European Journal of Pharmacology, 44 (1977) 81--84 © Elsevier/North-Holland Biomedical Press
81
Short c o m m u n i c a t i o n DELAYED APPEARANCE OF PROPRANOLOL SENSITIVITY IN CULTURED HEART CELLS M.J.A. WALKER Department of Pharmacology, University of British Columbia, Faculty of Medicine, Vancouver, B.C. Canada V6T 1W5
Received 7 March 1977, accepted 15 April 1977
M.J.A. WALKER, Delayed appearance of propranolol sensitivity in cultured heart cells, European J. Pharmacol. 44 (1977) 81--84. Examination of the appearance of adrenaline responsiveness and propranolol sensitivity with culture age in myoblasts cultured from neonatal rat hearts shows that propranolol sensitivity lags behind adrenergic responsiveness. Such a finding may illustrate subtle differences in sites for agonist and antagonist at the fl-receptor of these cells. Adrenaline
Propranolol
Cultured heart cell
1. Introduction Discussion of the ~-adrenergic receptor sometimes assumes an identical binding site (Patil et al., 1974) and activation state, for both agonist and antagonists. Examination of adrenergic responsiveness for varying agonists, antagonists and tissues may help establish the validity of such a premise (Patil et al., 1974). The appearance of adrenaline responsiveness in cultured heart myoblasts is documented for both chick (Ertel et al., 1971), neonatal rat (Kaumann and Wittman, 1975) and mice (Boder and Johnson, 1972) cells, but the effectiveness of adrenergic receptor blocking drugs has only recently been documented or quantitated (Kaumann and Wittman, 1975, and Fayet et al., 1974). During studies on the relationship between the develo p m e n t of adrenaline accumulation and the appearance of responsiveness to the same agonist, I a t t e m p t e d to quantitate the appearance of d,l-propranolol sensitivity in the same preparation (Walker, 1974); investigation showed t h a t adrenaline responsiveness and
propranolol sensitivity developed at different rates.
2. Materials and methods Cultures of neonatal rat heart myoblasts were prepared from ventricles of 3--5 day old rats by trypsin digestion. Dispersed cells were suspended in a tissue culture medium (pH 7.4) containing 20% F-10 media (Colorado Serum Co.), 4% foetal bovine serum (Microbiological Associates) and 1% antibiotic solution (6 mg/ml penicillin G + 10 mg/ml streptomycin) in a Hank basic salt solution which lacked KC1 and NaHCO3. Cells were cultured in Falcon flasks at 37°C and such cultures were examined for adrenaline responsiveness and d,l-propranolol sensitivity at varying periods after the initial seeding. Adrenaline responsiveness was measured in terms of the positive chronotropic response of single cells with rates being determined electronically from density changes in a television screen image by means of a photo-resistor network and appropriate Grass amplifiers
82
M.J.A. W A L K E R a it j~
150
125
i:K
I00
75 5O
"
,
,
,
,
l
2
3
4
,'// lll'~ 5
9
15
b
(tachograph). Adrenaline was added to the tissue culture media in single or cumulative doses and the activity of single cells recorded. Either the response of a single cell t o varying doses of adrenaline was recorded, or the rates of r a n d o m l y selected cells were recorded for each dose level. The latter technique was used rout i nel y to give estimates of adrenaline responsiveness in cultures of different culture ages. In general, dose--response curves were obtained by the cumulative m e t h o d with a p p r o x i m a t e l y 5 min exposure to each concentration of adrenaline; such curves did n o t differ from those obtained by single dose techniques• Sensitivity to d,l-propranolol was det erm i ned by a three-point parallel assay (Schild, 1947) using a cumulative dose schedule and allowing an equilibration period of 10 min for each new dose level• Three dose levels (10 -7, 10 -6 and 10 -s M) were used with drugs added as 1--10 pl stock/ml media, d-Propranolol and d,l-practolol were also investigated at the two highest dose levels.
3
3. Results
/ ; •
i
l
[. .....
As illustrated in fig. l a and b, b o t h adrenaline responsiveness and propranolol sensitivity increased with culture age. With either drug there was little effect in the first few hours of culture b u t measurements of rates were somewhat difficult during this period because cells were still in the process of flattening and t herefore difficult to record; by 24 h all cells had assumed a flattened appearance and the recording of rates presented no difficulties. Adrenaline responsiveness continued to increase with age, for all dose levels,
[o.I
V'" 1
2
3
4
5
Fig. l a a n d l b . Fig. l a s h o w s t h e d e v e l o p m e n t o f a d r e n a l i n e sensitivity in c u l t u r e d n e o n a t a l r a t h e a r t cells. Each p o i n t is t h e m e a n (_+S.E.M.) of t h e m e a n s f r o m 5--6 d i f f e r e n t c u l t u r e s s a m p l e d at t h e t i m e indic a t e d a n d w h e r e t i m e zero was t h e t i m e o f initial seeding• R a t e s were m e a s u r e d e l e c t r o p h o t o m e t r i c a l l y f r o m single cells o f f images o n a T.V. screen• A, Control; E~, 1 0 - 5 ; ", 1 0 - 6 ; e, 10 -7 M adrenaline• O r d i n a t e :
b e a t s / m i n ; abscissa: c u l t u r e age (days). In fig. l b a parallel assay design (3 p o i n t s , a n d d , l - p r o p r a n o l o l a d d e d c u m u l a t i v e l y } was used to calculate dose ratios; 5--6 c u l t u r e s were e x a m i n e d w i t h each p o i n t t h e m e a n (-+S.E.M.)• T h e lines s h o w n are n o t calculated best-fit lines. T h e o p e n s y m b o l s are values for d,1p r a c t o l o l a n d are t h e m e a n o f 2--4 estimates. ~, 1 0 - s ; ©, 1 0 - 6 ; A, 10 -7 M p r o p r a n o l o l . O r d i n a t e : log dose r a t i o ; abscissa: c u l t u r e age (days}.
P R O P R A N O L O L S E N S I T I V I T Y IN C U L T U R E D H E A R T C E L L S
until cultures were three days old. The growth of propranolol sensitivity however lagged behind that of adrenaline responsiveness such that sensitivity continued to grow in the presence of a fully expressed adrenaline response (fig. lb). The differences in development rates for adrenaline responsiveness and propranolol sensitivity can be replotted in a variety of ways *. For example, dose response curves for each day of culture age can be plotted for adrenaline while similar age dependent Schild plots can be constructed for propranolol. Such conventional pharmacological plots show, in both cases, incrases in slope with age but constant pA~ or pD2 values (respective values: 7.9 for propranolol and 8.2 for adrenaline). The increase in slope of Schild plots for propranolol with age fits a simple exponential with 1/T equal to 0.56 -+ 0.05 S.D. and a m a x i m u m slope of 1.82 (1.78--1.93: 95% confidence limits). d-Propranolol was w i t h o u t effect on adrenaline responses at 10 -6 M whereas 10 -s M produced a non-parallel shift of dose-response curves and a dose ratio of 3.0 which did not vary with culture age. Experiments with d,l-practolol in 2.5 and 4 day old cultures gave dose ratio values which fell on the d,l-propranolol curves shown in fig. l b . In view of the anomalies observed in cultured cells adrenaline responsiveness and propranolol sensitivity have been investigated in source tissue (neonatal rat hearts) and such investigations (Walker and Au, 1975) showed normal responsiveness in atrial tissue for rate and force but a lack of responsiveness in source tissue ventricles. Where d,l-propranolol sensitivity was measured, a mean pA2 value of 7.92 (range in tissues 7.55--8.49) was calculated and Schild plot slopes n o t statistically different from 1.0 were obtained. The reason for the lack of adrenaline responsiveness in
* To avoid r e p e t i t i o n and for t h e sake o f brevity such p l o t s are n o t s h o w n .
83
early ventricles is at present under investigation.
4. Discussion The effects of d-propranolol and d,l-practolol indicate that the results with antagonists are dependent on fi-receptor properties whilst the results with source tissue indicate t h a t the process of cell culture induces changes in cells with regard to adrenaline sensitivity. The different development rates for adrenaline responsiveness and propranolol sensitivity are not easily reconciled with a receptor in which propranolol and adrenaline compete for identical sites on cultured rat heart cells. The possibility of multiple binding sites for agonists and antagonists at the/3 receptor has been discussed but little pharmacological evidence is available while binding studies have not clarified the situation (e.g. Ari~ns, 1967; Bilezikian and Aurbach, 1973). The change in dose--response curve, and Schild plot, slopes with age for both adrenaline and propranolol indicate the the " m a t u r a t i o n " process is not simply a change in association constants and, for a conventional receptor model (I + R ~IR), the increase of slope to a m a x i m u m of 1.8 argues for changing and anomalous stoichiometrics in the presence of a fully developed adrenaline response. It must be noted, however, that in vitro neonatal rat heart tissue gave slopes of 1.0 and the literature reports values of 0.87 for 2 day and 1.05 in 4 day old cultured rat heart cells (Kaumann and Wittmann, 1975) and 1.07 for chick cells (age not given and calculated from data, Fayet et al., 1974). The increase in slope for adrenaline responsiveness might reflect a maturation of the transduction or response systems and not receptor development, but this does not resolve the difficulty with propranolol for, even in the presence of a fully expressed adrenaline response, sensitivity to propranolol still develops. The difficulties would be resolved if differences in recognition sites exist on the fl-receptor for adrenaline and propranolol.
84 Acknowledgement Research was funded by the British Columbla Heart Foundation.
References Ari~ns, E.J., 1967, The structure--activity relationships of ~-adrenergic drugs and beta adrenergic blocking drugs, Ann. N.Y. Acad. Sci. 139,606. Bilezikian, J.P. and G.D. Aurbach, 1973, A ~-adrenergic receptor of the turkey erythrocyte. I. Binding of catecholamine and relationship to adenylate cyclase activity, J. Biol. Chem. 248, 557. Boder, G.B. and I.S. Johnson, 1972, Comparative effects of some cardioactive agent on automaticity of cultured heart cells, J. Mol. Cell. Cardiol. 4, 453. Ertel, R.J., D.E. Clarke, J.C. Chao and F.R. Francke, 1971, Autonomic receptor mechanisms in embryonic chick myocardial cell cultures, J. Pharmacol. Exptl. Therap. 178, 73.
M.J.A. WALKER Fayet, G., F. Couraud, F. Miranda, and S. Lissitzky, 1974, Electrooptical system for monitoring activity of heart cells in culture; application to the study of several drugs and scorpion toxins, European J. Pharmacol. 27, 165. Kaumann, A.J. and R. Wittmann, 1975, Apparent equilibrium constant between ~-adrenoceptors and a competitive antagonist on a cultured pacemaker cell of mammalian heart, Naunyn-Schmiedeb. Arch. Pharmakol. 287, 23. Patil, P.N., D.D. Miller and U. Trendelenburg, 1974, Molecular geometry and adrenergic drug activity, Pharmacol. Rev. 2 6 , 3 2 3 . Schild, H.O., 1947, pA. A new scale for the measurement of drug antagonism, Brit. J. Pharmacol. 2, 189. Walker, M.J.A., 1974, Adrenaline sensitivity and binding in cultured neonatal rat heart cells, Proc. Can. Fed. Biol. Sci. 17, 20. Walker, M.J.A., and T.L.S. Au, 1975, Adrenaline responsiveness in intact and cultured neonatal rat heart cells, 6th Intern. Congr. Pharmacol., Helsinki, Abstract 1432.
81
Short c o m m u n i c a t i o n DELAYED APPEARANCE OF PROPRANOLOL SENSITIVITY IN CULTURED HEART CELLS M.J.A. WALKER Department of Pharmacology, University of British Columbia, Faculty of Medicine, Vancouver, B.C. Canada V6T 1W5
Received 7 March 1977, accepted 15 April 1977
M.J.A. WALKER, Delayed appearance of propranolol sensitivity in cultured heart cells, European J. Pharmacol. 44 (1977) 81--84. Examination of the appearance of adrenaline responsiveness and propranolol sensitivity with culture age in myoblasts cultured from neonatal rat hearts shows that propranolol sensitivity lags behind adrenergic responsiveness. Such a finding may illustrate subtle differences in sites for agonist and antagonist at the fl-receptor of these cells. Adrenaline
Propranolol
Cultured heart cell
1. Introduction Discussion of the ~-adrenergic receptor sometimes assumes an identical binding site (Patil et al., 1974) and activation state, for both agonist and antagonists. Examination of adrenergic responsiveness for varying agonists, antagonists and tissues may help establish the validity of such a premise (Patil et al., 1974). The appearance of adrenaline responsiveness in cultured heart myoblasts is documented for both chick (Ertel et al., 1971), neonatal rat (Kaumann and Wittman, 1975) and mice (Boder and Johnson, 1972) cells, but the effectiveness of adrenergic receptor blocking drugs has only recently been documented or quantitated (Kaumann and Wittman, 1975, and Fayet et al., 1974). During studies on the relationship between the develo p m e n t of adrenaline accumulation and the appearance of responsiveness to the same agonist, I a t t e m p t e d to quantitate the appearance of d,l-propranolol sensitivity in the same preparation (Walker, 1974); investigation showed t h a t adrenaline responsiveness and
propranolol sensitivity developed at different rates.
2. Materials and methods Cultures of neonatal rat heart myoblasts were prepared from ventricles of 3--5 day old rats by trypsin digestion. Dispersed cells were suspended in a tissue culture medium (pH 7.4) containing 20% F-10 media (Colorado Serum Co.), 4% foetal bovine serum (Microbiological Associates) and 1% antibiotic solution (6 mg/ml penicillin G + 10 mg/ml streptomycin) in a Hank basic salt solution which lacked KC1 and NaHCO3. Cells were cultured in Falcon flasks at 37°C and such cultures were examined for adrenaline responsiveness and d,l-propranolol sensitivity at varying periods after the initial seeding. Adrenaline responsiveness was measured in terms of the positive chronotropic response of single cells with rates being determined electronically from density changes in a television screen image by means of a photo-resistor network and appropriate Grass amplifiers
82
M.J.A. W A L K E R a it j~
150
125
i:K
I00
75 5O
"
,
,
,
,
l
2
3
4
,'// lll'~ 5
9
15
b
(tachograph). Adrenaline was added to the tissue culture media in single or cumulative doses and the activity of single cells recorded. Either the response of a single cell t o varying doses of adrenaline was recorded, or the rates of r a n d o m l y selected cells were recorded for each dose level. The latter technique was used rout i nel y to give estimates of adrenaline responsiveness in cultures of different culture ages. In general, dose--response curves were obtained by the cumulative m e t h o d with a p p r o x i m a t e l y 5 min exposure to each concentration of adrenaline; such curves did n o t differ from those obtained by single dose techniques• Sensitivity to d,l-propranolol was det erm i ned by a three-point parallel assay (Schild, 1947) using a cumulative dose schedule and allowing an equilibration period of 10 min for each new dose level• Three dose levels (10 -7, 10 -6 and 10 -s M) were used with drugs added as 1--10 pl stock/ml media, d-Propranolol and d,l-practolol were also investigated at the two highest dose levels.
3
3. Results
/ ; •
i
l
[. .....
As illustrated in fig. l a and b, b o t h adrenaline responsiveness and propranolol sensitivity increased with culture age. With either drug there was little effect in the first few hours of culture b u t measurements of rates were somewhat difficult during this period because cells were still in the process of flattening and t herefore difficult to record; by 24 h all cells had assumed a flattened appearance and the recording of rates presented no difficulties. Adrenaline responsiveness continued to increase with age, for all dose levels,
[o.I
V'" 1
2
3
4
5
Fig. l a a n d l b . Fig. l a s h o w s t h e d e v e l o p m e n t o f a d r e n a l i n e sensitivity in c u l t u r e d n e o n a t a l r a t h e a r t cells. Each p o i n t is t h e m e a n (_+S.E.M.) of t h e m e a n s f r o m 5--6 d i f f e r e n t c u l t u r e s s a m p l e d at t h e t i m e indic a t e d a n d w h e r e t i m e zero was t h e t i m e o f initial seeding• R a t e s were m e a s u r e d e l e c t r o p h o t o m e t r i c a l l y f r o m single cells o f f images o n a T.V. screen• A, Control; E~, 1 0 - 5 ; ", 1 0 - 6 ; e, 10 -7 M adrenaline• O r d i n a t e :
b e a t s / m i n ; abscissa: c u l t u r e age (days). In fig. l b a parallel assay design (3 p o i n t s , a n d d , l - p r o p r a n o l o l a d d e d c u m u l a t i v e l y } was used to calculate dose ratios; 5--6 c u l t u r e s were e x a m i n e d w i t h each p o i n t t h e m e a n (-+S.E.M.)• T h e lines s h o w n are n o t calculated best-fit lines. T h e o p e n s y m b o l s are values for d,1p r a c t o l o l a n d are t h e m e a n o f 2--4 estimates. ~, 1 0 - s ; ©, 1 0 - 6 ; A, 10 -7 M p r o p r a n o l o l . O r d i n a t e : log dose r a t i o ; abscissa: c u l t u r e age (days}.
P R O P R A N O L O L S E N S I T I V I T Y IN C U L T U R E D H E A R T C E L L S
until cultures were three days old. The growth of propranolol sensitivity however lagged behind that of adrenaline responsiveness such that sensitivity continued to grow in the presence of a fully expressed adrenaline response (fig. lb). The differences in development rates for adrenaline responsiveness and propranolol sensitivity can be replotted in a variety of ways *. For example, dose response curves for each day of culture age can be plotted for adrenaline while similar age dependent Schild plots can be constructed for propranolol. Such conventional pharmacological plots show, in both cases, incrases in slope with age but constant pA~ or pD2 values (respective values: 7.9 for propranolol and 8.2 for adrenaline). The increase in slope of Schild plots for propranolol with age fits a simple exponential with 1/T equal to 0.56 -+ 0.05 S.D. and a m a x i m u m slope of 1.82 (1.78--1.93: 95% confidence limits). d-Propranolol was w i t h o u t effect on adrenaline responses at 10 -6 M whereas 10 -s M produced a non-parallel shift of dose-response curves and a dose ratio of 3.0 which did not vary with culture age. Experiments with d,l-practolol in 2.5 and 4 day old cultures gave dose ratio values which fell on the d,l-propranolol curves shown in fig. l b . In view of the anomalies observed in cultured cells adrenaline responsiveness and propranolol sensitivity have been investigated in source tissue (neonatal rat hearts) and such investigations (Walker and Au, 1975) showed normal responsiveness in atrial tissue for rate and force but a lack of responsiveness in source tissue ventricles. Where d,l-propranolol sensitivity was measured, a mean pA2 value of 7.92 (range in tissues 7.55--8.49) was calculated and Schild plot slopes n o t statistically different from 1.0 were obtained. The reason for the lack of adrenaline responsiveness in
* To avoid r e p e t i t i o n and for t h e sake o f brevity such p l o t s are n o t s h o w n .
83
early ventricles is at present under investigation.
4. Discussion The effects of d-propranolol and d,l-practolol indicate that the results with antagonists are dependent on fi-receptor properties whilst the results with source tissue indicate t h a t the process of cell culture induces changes in cells with regard to adrenaline sensitivity. The different development rates for adrenaline responsiveness and propranolol sensitivity are not easily reconciled with a receptor in which propranolol and adrenaline compete for identical sites on cultured rat heart cells. The possibility of multiple binding sites for agonists and antagonists at the/3 receptor has been discussed but little pharmacological evidence is available while binding studies have not clarified the situation (e.g. Ari~ns, 1967; Bilezikian and Aurbach, 1973). The change in dose--response curve, and Schild plot, slopes with age for both adrenaline and propranolol indicate the the " m a t u r a t i o n " process is not simply a change in association constants and, for a conventional receptor model (I + R ~IR), the increase of slope to a m a x i m u m of 1.8 argues for changing and anomalous stoichiometrics in the presence of a fully developed adrenaline response. It must be noted, however, that in vitro neonatal rat heart tissue gave slopes of 1.0 and the literature reports values of 0.87 for 2 day and 1.05 in 4 day old cultured rat heart cells (Kaumann and Wittmann, 1975) and 1.07 for chick cells (age not given and calculated from data, Fayet et al., 1974). The increase in slope for adrenaline responsiveness might reflect a maturation of the transduction or response systems and not receptor development, but this does not resolve the difficulty with propranolol for, even in the presence of a fully expressed adrenaline response, sensitivity to propranolol still develops. The difficulties would be resolved if differences in recognition sites exist on the fl-receptor for adrenaline and propranolol.
84 Acknowledgement Research was funded by the British Columbla Heart Foundation.
References Ari~ns, E.J., 1967, The structure--activity relationships of ~-adrenergic drugs and beta adrenergic blocking drugs, Ann. N.Y. Acad. Sci. 139,606. Bilezikian, J.P. and G.D. Aurbach, 1973, A ~-adrenergic receptor of the turkey erythrocyte. I. Binding of catecholamine and relationship to adenylate cyclase activity, J. Biol. Chem. 248, 557. Boder, G.B. and I.S. Johnson, 1972, Comparative effects of some cardioactive agent on automaticity of cultured heart cells, J. Mol. Cell. Cardiol. 4, 453. Ertel, R.J., D.E. Clarke, J.C. Chao and F.R. Francke, 1971, Autonomic receptor mechanisms in embryonic chick myocardial cell cultures, J. Pharmacol. Exptl. Therap. 178, 73.
M.J.A. WALKER Fayet, G., F. Couraud, F. Miranda, and S. Lissitzky, 1974, Electrooptical system for monitoring activity of heart cells in culture; application to the study of several drugs and scorpion toxins, European J. Pharmacol. 27, 165. Kaumann, A.J. and R. Wittmann, 1975, Apparent equilibrium constant between ~-adrenoceptors and a competitive antagonist on a cultured pacemaker cell of mammalian heart, Naunyn-Schmiedeb. Arch. Pharmakol. 287, 23. Patil, P.N., D.D. Miller and U. Trendelenburg, 1974, Molecular geometry and adrenergic drug activity, Pharmacol. Rev. 2 6 , 3 2 3 . Schild, H.O., 1947, pA. A new scale for the measurement of drug antagonism, Brit. J. Pharmacol. 2, 189. Walker, M.J.A., 1974, Adrenaline sensitivity and binding in cultured neonatal rat heart cells, Proc. Can. Fed. Biol. Sci. 17, 20. Walker, M.J.A., and T.L.S. Au, 1975, Adrenaline responsiveness in intact and cultured neonatal rat heart cells, 6th Intern. Congr. Pharmacol., Helsinki, Abstract 1432.
