensive series of competitive antagonists have been determined on isolated atria (Blinks, 1967 ; Kaumann and Blinks, 1967 ; Kaumann, 1.973) Positive chronotropie effects of cateeholamines were matched to those obtained in the presence of competitive antagonists. Apparent equi- librium dissociation constants for the inhibitors were then calculated from equiefi~etive concentration-ratios of cateeholamine in the presence and absence of the competitive antagonist. With the concentration- ratio approach (Waud, 1968) the assumption is made that the concen- trations of catecholamine and fl-adrenoeeptor blocker in the receptor region are identical to the concentrations in the bathing solution. Cap- ture of eatecholamine by nerve endings and other tissues (Iversen, 1967) us it diffuses through the extraeellular space of the atrium may result in a gradient of amine between the central and the superficial cell layers. This gradient ma,y not necessarily be linear w-ithin the concentration range of catecholamine required to estimate the apparent affinities of antagonists (Kaumann, 19~ 0, 1972). Another phenomenon which may

Send offprlnt requests to A. J. Kaumann's present adress: Departamento de Matems Faeultad de Farmaeia y Bioqulmica, Junin 956, planta baja, Buenos Aires, Argentina.

* This work was supported by a grant of the Deutsche Forsehungsgemein- sehaft within the research project "SFB 30, Cardiologie".

** Established investigator of the Consejo Naeional de Investigaeiones Cientifi- casy T6cnicas of Argentina..

24 A. J. Kaumann and R. Wittmann

also v a r y wi th the concen t ra t ion of ca techo lamines in i so la ted a t r i a is the possible p roduc t i on of p a c e m a k e r shifts (West, 1955). On de t e rmin ing concen t ra t ion- ra t ios of agonis ts the ma in a s sumpt ion a b o u t an tagon i s t s is t h a t t h e y should be in equi l ib r ium wi th the receptors . Equ i l ib r i a be- tween m a n y f l -adrcnoceptor blockers and recep tors arc ach ieved r a the r s lowly on i so la ted t issues, p r e s u m a b l y because there exists a diffusion de l ay th rough the t issues and perhaps because of the i r unspecific lipo- phi l ia (Pot te r , 1967). Because of the slow onse t of b lockade , t ime-de- p e n d e n t changes in sens i t iv i ty of the t issues to ca techolamincs m a y de- velop. Er ro r s in the e s t ima te of the a p p a r e n t equi l ibr ium cons t an t for a compe t i t ive f l -adrenoceptor an t agon i s t due to these fac tors can be avoi- ded b y using single cu l tu red h e a r t cells where the or iginal a s sumpt ions s t r i c t l y app ly .

As a first a p p r o a c h we inves t i ga t ed the mode of a n t a g o n i s m of the pos i t ive chronot ropic effects of ca techolamines b y the p o t e n t f l -adreno- cep tor an t agon i s t ( - ) -KL255 on spon taneous ly bea t ing cells. ( - ) -KL255 was se lec ted because of i ts high aff in i ty for the f l -adrenoceptors , i t s r e l a t i ve ly low t o x i c i t y and i ts l ack of s y m p a t h o m i m e t i c act i~dty (Pendlc- t o n et al., 1970; K a u m a n n et al., 1970; K a u m a n n , 1972 Table 1, p. 144; K a u m a n n and B i rnbaumcr , 1973, 1974; B i r n b a u m c r et al., 1974).

Methods Cell cultures were made from hearts of newborn rats and kittens. All proce-

dures were carried out under sterile conditions. 6 to 12 hearts of 2 to 4 days old newborn Wistar rats were immersed into a modified, sterile Krebs solution contain- ing 250000 U/l penicillin, 0.1 g/1 streptemyocin, 10 mg/1 phenol red, 4• 10 -5 M ethylencdiamine tetraacetic acid (EDTA) (disodium salt), pH 7.2. The Krebs solution had the following composition (mEq per liter) Na § 140; K +, 5; Ca++, 4.5; Mg ++, 2; Cl-, 98.5; SO~--, 2; HCO3-, 29; HP04--, 2; (millmiolar concentration) fumarate, 10; pyruvate, 5; l-glutamate, 5; glucose, 10. Water was deionized and redistilled in glass. The Krebs solution was equilibrated with 950/0 O 3 and 50/0 CO2. The hearts were washed free of blood cells with Krebs solution at room temperature. The hearts were further washed in a Ca++-free Krebs solution with 4 • 10 -5 M EDTA and cut into small pieces. Single heart cell's were obtained by treatment with 0.15~ trypsin, pH 7.8 in Ca++-free Krebs solution. 5 successive trypsinizations were made, lasting 10 rain each. The trypsin-containing solution was renewed after each step. The supernatant of the third and following steps were centrifuged at about 200 • g and the cells separated by filtration through a G 1 glass filter (Schott, Mainz). Cells were centrifuged for another 10 rain at 200 • in culture medium to wash away residual trypsin. The cell suspension was diluted to yield about 5 • 105 cells/ml and distributed into 5 ml samples in tissue culture flasks of 25 cm ~ area (Falcon). Way- mouth's (1959) medium (l~low Lab.) supplemented with 250000 U/1 penicillin, 0.1 g/1 streptomyocin, 10 mg/ml phenol red and 10~ horse serum was used. Cul- tures were grown at 35~ in a mixture of room air with enough CO S to maintain a p i t of 7.2. To remove red blood cells and cell debris the culture medium was changed after the first 24 hrs and every 3 to 5 days, thereafter. Cells of kitten hearts were ob- tained either from newborn animals (less than 48 hrs after birth) or from the uterus of a pregnant cat, about 1 to 3 days before delivery. Cultures with heart cells from kittens were prepared essentially as from rats.

fl-Adrenoeeptors in Cultured Heart Cells 25

The rate of spontaneously beating cells was counted usually with the help of a 53226 B time counter DVM, Hewlett-Packard, during an acoustic signal delivered by an electronic buzzer timed to sound exactly 10 sec each minute. Whenever the rate of contractions was counted, the microscope lamp was switched on for only 15 sec/min to avoid excessive heating of the cultures.

Drugs were added with 5 ~l and l0 ~l Hamilton syringes. Multiple vigorous stirrings of the medium after each drug addition were achieved by sucking and rein- troducing repeatedly 1/3 to 1/2 of the medium through a sterile plastic cannula. Since the cells were solidly anchored to the bottom of the culture vessels, their shape remained unchanged despite high speed passages of debris and cell fragments during the flushes, as observed through the microscope.

The drugs used were: (-)-isoprenaline bitartrate dihydrate and (-)-noradrenaline bitartrate monohydrate (Sterling-Winthrop); 1-(6'-chloro-3'-methylphenoxy)-terc- (butylaminopropane-2-ol) [(-)-KL255] (Schwarz). The stock solutions of catechol- amines 0.01 M were acidified with 0.1 N HCI to a pI-[ of about 3 during the initial dilution and left refrigerated. For each experiment, appropriate dilutions of the eateeholamines were made with deionized, redistilled water containing 0.04 mM EDTA.

Results and Discussion

As soon as the cells became s tuck to the b o t t o m of the culture vessel, spontaneous contract ions were observed (Fig. 1). This occured at about l0 hrs with ra t cells and 3 to 4 days after init iating the cultures with ki t ten cells. The rate of spontaneous ly beating ra t cells ranged from 18 to 162 beats /min in 15 cultures. I n 3 to 5 days old cultures, the ra te changed less t han 18 beats/rain in each culture while observed during 3 to 4 hrs. The spontaneous ra te of beat ing of the k i t ten cells ranged from 19 to 143 beats/rain in 6 cultures. W h e n observed during 3 to 4 hrs, the ra te oscillated by up to 48 beats /min during tha t period. The earliest adminis t ra t ion of eateeholamines were made in 26 hrs old ra t cell cul- tures or 4 days old k i t ten cell cultures. I n these and all older cultures as well, definite responses to eatecholamines were observed, indicating t h a t single cells had/~-adrenoeeptors. Increments of ra te with eateeholamines appeared to be larger with cells beat ing at relat ively low rates. These cells were selected for quant i ta t ive studies. Concent ra t ion-dependent effects were observed from 10 -11 M (-)-isoprenaline onwards (Fig.2). Concen- t ra t ions of (-)-noradrenaline had to be one order of magni tude greater to obtain effects similar to those with (-)-isoprenaline. The onset of the positive ehronotropie effect of cateeholamines was fast and equilibrium was observed in less than 3 min.

Sensitivities to eateeholamines of ra t and k i t ten cells were similar. The effects of eatecholamines were t e rmina ted by changing the culture medium and were reproducible up to 3 t imes at intervals of 30 rain. Posit ive ehronotropie effects of cateeholamines on cul tured hear t cells have been observed by others (Wollenberger, 1964; Er te l et al., 1971; Boder and Johnson, 1972). However , two groups were unable to obtain any effects with eateeholamines on cul tured chicken hear t cells (Kauf- m a n n et al., 1969; Sperelakis, 1972); this has been a t t r ibu ted to t ryp t ic digestion of the ~-adrenoceptors (Kaufmann et al., 1969). Since we also

26 A. J. K a u m a n n and R. W i t t m a n n

Fig. 1. Top: Cultured four days old, beat ing pacemaker cells of the r a t observed th rough an inver ted phase microscope. The middle cell was cell No. 4 of Table 1. During photography this cell and its r ight elongated neighbour cell were contracting a t independent rates. The ent ire cytoplasm of bo th cells was moving, th&reby blurr ing the image. Bot tom: 30 days old contract ing cells of k i t t en heart . The top 2 cell clusters were uni ted by a small contract ing bridge-like fiber, l~ounded cells suggesting recent division can be observed a t the left upper cluster. This culture vessels was vi r tual ly covered wi th a monolayer of cell on the 26th culture day. I t was possible to t ransfer successfully cells from such cultures to grow ~ new culture. CellNo. ]e of Table 1 was from such a t ransferred culture. Calibration bars are 50tzm

1 6 8

15C - -

132 - -

I J J

7

. ~ 96

6 0

42 _ e I _, 11

=<

i I

o: J i i

10 9 8 7

-LOG. [(-)-ISOPRENALINE} MOLAR

i 3 g

e~ I--

z 1 o

q

m

9 . 6 2 /

10

/ /

I I I I 9 8 7 6

-LOG. [ ( - ) -KL2ss] MOLAR

Fig.2. Competit ive inhibi t ion by (-)-KL255 of the positive chronotropic action of (-)- isoprenaline on a 4 days old, cultured, r a t hea r t cell wi th pacemaker activity. Top: Cumulat ive concentration-effect curves to (-)-isoprcnaline. Each symbol is a mean of 5 successive equil ibrium counts during the last 5 rain preceding the next condition. The incubat ion medium was not replaced during the experiment. 4 suc- cessive curves to (-)-isoprenaline were determined, wi thout (o) and in the presence of 1 • 10 -~ M (~), 2 • 10 -7 iK (,) and 2 • 10 -G ~ (c) (-)-KL255, in t h a t sequence, respectively. The first new addit ion of (-)-isoprenaline was made 11 to 13 rain after each (-)-KL255 adminis trat ion. The to ta l volume of all adminis tered drugs was 2 . 4 0 of the volume of the incubat ion medium. Bot tom: Double log plot from con- centra t ion ratios of (-)-isoprenaline at the 118 (A), 119 (,), and 113 (D) bea ts /minute level, wi th 1 • 10 -9 M, 2 • 10 -7 M and 2 • 10 -6 ~{ (-)-KL255, respectively. Equi- effective concentrations of (-)-isoprenaline wi thout (-)-KL255 were obtained by interpolat ion to the first concentrat ion effect curve. For fu r ther detail of this ex-

per iment see cell No. 1 of Table

2 8 A. J. Kaumann and R. Wittmann

4 ~

r~

r~

8

X

0

0

o

v v ~

aaa;aa. x X X x x X

+ + + + + + .~.~.~.~.~.~ N N N ~ N N

.2..' - -

X X

§

~ x

.z..' ..k' .z..'

I ' I " ' 7

x x x

+ + - I -

~-Adrenoceptors in Cultured Heart Cells 29

o o o ~ ~ ~ X x X X X ~ ~ § 2 4 7

.~.~.~ z ~ ~ o

v

~D

30 A. J. Kaumann and R. Wittmann

used trypsinizations to disaggregate heart cells, but observed responses to eateeholamines, the fl-adrenoceptors apparently were unaffected by the same high concentrations of trypsin used on chicken hearts (Kaufmann et al., 1969). However, despite the fact that we consistently observed effects with eateeholamines, the affinity of the fl-adrenoceptors for the amines might have been modified by the t reatment with trypsin. In order to explore this possibility, affinity constants between cateehol- amines and/3-adrenoceptor should be estimated on single cells and compa- red with constants estimated on other systems. Since apparent affinity constants between catecholamines and/~-adrenoeeptors cannot yet be determined, we at tempted to investigate the mode of blockade of a pure antagonist. The experiment of Fig. 2 (top) shows that (-)-KL255 shifted surmountably the concentration-effect curve of (-)-isoprenaline towards greater amine concentrations. This suggested that the catecholamine- induced increase of pacemaker rate was blocked competitively by the fl-adrenoceptor blocker. Conventional theory was applied to examine the competitive behavior of (-)-KL255 and (-)-isoprenaline. A slope of unity of the double log plot of Fig.2 (bottom) indicates simple competitive blockade (Arunlakshana and Schild, 1959). Over a 2000-fold concen- trat ion range of ('-)-KL255 the slope of the line was near unity. In another experiment (cell No. 3 of Table 1) the slope of the double log plot was 0.87 for a 500-fold (-)-KL255 concentration range. Since (-)-KL255 antagonized (-)-isoprenaline competitively, the apparent dissociation constant Ks of the (-)-KL255-receptor complex was estimated (Table 1).

From the antagonism of (-)-KL255 to the ehronotropic effects of (-)-isoprenaline a KB of 3.6 ~ 3.7 • -1~ M (mean • 2 S.D. from 7 determinations) was estimated for (-)-KL255 on 3 rat cells. We do not have estimates of KB for the (-)-KL255-fl-adrenoeeptor complex on rat pacemakers in isolated atria for comparison. However, Pendleton et al. (1970) found on rat atria that ( • antagonized the ehronotropie effects of (-)-isoprenaline essentially in a simple competitive manner. A Ks of 1.1 • -9 M can be estimated for racemic KL255 from their data. Since the affinity of (-)-KL255 for the fl-adrenoceptors is two orders of magnitude higher than that of (~-)-KL255 (Kaumann st al., 1970; Kaumann and Birnbaumer, 1973, 1974; Birnbaumer et al., 1974) it can be assumed tha t with the experimental conditions of Pendleton et aI. the KB of (-)-KL255 would be 5.5 • l0 -1~ M because most of the affinity of the racemate of KL255 is due to its (-)-isomer. This assumed value of KB for (-)-KL255 is notIsignificantly different from our KB'S of (-)- KL255 on cultured rat heart cells. Table 1 shows also that the KB of, (-)-KL255 is similar whether (-)-isoprenaline or (-)-noradrenaline was used, which is expected if we assume that all three drugs compete for the same receptor.

Using l0 -9 M to 10 -s M (-)-KL255, a mean KB of 2.4 • 10 -1~ M was estimated for (-)-KL255 as antagonist of the chronotropic effects of

fl-Adrenoeeptors in Cultured Heart Cells 31

(-)-isoprenaline on kitten atria (Kanmann et al., 1970; Birnbaumer et al., 1974). Itydroeortisone potentiates cardiac effects of (-)-isoprenaline on kitten right and left atria, presumably by preventing extraneuronal uptake of the eatecholamine (Kaumann, 1972). 10 .7 M (-)-KL255 shifts concentration-effect curves of (-)-isoprenaline to the right by two orders of magnitude, even in the presence of hydroeortisone ; Ke 's of 3 • 10 -1~ M and 5 • 10 -1~ M were estimated for (-)-KL255 in the presence and absence of hydroeortisone, respectively (Kaumann, 1972, Table 1). I t appears theretbre that extraneuronal uptake of (-)-isoprenaline has little influence on the estimate of Ke 's for/~-adx'enoceptor blockers on isolated atria of kittens. I t remains to be established experimentally whether or not ex- traneuronal uptake of eateeholamines exists in monolayers of cultured heart cells of rats and kittens. The similarity of the KB'S for (-)-KL255 on rat and kitten heart cells suggests that the fl-adrenoeeptors are similar in both species. A mean KB of 4.1 • 10 -1~ IV[ was estimated for the (-)- KL255-/~-adrenoeeptor complex coupled to the adenylyl eyelase in cell- free membrane particles (Kaumann and Birnbaumer, 1974) indicating that this/~-adrenoeeptor is chemically the same as the receptor which mediates ehronotropie effects of eateeholamines.

The kinetics of the onset of effect of (-)-KL255 were relatively fast. In the experiments of Fig. 2 it took 8 min (half time 2 min) to reduce the equilibrium effect of 8 • 10 -11 M (-)-isoprenaline to a new equilibrium level with 1 • 10 -s M (-)-KL255 which occupied 82 ~ of the receptors of the cell. With a concentration of (-)-KL255 required to achieve 900/0 receptor occupancy 70 min (half time 18 rain) are necessary for equili- briton blockade of (-)-isoprenaline on isolated left and right atria (Kau- mann and Blinks, unpublished observations). This slow onset of blockade in isolated tissues may be related to a diffusion delay through ist cells. However, rates of onset of receptor occupancy in cell-free membrane particles (Kaumann and Birnbaumer, 1974) as measured with the adenylyl eyelase assay are still about 4 times faster than in cultured cells. I t remains to be established whether the slow onset of blockade in single cells is due to mixing being rate-limiting in our system.

Acknowledgements. A. J. Kaumann is grateful for the hospitality of Prof. R. Kaufmann. This work was made while A. J. K. was a visiting scientist of the Deut- seher Akademiseher Austausehdienst.

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32 A. J. Kaumann and R. Wittmann

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