- Email: [email protected]
Isoprenaline stimulation of cyclic AMP production by isolated cells from adult rat myocardium
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOPRENALINE STIMULATION OF CYCLICAMPPRODUCTION BY ISOLATED CELLS FROM ADULT RAT MYOCARDIUM T. Powell and V: W. T w i s t D e p a r t m e n t of Physics as Applied to Medicine The M i d d l e s e x H o s p i t a l Medical School London, W1P 6DB
Received August ii,1976 SUMMARY When 1.25 x 10- 6 tool/1 i s o p r e n a l i n e is added to s u s p e n s i o n s of adult r a t m y o c a r d i a l c e l l s in c a l c i u m - f r e e b i c a r b o n a t e buffer, t h e r e is a 1 . 6 - f o l d i n c r e a s e above c o n t r o l of c y c l i c AMP l e v e l s 30 seconds a f t e r d r u g addition. The p h o s p h o d i e s t e r a s e inhibitor methylisobutylxanthine (MIX) at 0 . 5 m m o l / 1 i n c r e a s e s tile c o n t r o l l e v e l of cAMP and a l s o the l e v e l of s t i m u l a t i o n to 3-fold. When MIX is p r e s e n t , Ca -H" (1 mmol/1) has no effect on e i t h e r c o n t r o l or s t i m u l a t e d cAMP, w h e r e a s o m i s s i o n of Mg"H- r e s u l t s in a l o w e r i n g of c o n t r o l cAMP, with the l e v e l s a f t e r s t i m u l a t i o n unchanged. P r o p r a n o l o l at 8.45 x 10-5 mol/1 a b o l i s h e s s t i m u l a t i o n by i s o p r e n a l i n e .
INTRODUCTION T h e r e is now s u b s t a n t i a l evidence for the contention that f l - a d r e n e r g i c s t i m u l a t i o n of the m y o c a r d i u m p r o m o t e s i n c r e a s e d s a r c o l e r r m a l adenyl c y c l a s e a c t i v i t y (1-3).
Although m a n y studies have been r e p o r t e d of
c a t e c h o l a m i n e - s t i m u l a t e d c y c l i c AMP production in both i s o l a t e d p e r f u s e d h e a r t s (4, 5) and p l a s m a m e m b r a n e p r e p a r a t i o n s (6), d a t a on i s o l a t e d m a t u r e m y o c a r d i a l c e l l s have been p r e c l u d e d until now b y the a b s e n c e of a suitable preparation.
We r e p o r t h e r e on the s t i m u l a t i o n by i s o p r e n a l i n e of cAMP
production in s i n g l e - c e l l s u s p e n s i o n s obtained f r o m adult r a t m y o c a r d i u m . A l s o d e s c r i b e d a r e the effects of p h o s p h o d i e s t e r a s e inhibition and changes in both c a l c i u m and m a g n e s i u m buffer c o n c e n t r a t i o n s on the l e v e l of c a t e c h o l a m i n e stimulation.
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any/orm reserved.
1218
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS A l l c h e m i c a l r e a g e n t s w e r e ' A n a l a r ' g r a d e or equivalent. D L i s o p r e n a l i n e sulphate was obtained f r o m W a r d Blenkinsop, D L - p r o p r a n o l o l HC1 f r o m ICI and the p h o s p h o d i e s t e r a s e inhibitor 1 - m e t h y l - 3 - i s o b u t y l x a n t h i n e (MIX) f r o m A l d r i c h C h e m i c a l s . Purified s u s p e n s i o n s of i s o l a t e d m y o c a r d i a l c e l l s w e r e obtained by t i m e d in v i t r o p e r f u s i o n s of adult r a t h e a r t s with c a l c i u m - f r e e K r e b s b i c a r b o n a t e buffer containing c r u d e c o l l a g e n a s e , a s d e s c r i b e d in d e t a i l p r e v i o u s l y (7, 8). C e l l s u s p e n s i o n s (5 ml) w e r e p r e i n c u b a t e d at 37°C in the a p p r o p r i a t e g a s s e d buffer m e d i u m containing 2~o (w/v) bovine s e r u m albumin (8) for 5 - 7 m i n u t e s and then i s o p r e n a l i n e added. S a m p l e s (1 ml) w e r e t a k e n at known t i m e s , added to 2 m l ethanol, v o r t e x m i x e d and then s t o r e d at -20°C for at l e a s t 16 h o u r s . C o n t r o l flasks w e r e included in e a c h e x p e r i m e n t containing an equivalent volume of blank d r u g vehicle. A f t e r s t o r a g e , s a m p l e s w e r e w a r m e d to 4°C and c e n t r i f u g e d at 2250 g for 20 minutes. A p o r t i o n (2 ml) of e a c h s u p e r n a t a n t w a s withdrawn and e v a p o r a t e d to d r y n e s s for 4 h o u r s at 37°C in a v a c u u m oven. The d r i e d r e s i d u e s w e r e r e c o n s t i t u t e d in 500 ~1 of 0.0S mol/1 T r i s - E D T A buffer (pH 7.5) and cAMP d e t e r m i n e d in duplicate 50 R1 s a m p l e s b y a s t a n d a r d s a t u r a t i o n a s s a y (9, 10). U n l e s s s t a t e d o t h e r w i s e , r e s u l t s a r e given a s m e a n ~- s t a n d a r d e r r o r of the m e a n with the n u m b e r of e x p e r i m e n t s following in p a r e n t h e s i s . Group m e a n s w e r e c o m p a r e d u s i n g Student's t - t e s t .
RESULTS F i g u r e 1 shows t y p i c a l r e s u l t s f r o m e x p e r i m e n t s in which m y o c y t e s
~ i
105cells/m[
300
37°C
l
lO-6mol/I Isoprenohnesulphole .0.Sin mol/L MIX 0 5mmol/I MIX
8
c~ 225
-~ 150
x lOL,cellslml
75 E c~
I
-A
1 -
Figure
1
-
2
4
TIME(m,ns)
C y c l i c AMP p r o d u c t i o n b y i s o l a t e d c a r d i a c m y o c y t e s in Ca ~ - - free b i c a r b o n a t e buffer.
1219
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
160f
~
.2mlns
/
,20secs ~: 6o g 801
z~ z~
~ ~o I 20 0
Figure 2
-9 -8 -7 -6 -5 4 -- LOG([SOPRENALINE(mol/I))-->
L o g - d o s e r e s p o n s e c u r v e s for c y c l i c AMP production. Values obtained f r o m time c o u r s e r e s p o n s e s at e a c h dose of i s o p r e n a l i n e and given a s p m o l cAMP a c c u m u l a t e d p e r ml. above control. 1 ml. c e l l s u s p e n s i o n s incubated at 37°C, r e a c t i o n s t e r m i n a t e d by addition of 2 m l ethanol. C e l l count, 8 x 104 r o d c e l l s p e r ml. c e l l suspension.
w e r e incubated in c a l c i u m - f r e e b i c a r b o n a t e buffer containing 0 . 5 mmol/1 MIX with 10-6 tool/1 i s o p r e n a l i n e added or an equivalent volume (50 ~1) of pH 4 saline (control).
Two types of r e s p o n s e s have been r e c o r d e d (cf. F i g u r e 1).
Of 20 e x p e r i m e n t s p e r f o r m e d with 0 . 5 mmol/1 MIX, 9 have shown a r e l a t i v e l y l i n e a r cAMP r e s p o n s e to i s o p r e n a l i n e , w h e r e a s 11 have exhibited s a t u r a t i o n a f t e r 2 - 3 minutes.
In 7 e x p e r i m e n t s w h e r e MIX was omitted f r o m the
incubation m e d i u m , a l l r e s p o n s e s s a t u r a t e a f t e r 1 - 2 minutes e x p o s u r e to the d r u g (1.25 x 10 - 6 mol/1).
When t h e r e s p o n s e is a l i n e a r , a f a m i l y of log-
dose r e s p o n s e c u r v e s a r e obtained ( F i g u r e 2) a l l having h a l f - m a x i m a l v a l u e s on the o r d e r of 10 -7 mol/1 i s o p r e n a l i n e , with t h r e s h o l d between 10 -9_ 10 -8 mol/1. F i g u r e 3 s u m m a r i z e s the d a t a f r o m a s e r i e s of e x p e r i m e n t s using 1.25 x 10-6 tool/1 i s o p r e n a l i n e as s t i m u l a n t , cAMP l e v e l s being m e a s u r e d 30 seconds a f t e r d r u g addition.
With no p h o s p h o d i e s t e r a s e inhibitor p r e s e n t
1220
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(n=5)
120
(n=11)
(n=4)
100
lO6mol/[ ~] 125 Isoprenohne
80
17Control
~ 6o (n=3)
I
r•
0
0 1118 0 0
Figure 3
0.5 1.18 0 0
0.5 1,18 1.0 0
0 0
Addqtlons 0.5 MIX 7 Mg'* 1.180 Ccl..] (rnmol/I) 8.45 #r opr o nolol (t0-6moll[)
E f f e c t s of p h o s p h o d i e s t e r a s e inhibition, Ca -H', Mg++ and P r o p r a n o l o l on b a s a l and i s o p r e n a l i n e - s t i m u l a t e d cAMP production, m e a s u r e d 30 s e c o n d s a f t e r d r u g addition. Ca qqand Mj~_ ~ added a s c h l o r i d e and sulphate s a l t s , r e s p e c t i v e l y . In Mg ~ - - f r e e e x p e r i m e n t s , m a g n e s i u m o m i t t e d f r o m s e d i m e n t a t i o n p r o c e d u r e (8) a s w e l l as incubation m e d i u m . T e m p e r a t u r e 37°C. E a c h s t i m u l a t e d group c o m p a r e d with c o r r e s p o n d i n g c o n t r o l by Student's t - t e s t . *=0.05-¢P~0.01, ** = 0 . 0 1 ~ P-¢ 0.001, * * * = P . ~ 0.001.
in Ca -H'- f r e e b i c a r b o n a t e buffer, t h e r e a r e some I 0 . 5 + 2 . 2 p m o l cAMP p e r l05 r o d c e l l s ( n = 7 ) in c o n t r o l f l a s k s and t h i s is not s i g n i f i c a n t l y i n c r e a s e d s t a t i s t i c a l l y to 16. 6 + 9.3 p m o l / 1 0 5 c e l l s when i s o p r e n a l i n e i s p r e s e n t .
On
addition of 0 . 5 m m o l / l MIX, c o n t r o l cAMP i n c r e a s e d significantly to 36.7 + 4 . 0 p m o l / 1 0 5 c e l l s ( n = l l , pd= 0.001) and when i s o p r e n a l i n e is p r e s e n t t h e r e is a f u r t h e r significant i n c r e a s e to l l 2 + 10.6 p m o l / 1 0 5 c e l l s ( n = i i , P-=0.001). The f l E t h e r addition of 1 m m o l / l C a C l 2 to the m e d i u m does not a l t e r e i t h e r the c o n t r o l or s t i m u l a t e d l e v e l s of cAMP.
However, the o m i s s i o n of MgSO 4
r e s u l t s in a l o w e r i n g of b a s a l cAMP to a l e v e l c o m p a r a b l e with that found in the a b s e n c e of MIX with Mg-H-, w h e r e a s the l e v e l of s t i m u l a t e d cAMP is unchanged f r o m that o b s e r v e d with MIX in the p r e s e n c e o r absence of Ca M". l~:opranolol at 8.45 x 10-6 m o l / l a b o l i s h e s s t i m u l a t i o n by i s o p r e n a l i n e . Although the m e a n l e v e l of cAMP in f l a s k s with i s o p r e n a l i n e i s not
1221
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
s t a t i s t i c a l l y different from control when all the data a r e pooled, for each e x p e r i m e n t there is a highly significant positive c o r r e l a t i o n between the level of cAMP in a s t i m u l a t e d flask and the c o r r e s p o n d i n g control ( r = 0.951, n = 7, P = 0. 001).
The data of F i g u r e 3 have therefore been e x p r e s s e d as the r a t i o (K)
of cAMP after 30 seconds in the p r e s e n c e of i s o p r e n a l i n e to the control value at the s a m e t i m e and a r e p r e s e n t e d in F i g u r e 4.
Cyclic AMP is i n c r e a s e d
about 1.6 - fold by i s o p r e n a l i n e when MIX is absent, 3 - fold with 0. 5 mmol/1 MIX added, i r r e s p e c t i v e of whether or not c a l c i n m is p r e s e n t at 1 retool/l, and s t i m u l a t e d 10 - fold when Mg++ is omitted from the incubation medium.
All of
these v a l u e s differ significantly from unity (el. F i g u r e 4).
(n=Z~)
12
l:
R
6
2
(n=3)
0 0 118 0 0
Figure 4
0.5 1.18 0 0
0.5 118 10 0
)5
o 0
o
r~ 0.5 118
Additions MIX ] Mg**/(retool/I) 0 Ca**j 6.45 PropronoLol (106mo1/[)
Stimulation of cAMP production by i s o p r e n a l i n e . R = cAMP produced after 30 seconds stimulation. cAMP in control flask after 30 seconds. t - t e s t s p e r f o r m e d a s s u m i n g that R = 1, i . e . rio stimulation.
DISCUSSION The viable s i n g l e - c e l l suspension of c a r d i a c myocytes p e r m i t s examination of c a t e c h o l a m i n e - s t i m u l a t e d cyclic AMP production in a p r e p a r a t i o n where the spatial o r g a n i s a t i o n of the r e c e p t o r - e n z y m e complex r e s e m b l e s that found in the intact t i s s u e , without the p r e s e n c e of an extensive e x t r a c e l l u l a r space.
1222
The
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
receptor moiety is exposed to an approximately interstitial fluid environment, whereas the adenyl cyclase unit functions in a cytoplasmic milieu, assuming an intact cell sarcolemma. In suspensions hypersensitive to calcium, addition of this ion produces cellular contraction and damage (11,12) resulting in a preparation resembling broken-cell or plasma membrane systems.
Thus in the only previous report on
this subject, Moustafa and co-workers (12), using a calcium-intolerant preparation, reported that Ca"H- inhibited cAMP production in isolated cardiac myocytes at a concentration (0.5 mmol/1) known to inhibit adenyl cyclase activity in cell-free systems (6). The unchanged levels of basal cAMP found in this study when 1 mmol/1 calcium is included (Figure 3) indicate that in the present preparation intracellular free Ca -H- concentration is being maintained at about 10- 6 - 10 -7 tool/l, which is both typical for this type of tissue (13) and also in the range where adenyl cyclase activity is not inhibited by this cation (6).
Furthermore, it can be calculated
from the data of Moustafa et al (12) that about 10 pmol cAMP/105 cells were produced 30 seconds after the addition of 10-6 mol/1 isoprenaline In calcium-free buffer containing 2 mmol/1 theophylline and 0.5 mmol/1 EGTA. This is only 12~o of the response reported here and could be due to the fact that EGTA might also inhibit adenyl cyclase activity, independent of its metal-ion chelating ability (14). Adenyl cyclase has an absolute requirement for Mg-H- (15, 16) so that reduction in basal cyclic AMP in myocytes incubated in Mg-H-- free buffer (Figure 3) is due probably to cell depletion of Mg-H'. The half-time for magnesium efflux from rat ventricle is on the order of 3 hours at 37°C (17) so that the short incubation times used here would not be expected to reduce intracellular magnesium to a level where adenyl cyclase would be inhibited completely. Further evidence to support the contention that the sarcolemma is
1223
VoI. 72, No. 3, 1 9 7 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
functioning in a relatively typical manner is obtained from the dose-response curves (Figure 2 ) where the half-maximal values on the order of 10 -7 mol/1 agree weU with those found in other studies (2). The date obtained in the presence of MIX showing both linear and saturable responses to isoprenaline (Figure 1) probably reflect the complex feedback relationship between adenyl cyclase, cyclic AMP and Ca -H- (6) and might well depend upon the level of trace amounts of calcium which are unavoidable in the nominally calcinm-free incubations.
This would not
only influence the level of residual phosphodiesterase activity, which Ca"H" is known to stimulate at v e r y low concentrations (18, 19), but also transport of trace extracellular calcium might be facilitated by the phosphodiesterase inhibitor itself (20). The consistent finding of cyclic AMP saturating after 1 - 2 minutes where MIX is omitted (7 experiments) agrees well with data from both intact hearts (4) and also other single-cell systems (21).
Indeed, studies with these
minimally-tensioned isolated myocardial cells might well clarify many of the problems concerning plasma membrane regulation of fi-adrenergic activiation of adenyl cyclase. ACKNOWLEDGEMENTS This research is supported by a grant from The British Heart Foundation. We are indebted to Professor J.F. Tait and Mrs. S.A.S. Tait for both continual advice and also fl-counting facilities, and to Dr. B.L. Brown for instruction and reagents concerning cyclic AMP saturation assays.
REFERENCES
1. Robinson, G . A . , Sutherland, E . W . , and Butcher, E. (1971). Cyclic AMP, Academic Press, New York. 2. Lefkovitz, R.J., Limbird, L . E . , Mukherjee, C., and Caron, M.G. (1976). Biochim. Biophys. Acta., 457, 1-39. 3. Wollenberger, A. (1975) in Contraction and Relaxation in the Myocardium (Ed. W.G. Nayler) pp. 113 - 190, Academic Press, New York. 4. Kukovetz, W . R . , P/~ch, G., and Wurm, A. (1975). Adv. Cycl. Nucl. Res. 5, 395- 414. 5. Watanabe, A . M . , and Besch, H.R. (1975). Circ. R e s . , 37, 309-317.
1224
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6. Tada, M., Kirchberger, M.A., loria, I-A. M., and Katz, A.M. (1975) Circ. Res., 36, 8-17. 7. Gould, R.P. and Powe11, T. (1972). J. Physiol., 225, 16P-18P. B. Powell, T., and Twist, V.W. (1976). Biochem. Biophys. Kes. Commun., in press. 9. Brown, B . L . , Albano, J.D.M., Ekins, R . P . , Sgherzi, A.M., and Tampion, W. (1971). Biochem. J., 121, 561-562. 10. Brown, B . L . , Ekins, R . P . , and Albano, ].D.M. (1972). Adv. Cycl. Nucl. R e s . , 2, 25- 40. 11. F a r m e r , B.B., Jolly, W.W., Watanabe, A.M., and Besch, H.R. (1975). Fed. Proc., 84, 882. 12. Moustafa, E . , Skomedal, T . , Osnes, J-B., and ~Dye, I. (1976). Biochim. Biophys. Acta., 421, 411-415. 18. Reuter, H. (1974). Circ. Res., 84, 599-605. 14. Johnson, R . A . , and Sutherland, E.W. (1978). ]. Biol. Chem., 248, 5114- 5121. 15. Birnbaumer, L. (1978). Biochim. Biophys. Acta., 300, 129-158. 16. Drummond, G . I . , and Duncan, L. (1970). J. Biol. Chem., 245, 976-988. 17. Page, E . , and Polimeni, P.I. (1972). J. Physiol., 224, 121-189. 18. Teo, T . S . , and Wang, J.H. (1969). Biochem. Biophys. Res. Commun., 85, 5950 - 5955. 19. Katz, A.M., (1970). Physiol. Rev., 50, 68-158. 20. Nayler, W.G. (1967). Am. Heart J., 78, 379-894. 21. Tomasi, V., Poll, A., F e r r e t t i , E . , and Barnabei, O. (1975). Adv. Enz. Regulation, 13, 189-200.
1225
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOPRENALINE STIMULATION OF CYCLICAMPPRODUCTION BY ISOLATED CELLS FROM ADULT RAT MYOCARDIUM T. Powell and V: W. T w i s t D e p a r t m e n t of Physics as Applied to Medicine The M i d d l e s e x H o s p i t a l Medical School London, W1P 6DB
Received August ii,1976 SUMMARY When 1.25 x 10- 6 tool/1 i s o p r e n a l i n e is added to s u s p e n s i o n s of adult r a t m y o c a r d i a l c e l l s in c a l c i u m - f r e e b i c a r b o n a t e buffer, t h e r e is a 1 . 6 - f o l d i n c r e a s e above c o n t r o l of c y c l i c AMP l e v e l s 30 seconds a f t e r d r u g addition. The p h o s p h o d i e s t e r a s e inhibitor methylisobutylxanthine (MIX) at 0 . 5 m m o l / 1 i n c r e a s e s tile c o n t r o l l e v e l of cAMP and a l s o the l e v e l of s t i m u l a t i o n to 3-fold. When MIX is p r e s e n t , Ca -H" (1 mmol/1) has no effect on e i t h e r c o n t r o l or s t i m u l a t e d cAMP, w h e r e a s o m i s s i o n of Mg"H- r e s u l t s in a l o w e r i n g of c o n t r o l cAMP, with the l e v e l s a f t e r s t i m u l a t i o n unchanged. P r o p r a n o l o l at 8.45 x 10-5 mol/1 a b o l i s h e s s t i m u l a t i o n by i s o p r e n a l i n e .
INTRODUCTION T h e r e is now s u b s t a n t i a l evidence for the contention that f l - a d r e n e r g i c s t i m u l a t i o n of the m y o c a r d i u m p r o m o t e s i n c r e a s e d s a r c o l e r r m a l adenyl c y c l a s e a c t i v i t y (1-3).
Although m a n y studies have been r e p o r t e d of
c a t e c h o l a m i n e - s t i m u l a t e d c y c l i c AMP production in both i s o l a t e d p e r f u s e d h e a r t s (4, 5) and p l a s m a m e m b r a n e p r e p a r a t i o n s (6), d a t a on i s o l a t e d m a t u r e m y o c a r d i a l c e l l s have been p r e c l u d e d until now b y the a b s e n c e of a suitable preparation.
We r e p o r t h e r e on the s t i m u l a t i o n by i s o p r e n a l i n e of cAMP
production in s i n g l e - c e l l s u s p e n s i o n s obtained f r o m adult r a t m y o c a r d i u m . A l s o d e s c r i b e d a r e the effects of p h o s p h o d i e s t e r a s e inhibition and changes in both c a l c i u m and m a g n e s i u m buffer c o n c e n t r a t i o n s on the l e v e l of c a t e c h o l a m i n e stimulation.
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any/orm reserved.
1218
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS A l l c h e m i c a l r e a g e n t s w e r e ' A n a l a r ' g r a d e or equivalent. D L i s o p r e n a l i n e sulphate was obtained f r o m W a r d Blenkinsop, D L - p r o p r a n o l o l HC1 f r o m ICI and the p h o s p h o d i e s t e r a s e inhibitor 1 - m e t h y l - 3 - i s o b u t y l x a n t h i n e (MIX) f r o m A l d r i c h C h e m i c a l s . Purified s u s p e n s i o n s of i s o l a t e d m y o c a r d i a l c e l l s w e r e obtained by t i m e d in v i t r o p e r f u s i o n s of adult r a t h e a r t s with c a l c i u m - f r e e K r e b s b i c a r b o n a t e buffer containing c r u d e c o l l a g e n a s e , a s d e s c r i b e d in d e t a i l p r e v i o u s l y (7, 8). C e l l s u s p e n s i o n s (5 ml) w e r e p r e i n c u b a t e d at 37°C in the a p p r o p r i a t e g a s s e d buffer m e d i u m containing 2~o (w/v) bovine s e r u m albumin (8) for 5 - 7 m i n u t e s and then i s o p r e n a l i n e added. S a m p l e s (1 ml) w e r e t a k e n at known t i m e s , added to 2 m l ethanol, v o r t e x m i x e d and then s t o r e d at -20°C for at l e a s t 16 h o u r s . C o n t r o l flasks w e r e included in e a c h e x p e r i m e n t containing an equivalent volume of blank d r u g vehicle. A f t e r s t o r a g e , s a m p l e s w e r e w a r m e d to 4°C and c e n t r i f u g e d at 2250 g for 20 minutes. A p o r t i o n (2 ml) of e a c h s u p e r n a t a n t w a s withdrawn and e v a p o r a t e d to d r y n e s s for 4 h o u r s at 37°C in a v a c u u m oven. The d r i e d r e s i d u e s w e r e r e c o n s t i t u t e d in 500 ~1 of 0.0S mol/1 T r i s - E D T A buffer (pH 7.5) and cAMP d e t e r m i n e d in duplicate 50 R1 s a m p l e s b y a s t a n d a r d s a t u r a t i o n a s s a y (9, 10). U n l e s s s t a t e d o t h e r w i s e , r e s u l t s a r e given a s m e a n ~- s t a n d a r d e r r o r of the m e a n with the n u m b e r of e x p e r i m e n t s following in p a r e n t h e s i s . Group m e a n s w e r e c o m p a r e d u s i n g Student's t - t e s t .
RESULTS F i g u r e 1 shows t y p i c a l r e s u l t s f r o m e x p e r i m e n t s in which m y o c y t e s
~ i
105cells/m[
300
37°C
l
lO-6mol/I Isoprenohnesulphole .0.Sin mol/L MIX 0 5mmol/I MIX
8
c~ 225
-~ 150
x lOL,cellslml
75 E c~
I
-A
1 -
Figure
1
-
2
4
TIME(m,ns)
C y c l i c AMP p r o d u c t i o n b y i s o l a t e d c a r d i a c m y o c y t e s in Ca ~ - - free b i c a r b o n a t e buffer.
1219
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
160f
~
.2mlns
/
,20secs ~: 6o g 801
z~ z~
~ ~o I 20 0
Figure 2
-9 -8 -7 -6 -5 4 -- LOG([SOPRENALINE(mol/I))-->
L o g - d o s e r e s p o n s e c u r v e s for c y c l i c AMP production. Values obtained f r o m time c o u r s e r e s p o n s e s at e a c h dose of i s o p r e n a l i n e and given a s p m o l cAMP a c c u m u l a t e d p e r ml. above control. 1 ml. c e l l s u s p e n s i o n s incubated at 37°C, r e a c t i o n s t e r m i n a t e d by addition of 2 m l ethanol. C e l l count, 8 x 104 r o d c e l l s p e r ml. c e l l suspension.
w e r e incubated in c a l c i u m - f r e e b i c a r b o n a t e buffer containing 0 . 5 mmol/1 MIX with 10-6 tool/1 i s o p r e n a l i n e added or an equivalent volume (50 ~1) of pH 4 saline (control).
Two types of r e s p o n s e s have been r e c o r d e d (cf. F i g u r e 1).
Of 20 e x p e r i m e n t s p e r f o r m e d with 0 . 5 mmol/1 MIX, 9 have shown a r e l a t i v e l y l i n e a r cAMP r e s p o n s e to i s o p r e n a l i n e , w h e r e a s 11 have exhibited s a t u r a t i o n a f t e r 2 - 3 minutes.
In 7 e x p e r i m e n t s w h e r e MIX was omitted f r o m the
incubation m e d i u m , a l l r e s p o n s e s s a t u r a t e a f t e r 1 - 2 minutes e x p o s u r e to the d r u g (1.25 x 10 - 6 mol/1).
When t h e r e s p o n s e is a l i n e a r , a f a m i l y of log-
dose r e s p o n s e c u r v e s a r e obtained ( F i g u r e 2) a l l having h a l f - m a x i m a l v a l u e s on the o r d e r of 10 -7 mol/1 i s o p r e n a l i n e , with t h r e s h o l d between 10 -9_ 10 -8 mol/1. F i g u r e 3 s u m m a r i z e s the d a t a f r o m a s e r i e s of e x p e r i m e n t s using 1.25 x 10-6 tool/1 i s o p r e n a l i n e as s t i m u l a n t , cAMP l e v e l s being m e a s u r e d 30 seconds a f t e r d r u g addition.
With no p h o s p h o d i e s t e r a s e inhibitor p r e s e n t
1220
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(n=5)
120
(n=11)
(n=4)
100
lO6mol/[ ~] 125 Isoprenohne
80
17Control
~ 6o (n=3)
I
r•
0
0 1118 0 0
Figure 3
0.5 1.18 0 0
0.5 1,18 1.0 0
0 0
Addqtlons 0.5 MIX 7 Mg'* 1.180 Ccl..] (rnmol/I) 8.45 #r opr o nolol (t0-6moll[)
E f f e c t s of p h o s p h o d i e s t e r a s e inhibition, Ca -H', Mg++ and P r o p r a n o l o l on b a s a l and i s o p r e n a l i n e - s t i m u l a t e d cAMP production, m e a s u r e d 30 s e c o n d s a f t e r d r u g addition. Ca qqand Mj~_ ~ added a s c h l o r i d e and sulphate s a l t s , r e s p e c t i v e l y . In Mg ~ - - f r e e e x p e r i m e n t s , m a g n e s i u m o m i t t e d f r o m s e d i m e n t a t i o n p r o c e d u r e (8) a s w e l l as incubation m e d i u m . T e m p e r a t u r e 37°C. E a c h s t i m u l a t e d group c o m p a r e d with c o r r e s p o n d i n g c o n t r o l by Student's t - t e s t . *=0.05-¢P~0.01, ** = 0 . 0 1 ~ P-¢ 0.001, * * * = P . ~ 0.001.
in Ca -H'- f r e e b i c a r b o n a t e buffer, t h e r e a r e some I 0 . 5 + 2 . 2 p m o l cAMP p e r l05 r o d c e l l s ( n = 7 ) in c o n t r o l f l a s k s and t h i s is not s i g n i f i c a n t l y i n c r e a s e d s t a t i s t i c a l l y to 16. 6 + 9.3 p m o l / 1 0 5 c e l l s when i s o p r e n a l i n e i s p r e s e n t .
On
addition of 0 . 5 m m o l / l MIX, c o n t r o l cAMP i n c r e a s e d significantly to 36.7 + 4 . 0 p m o l / 1 0 5 c e l l s ( n = l l , pd= 0.001) and when i s o p r e n a l i n e is p r e s e n t t h e r e is a f u r t h e r significant i n c r e a s e to l l 2 + 10.6 p m o l / 1 0 5 c e l l s ( n = i i , P-=0.001). The f l E t h e r addition of 1 m m o l / l C a C l 2 to the m e d i u m does not a l t e r e i t h e r the c o n t r o l or s t i m u l a t e d l e v e l s of cAMP.
However, the o m i s s i o n of MgSO 4
r e s u l t s in a l o w e r i n g of b a s a l cAMP to a l e v e l c o m p a r a b l e with that found in the a b s e n c e of MIX with Mg-H-, w h e r e a s the l e v e l of s t i m u l a t e d cAMP is unchanged f r o m that o b s e r v e d with MIX in the p r e s e n c e o r absence of Ca M". l~:opranolol at 8.45 x 10-6 m o l / l a b o l i s h e s s t i m u l a t i o n by i s o p r e n a l i n e . Although the m e a n l e v e l of cAMP in f l a s k s with i s o p r e n a l i n e i s not
1221
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
s t a t i s t i c a l l y different from control when all the data a r e pooled, for each e x p e r i m e n t there is a highly significant positive c o r r e l a t i o n between the level of cAMP in a s t i m u l a t e d flask and the c o r r e s p o n d i n g control ( r = 0.951, n = 7, P = 0. 001).
The data of F i g u r e 3 have therefore been e x p r e s s e d as the r a t i o (K)
of cAMP after 30 seconds in the p r e s e n c e of i s o p r e n a l i n e to the control value at the s a m e t i m e and a r e p r e s e n t e d in F i g u r e 4.
Cyclic AMP is i n c r e a s e d
about 1.6 - fold by i s o p r e n a l i n e when MIX is absent, 3 - fold with 0. 5 mmol/1 MIX added, i r r e s p e c t i v e of whether or not c a l c i n m is p r e s e n t at 1 retool/l, and s t i m u l a t e d 10 - fold when Mg++ is omitted from the incubation medium.
All of
these v a l u e s differ significantly from unity (el. F i g u r e 4).
(n=Z~)
12
l:
R
6
2
(n=3)
0 0 118 0 0
Figure 4
0.5 1.18 0 0
0.5 118 10 0
)5
o 0
o
r~ 0.5 118
Additions MIX ] Mg**/(retool/I) 0 Ca**j 6.45 PropronoLol (106mo1/[)
Stimulation of cAMP production by i s o p r e n a l i n e . R = cAMP produced after 30 seconds stimulation. cAMP in control flask after 30 seconds. t - t e s t s p e r f o r m e d a s s u m i n g that R = 1, i . e . rio stimulation.
DISCUSSION The viable s i n g l e - c e l l suspension of c a r d i a c myocytes p e r m i t s examination of c a t e c h o l a m i n e - s t i m u l a t e d cyclic AMP production in a p r e p a r a t i o n where the spatial o r g a n i s a t i o n of the r e c e p t o r - e n z y m e complex r e s e m b l e s that found in the intact t i s s u e , without the p r e s e n c e of an extensive e x t r a c e l l u l a r space.
1222
The
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
receptor moiety is exposed to an approximately interstitial fluid environment, whereas the adenyl cyclase unit functions in a cytoplasmic milieu, assuming an intact cell sarcolemma. In suspensions hypersensitive to calcium, addition of this ion produces cellular contraction and damage (11,12) resulting in a preparation resembling broken-cell or plasma membrane systems.
Thus in the only previous report on
this subject, Moustafa and co-workers (12), using a calcium-intolerant preparation, reported that Ca"H- inhibited cAMP production in isolated cardiac myocytes at a concentration (0.5 mmol/1) known to inhibit adenyl cyclase activity in cell-free systems (6). The unchanged levels of basal cAMP found in this study when 1 mmol/1 calcium is included (Figure 3) indicate that in the present preparation intracellular free Ca -H- concentration is being maintained at about 10- 6 - 10 -7 tool/l, which is both typical for this type of tissue (13) and also in the range where adenyl cyclase activity is not inhibited by this cation (6).
Furthermore, it can be calculated
from the data of Moustafa et al (12) that about 10 pmol cAMP/105 cells were produced 30 seconds after the addition of 10-6 mol/1 isoprenaline In calcium-free buffer containing 2 mmol/1 theophylline and 0.5 mmol/1 EGTA. This is only 12~o of the response reported here and could be due to the fact that EGTA might also inhibit adenyl cyclase activity, independent of its metal-ion chelating ability (14). Adenyl cyclase has an absolute requirement for Mg-H- (15, 16) so that reduction in basal cyclic AMP in myocytes incubated in Mg-H-- free buffer (Figure 3) is due probably to cell depletion of Mg-H'. The half-time for magnesium efflux from rat ventricle is on the order of 3 hours at 37°C (17) so that the short incubation times used here would not be expected to reduce intracellular magnesium to a level where adenyl cyclase would be inhibited completely. Further evidence to support the contention that the sarcolemma is
1223
VoI. 72, No. 3, 1 9 7 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
functioning in a relatively typical manner is obtained from the dose-response curves (Figure 2 ) where the half-maximal values on the order of 10 -7 mol/1 agree weU with those found in other studies (2). The date obtained in the presence of MIX showing both linear and saturable responses to isoprenaline (Figure 1) probably reflect the complex feedback relationship between adenyl cyclase, cyclic AMP and Ca -H- (6) and might well depend upon the level of trace amounts of calcium which are unavoidable in the nominally calcinm-free incubations.
This would not
only influence the level of residual phosphodiesterase activity, which Ca"H" is known to stimulate at v e r y low concentrations (18, 19), but also transport of trace extracellular calcium might be facilitated by the phosphodiesterase inhibitor itself (20). The consistent finding of cyclic AMP saturating after 1 - 2 minutes where MIX is omitted (7 experiments) agrees well with data from both intact hearts (4) and also other single-cell systems (21).
Indeed, studies with these
minimally-tensioned isolated myocardial cells might well clarify many of the problems concerning plasma membrane regulation of fi-adrenergic activiation of adenyl cyclase. ACKNOWLEDGEMENTS This research is supported by a grant from The British Heart Foundation. We are indebted to Professor J.F. Tait and Mrs. S.A.S. Tait for both continual advice and also fl-counting facilities, and to Dr. B.L. Brown for instruction and reagents concerning cyclic AMP saturation assays.
REFERENCES
1. Robinson, G . A . , Sutherland, E . W . , and Butcher, E. (1971). Cyclic AMP, Academic Press, New York. 2. Lefkovitz, R.J., Limbird, L . E . , Mukherjee, C., and Caron, M.G. (1976). Biochim. Biophys. Acta., 457, 1-39. 3. Wollenberger, A. (1975) in Contraction and Relaxation in the Myocardium (Ed. W.G. Nayler) pp. 113 - 190, Academic Press, New York. 4. Kukovetz, W . R . , P/~ch, G., and Wurm, A. (1975). Adv. Cycl. Nucl. Res. 5, 395- 414. 5. Watanabe, A . M . , and Besch, H.R. (1975). Circ. R e s . , 37, 309-317.
1224
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6. Tada, M., Kirchberger, M.A., loria, I-A. M., and Katz, A.M. (1975) Circ. Res., 36, 8-17. 7. Gould, R.P. and Powe11, T. (1972). J. Physiol., 225, 16P-18P. B. Powell, T., and Twist, V.W. (1976). Biochem. Biophys. Kes. Commun., in press. 9. Brown, B . L . , Albano, J.D.M., Ekins, R . P . , Sgherzi, A.M., and Tampion, W. (1971). Biochem. J., 121, 561-562. 10. Brown, B . L . , Ekins, R . P . , and Albano, ].D.M. (1972). Adv. Cycl. Nucl. R e s . , 2, 25- 40. 11. F a r m e r , B.B., Jolly, W.W., Watanabe, A.M., and Besch, H.R. (1975). Fed. Proc., 84, 882. 12. Moustafa, E . , Skomedal, T . , Osnes, J-B., and ~Dye, I. (1976). Biochim. Biophys. Acta., 421, 411-415. 18. Reuter, H. (1974). Circ. Res., 84, 599-605. 14. Johnson, R . A . , and Sutherland, E.W. (1978). ]. Biol. Chem., 248, 5114- 5121. 15. Birnbaumer, L. (1978). Biochim. Biophys. Acta., 300, 129-158. 16. Drummond, G . I . , and Duncan, L. (1970). J. Biol. Chem., 245, 976-988. 17. Page, E . , and Polimeni, P.I. (1972). J. Physiol., 224, 121-189. 18. Teo, T . S . , and Wang, J.H. (1969). Biochem. Biophys. Res. Commun., 85, 5950 - 5955. 19. Katz, A.M., (1970). Physiol. Rev., 50, 68-158. 20. Nayler, W.G. (1967). Am. Heart J., 78, 379-894. 21. Tomasi, V., Poll, A., F e r r e t t i , E . , and Barnabei, O. (1975). Adv. Enz. Regulation, 13, 189-200.
1225