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Opposite effects of adrenaline and ouabain on the resting potential of rat atrial cells
Life Sciences, Vol. 39, pp. 2541-2550 Prlnted in the U.S.A.
AND
OPPOSITE OUABAIN OF
Jacques
Pergamon Journals
EFFECTS OF ADRENALINE ON THE RESTING POTENTIAL RAT ATRIAL CELLS
D1acono, Jean Di4trich and H41~ne Lajoix
Laboratoire de Physiologie Compar4e BStiment 443, Universit6 de Parls - Sud 91404 O r s a y , F r a n c e (Recelved in final form September 17, 1986) Summary In the left rat atrium changes in diastolic potential ( E m a x ) evoked by sudden stin~ulation traln w e r e modified by adrenaline and ouabain. T h e early stimulation depolarization phase ( S D P ) of E n~ax occuring on stimulation was shortened by adrenaline , but lengthened and strongly enhanced by ouabain. The stimulation repolarization phase ( S R P ) following S D P was m a r k e d l y inhibited by ouabain, while accelerated and increased by adrenaline. In continuously stimulated atria E m a x w a s decreased by ouabain and a u g m e n t e d by adrenaline. The adrenaline-induced hyperpola rization was reduced or suppressed in the presence of 10-4 M or 10-3 M ouabain, respectively. The present data suggest that adrenaline could stinlulate the electrogenic s o d i u m p u m p in the rat atrium. C o n n e l y (I) and Straub (Z) w e r e probably the first to propose that an active transport of s o d i u m influenced the rnenlbrane potential in myelinated nerve fibres f r o m the frog. T h e participation of the s o d i u m p u m p wlth the resting potential was den~onstrated then in cardiac tissues (3, 4, 5). Activation of the s o d i u m pun~p is followed by m e m b r a n e hyperpolarlzation (6). This hyperpolarization as well as the active N a efflux are inhibited by cardiac glycosides (7).In Purkinje fibres overdriven by m o r e rapidly applied stimuli, the m a x i m u m diastolic potential ( E m a x ) increased w h e reas the subsequent spontaneous discharge w a s temporarily suppressed (8). T he stimulation hyperpolarization phase can be p r e c e d e d by transient fall of E m a x (9).A s overdrive-induced hyperpolarization was abolished by replacing s o d i u m with lithium or by adding 2-4 dinitrophenol, Vassalle (I0) suggested that the E m a x increase w a s a consequence of electrogenic sodiurn p u m p activation. This hypothesis w a s strengthened by similar results obtained f r o m guinea-pig (ll),rabbit (IZ) and rat (13)left atria submitted to stinlulus trains. Contrarily to cardiac glycosides that depolarlze m y o c a r d i u m cell (14),adrenaline increases the resting potential in atrial or Purkinje cells (15, 16). This hyperpolarization w a s also attributed to e n h a n c e m e n t of active N a - K transports (17, 18).Catecholamine-induced stimulation of the s o d i u m p u m p w a s suggested in different m u s c l e tissues such as, for example, Pur kinje fibres (19) or soleus m u s c l e (g0).However, recent studies s h o w that
0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
2542
Adrenallne and 0uabaln
in Rat Atrlum
Vol. 39, No. 26, 1986
hyperpolarization e v o k e d by adrenaline in c o r o n a r y sinus cells (21) or P u r kinje fibres (Z2) m i g h t be attributed to an i n c r e a s e in the p e r m e a b i l i t y of the cell m e m b r a n e to p o t a s s i u m ions. T h e a i m of our e x p e r i m e n t s w a s to d e t e r m i n e w h e t h e r adrenaline h y p e r p o l a r i z e s the rat a t r i u m cell t h r o u g h stimulation of an electrogenic sod i u m p u m p or an i n c r e a s e in m e m b r a n e c o n d u c t a n c e to p o t a s s i u m ions. T h e refore, the effects of adrenaline a n d ouabain on E m a x of rat atrial cells sti m u l a t e d either periodically or continuously w e r e studied. A l t h o u g h rat r n y ocardiurn does not present a great sensitivity to c a r d i a c g l y c o s l d e s , h i g h d o ses of ouabain strongly inhibit the sarcolerrlmal N a - K ATPase involved in the active N a a n d K transport (23, 24). T h e e x p e r i m e n t a t i o n d e m o n s t r a t e s : i) opposite effects of adrenaline and o u a b a i n on the late repolarization p h a s e of E m a x that followed application of stimulus train, 2) opposite effects of adrenaline a n d ouabain on E m a x in regularly stimulated atrla a n d 3) antagonist action of o u a b a i n on the a d r e n a l i n e - i n d u c e d hyperpolarization of E m a x . T h e s e results suggest that adrenaline stimulates the electrogenic sodium p u m p in rat atrial cells.
Methods M a l e W1star rats w e i g h i n g f r o m 300 to 4 0 0 g w e r e u s e d in our exp e r i m e n t s . In brief, a n l m a l s w e r e anesthetized with s o d i u m pentobarbital (50 mg/Kg).The heart w a s r e m o v e d f r o m the chest a n d i m m e r s e d for less than one m i n u t e in n o r m a l T y r o d e ' s solution m a i n t a i n e d at r o o m t e m p e r a t u r e and saturated with 100T0 oxygen. T h e aorta w a s then canulated a n d the heart perfused for o n e m i n u t e a c c o r d i n g to Langendorff's m e t h o d . After canula r e m oval, the left a t r i u m w a s rapidly dissected free in p r e s e n c e of o x y g e n a t e d solution, t r a n s f e r r e d to a s m a l l c h a m b e r (0,5 ml) a n d s u p e r f u s e d at 6 m l / minute. The temperature w a s thermostatlcally m o n i t o r e d at 35"C. After a 30 m i n u t e equilibration p e r i o d the intracellular potential w as r e c o r d e d by m e a n s of m i c r o e l e c t r o d e s having resistance f r o m i0 to 15 M~ ( tip potential less than 5 m V ) a n d filled with 3 M K C I solution. T w o unpolarizable c a l o m e l electrodes w e r e used. T h e first one w a s inserted betwe e n the m i c r o e l e c t r o d e a n d the cathode follower input. T h e s e c o n d one m a i n tained extracellular spaces at r e f e r e n c e potential. Eventual slightasyrnn~etry of potential b e t w e e n c a l o m e l electrodes w a s electrically r e m o v e d . Atria w e r e stirnulated at 4 H z f r e q u e n c y either for periods of 4 m i n u t e s or continuously b y rectangular stimuli ( 4 m s ) 20~0 higher than the threshold. B e t w e e n two stimulus trains atria w e r e m a i n t a i n e d at rest for about ten m i n u t e s . In sonde cases action potentials w e r e triggered at f r e q u e n c y of 0.4 Hz. Electrical events w e r e displayed on a dual b e a m T e k t r o n i x oscilloscope. Isolated action potentials a n d their d V / d t w e r e r e c o r d e d on a stationary film0 D u r i n g investigations with 4 I Iz stimulus trains the horizontal s w e e p w a s stopped and vertical m o v e m e n t s of the b e a m w e r e r e c o r d e d on 5 m m / s continuously m oving film. A s vertical a m p h f i c a t i o n u s e d h e r e w a s Z m V / c m , only the Io w e r part of action potentials w a s displayed on the s c r e e n a n d r e c o r d e d o M o st of the results are p r e s e n t e d as m e a n s _4- s t a n d a r d e r r o r of m e a n s . W h e n e v e r possible the significance of m e a n difference w a s c h e c k e d by Student's t-test, T h e n o r m a l T y r o d e ' s solution contained ( in m M ) : N a C I 143, K C I 5.6, C a C I ~ .< g. 16, M g C I ~ 0.25, T r i s - H C l buffer 5, glucose I I ( p H 7.4 ) a n d w a s saturated withl00~/0 oxygen. D r u g s w e r e freshly m a d e a n d u s e d at final concentrations ( M ) of : o u a b a i n ( C a l b i o c h e m , B e h r i n g C o r p . , L a Jo-
Vol. 39, No. 26, 1986
Adrenallne and Ouabaln in Rat Atrium
2543
lla, USA) 10-4 and 10-3 ; 1-adrenaline ( Merk, Darmstadt, Germany ) 10-6. Ascorbic acid was added to adrenaline-containing solutions to help prevent o x i d a t i o n . In o r d e r t o a v o i d a c c u m u l a t i o n phenomena the drugs were used only one time except in 4 experiments where reversibility was checked.
Results Effects tential:
of adrenaline
and ouabain
on rate-related
changes
in membrane
po -
Left atria were stimulated at 4 Hz for 4 minutes. The stimulation wa s t h e n r e m o v e d f o r a t l e a s t 10 m i n u t e s . C h a n g e s i n m e m b r a n e potential were recorded d u r i n g a n d a f t e r s t i m u l u s t r a i n a s s h o w n i n F i g . 1. E a c h t r a c e begins with the steady resting potential from quiescent atriai cell. This potentialis extrapolated to the right part of the recording by a thin horizontal l i n e . On s t i m u l a t i o n , d a r k a r e a s show the lower part of the action potentials. The oscilloscope high sensitivity selected here ( see Methods ) explains the f a c t t h a t t h e u p p e r e d g e o f t h e d a r k a r e a i s s t r a i g h t . In c o n t r o l e x p e r i m e n t
lOmV [
_
_
1 mm
B h
FIG. 1 Rate-related changesir membrane potential occurlng on stlmula~ion ( 4 H z ) f r o m q u i e s c e n c e in rat atrium. Each trace shows t h e r e s t i n g p o t e n t i a l b e f o r e , d u r i n g a n d a f t e r s t i m u l a t i o n . A: s t i mulatlon of the same atrlun, in control conditions (a) a n d i n p r esence of 10-6 M adrenahne (b). B: s t i m u l a t i o n of a n o t h e r a t r i um in control medium (a) a n d i n p r e s e n c e of 10-4 M ouabaln (b). The two controls ( A a a n d Ba) s h o w h a b i t u a l s l i g h t r e s p o n s e differences observed from one atrium to an other.
( F i g . 1 A a ), E m a x f i r s t d e c r e a s e d ( stimulation depolarization phase SDP) then increased ( stimulation repolartzation phase SRP ) to a value slightly n e g a t i v e to t h e c o n t r o l r e s t i n g potential. When stimulation was Interrupted , the resting potential augmented transiently before returning to the control steady level. Ten minutes after stimulus removal the same atrium, always
2544
Adrenallne and Ouabaln in Rat Atrlum
Vol. 39, No. 25, 1986
maintained at rest, was exposed to 10-6 M adrenaline f o r 10 m i n u t e s . A n e w stimulation train was then Initiated in presence of adrenaline ( F~g. 1 Ab)o It c a n b e n o t i c e d t h a t a d r e n a l i n e induced more rapid developments of SDP and SRP. Both phases were also amplified by adrenaline xn t h i s p r e p a r a t i o n . T h e sarr~e s t i m u l a t i o n protocol was apphed in an other series of atria but 1 0 - 4 M o u a b a i n w a s a d d e d to t h e p e r f u s i o n solution before the second stlniulation train. Fig. 1 B illustrates one of these experiments. The stimulation period in presence of ouabain was characterized by very large SDPfollo~¢ed by extremely weak SRP ( Fig. 1 Bb ) with respect t o c o n t r o l ( F i g . 1 Ba). Post-stimulation hyperpolarization was hardly observed in this case. T h e S R P can be fitted by single exponential c u r v e particularly after the first m i n u t e of stimulation. M e a n results f r o m 8 e x p e r i m e n t s s h o w that the S R P tin,e constant w a s r e d u c e d f r o m 48 to 30 s e c o n d s by adrenaline(Pig. 2, left part). T h e c h a n g e s in action potential induced by adrenaline w e r e also studied since they rnight explain s o m e of the features found (eog., the lack of d e c r e a s e of the initial S D P a m p l i t a d e ). in atria regularly p a c e d at
50. • Rorgllml o
E
_-1o
Il dr ol ol l l aN
\\
i
,r_,
1
I
E
~
os
E
01
o
t0mo (re,n)
FIG. 2 Left pannel: s e m i - l o g a r i t h m i c plots of S R P occuring on s t i m u lation ( 4 H z ) f r o m quiescence. T h e difference b e t w e e n E m a x value at a given time, E m a x (t),a n d its steady state value, E m a x (s),is plotted v e r s u s stimulation time. T i m e constants ( r ) are given in seconds. Points are m e a n values f r o m 8 experlrr~ents. Right pannel: action potential and dV/dt obtained without (upper part) and with ( lower part ) 10-6 M adrenaline from 0.4 Hz stimulated atrium. The horizontal line indicates 0 mV potential The square side represents 20 mV and 100 V/s vertically and 20 ms horizontally. Stimulation artefact precedes the action potential.
Vol. 39, No. 25, 1986
Adrenaline and 0uabain in Rat Atrium
2545
0.4 H z adrenaline retarded the action potential repolarization phase, which presented m o r e evident plateau, but did not m o d i f y the m a x i m a l rate of rise ( dV/dt ) of the upstroke ( Fig. Z right part, Table I ).Estimation of the full action potential duration w a s difficult by reason of the asymptotic shape of its terminal repolarization phase. H o w e v e r , the action potential duration , at potential levels c o m p r i s e d between 10~0 and 8 5 % of the m a x i m a l ampli rude, w a s always lengthened by adrenaline.
TABLE I E f f e c t s of 1 0 - 6 M A d r e n a l i n e on R a t e - R e l a t e d C h a n g e s in E m a x and A c t i o n P o t e n t i a l U p s t r o k e
Control
SDP n=6
max
(mV)
SDP n=5
tp
( s ) 2Z. 0
SKID m a x n=5 dV/dt max n=9
(mV)
(a)
3.53 + 0.46
+ 1.8
5. Z1 -+ 0.45
(V/s) 160o33 +-11.58
Adrenaline
(b)
3.83 +- 0.75
12.6
-+ 0.4
7.80-+ 158.67
0.54
+-IZ. 14
b-a
+
(%)
8.4
P
=,0.7
-42.7
<0.01
+49.7
<0.01
1.0
>0.7
SDP max : m a x i m a l amplitude of S D P S D P tp : S D P time to peak SRP max : m a x i m a l amplitude of S R P dV/dt m a x : m a x i m a l slope of the action potential upstroke b-a : in per cent of a P - Student's t-test ~k : significantly different f r o m control M e a n s +- standard errors of m e a n s f r o m n experiments
T h e electrical effects of 10-6 M adrenaline on atria stimulated at 4 H z for 4 minutes are s u m m a r i z e d in Table I which s h o w s that : a)the m a ximal an~plitude of S D P ( S D P m a x ) r e m a i n e d statistically unchanged, b) the S D P time to peak ( S D P tp ) w a s reduced f r o m ZZ. 0 to IZ. 6 seconds and c) the m a x i m a l amplitude of S R P ( S R P m a x ), m e a s u r e d as the potential difference between the m i n i m a l and steady state values of E m a x during drive, w a s m a r k e d l y enhanced. O n the contrary, the effects of 10-4 M ouabain on changes in E m a x p r o d u c e d by identical stimulation trains w e r e roughly opposite. The amplitude and time to peak of S D P w e r e both enlarged as s h o w n in Fig. 1 B w h e r e a s S R P amplitude w a s strongly reduced in presence of the glycoside ( Table II).
2546
Adrenaline and Ouabaln
in Rat Atrlum
TABLE
Effects
of 10-4 M
Control
SRP n=4
max
(mV)
5.45
Ouabain
(a)
Same
on l~ate-l~elated C h a n g e s
(b)
b-a
2 . 2 0 - + 0.37
nomenclature
Effects of adrenallne a n d o u a b a i n continuously stimulated atria:
39, No. 26, 1986
II
Ouabain
+ 0.56
Vol.
as in Table
in E
max
(%)
P
- 59°6
~0.01
I
on the dlastolic potential
(
E
max
) of
Atria regularly p a c e d at 4 H z w e r e e x p o s e d to a d r e n a h n e ( 10-6 M ) or o u a b a i n ( 10-4 or 10-3 M ) o W h e n adrenaline alone w a s a d d e d to the perfuslc~n m e d i u m E max i n c r e a s e d in 4 m i n u t e s to a steady level in n~ean 3 . 8 2 - 0.43 m V negatlve to the control value ( F i g . 3, u p p e r part ).In an att e m p t to d e t e r m i n e w h e t h e r a d r e n a h n e - i n d u c e d h)q0erpolarlzation reflects stlmulation of the s o d i u m p u m p , the c a t e c h o l a m i n e action w a s studied in pre s e n c e of ouabain k n o w n to inhibit the m e m b r a n e Na-K ATPase activity in rat r n y o c a r d i u m . T h e Fig. 3 ( m i d d l e part) s h o w s that 10-4 M ouabaln depolarized E m a x flrst rapldly then m o r e slowly to a steady level 5.6 mV positive to the control diastohc potential. Addition of 10-6 Ivl a d r e n a h n e to ouabain w a s also followed by E m a x hyperpolarization. H o w e v e r , this effect ( about 0.9 m V ) w a s less n~arked than that o b s e r v e d in p r e s e n c e of a d r e n a h n e alone. In atrla flrst p e r f u s e d with 10-3 M ouabaln the d e c r e a s e in E m a x r e a c h e d about I0.4 n ~ V in 6 r~inutes ( F1go 3 , 1 o w e r part )oAdditlon of I0-6 M a d r e n a h n e could not n.odlfy, ln this case ,the effect of ouabain , so that a slow depolarlzation ( about 0o4 m V per m l n u t e ) perslsted even in p r e s e n c e of adrenallneo T h e s e effects w e r e totally r e v e r s i b l e o R e n ~ o v a l o f t h e two drugs c a u s e d E n~ax to h y p e r p o l a r i z e transiently b e y o n d the control value before slow return to its norn~al steady state level ,n about 30 n~inutes. In o r d e r to verify w h e t h e r adrenaline is still able to p r o d u c e hyperpolarization after previzus 10-3 M ouabaln exposure, four atria w e r e s u b m i t t e d to the perfuslon protocol of Fig. 3 ( l o w e r part ) and allowed to r e c o v e r in norn~aI medium for 30 to 40 minutes. At thls n~oment, addltlon of I0-6 M a d r e n a h n e w a s f~ollowed by E m a x hyperpolarization. This potentlal Increase reached 2.65 -' 0. 19 m V in 4 n~inutes, ioe. a value close to that obtained fror1~ atria e x p o s e d for the first t i m e to adrenaline (see a b o v e )o
Discussion In the present study c h a n g e s in dlastollc potential that con~e about during stin~ulation traln w e r e u s e d to approxlrnate the d e g r e e of N a - K pum p activation in the rat atrium. Orlginally, Vassalle (I0) d e s c r l b e d c h a n g e s in E m a x that o c c u r e d in Purklnje flbres durlng o v e r d r i v e periods a n d concluded that the S R P w a s p r o b a b l y due to enhancenlent of the s o d l u m p u m p a ctivityo S u b s e q u e n t studles have p u = s u e d in detall the basls for c h a n g e s in E ~sx o c c u r i n g on stl~ulatlon fror,~ q u l e s c e n c e {Ii, ig, 13, 25, 26, 27). S D P
Vol. 39, No. 26, 1986
Adrenaline and 0uabaln in Rat Atrium
2547
adr 10-6M ~
E i,u-4 ,el
E
w
~
w
I
[
:t--°4
-]
0
ouab 10 - 3 M adr 10-6 M ___~' +12
E÷ E
minutes
FIG.
3
Effects of 10-6 M adrenaline on E m a x of continuously 4 H z stim u l a t e d atrla in a b s e n c e ( u p p e r part ) and in p r e s e n c e o f 10-4 M ( m i d d l e part ) or 10-3 M ( l o wer part ) ouabaino E n]ax is the difference b e t w e e n E m a x value in p r e s e n c e of drugs a n d its steady state level in n o r m a l solution. M e a n values f r o m 5 to 8 e x p e r i m e n t s . B a r s represent S E M .
develops as potassium ( leaving the cell during drive ) accumulates in r e s t r icted intercellular s p a c e s ( 1 2 , 2 5 , 28). in r e t u r n , e x t r a c e l l u l a r concentration of potassium [K] decreases to a v a l u e c l o s e to t h e p r e s t i m u l a t l o n [K] during S R P ( i Z , 29, 30~. T h e s e s t u d i e s s t r e n g t h e n e d t h e i d e a t h a t c h a n g e s i n t r ° n s m e mbrane ionic gradients occuring on stimulation from quiescence ( or on overdriving spontaneously beating preparations ) activate the sodium-potassiu m p u m p w h i c h i n t u r n c a n r e s t o r e , at l e a s t i n p a r t , i n t r a - a n d e x t r a - c e l l u l a r Na and K concentrations. On t h i s p o i n t C o h e n et a l . (27) d e m o n s t r a t e d that the intracellular sodium activity was stimulation-rate dependent. When rate related changes in E max and[K]o are monitored u n d e r c o n d i t i o n s of s o d i u m p u m p i n h i b i t i o n , S D P a n d [K] are enhanced while subsequent SRP and [K]o recovery fail to develop~l~°12, 13, 3 1 ) . In o u r e x p e r i m e n t s SRP was reduced by 59.6% in the presence o f 1 0 - 4 M o u a b a i n ( T a b l e II ) . T h i s i s i n agreement with previous data indicating that the rat heart Na-K ATPase activity was diminished f r o m 4 2 % (Z3) to 70% (Z4) b y t h e s a m e d o s e o f o u a bain.
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Adrenaline and Ouabaln in Rat Atrlum
Vol. 39, No. 26, 1986
Stlmulation of the s o d i u m p u m p b y catecholarr.ines has long b e e n p r o posed° T h e increase of S R P m a x by adrenaline ( Table I ) confirr~s s a m e type results obtained f r o m Purkinje fibres (32) or papillary m u s c l e (33). If activation of the s o d i u m p u m p b y stimulation train is e n h a n c e d by adrenaline, two following effects can be e x p e c t e d : l) larger K influx into the cell a n d , consequently, m o r e rapid s u b s e q u e n t d e c r e a s e in [I~] L_ o , 2) larger h y p e r p o larizing p u m p current. T h e s e effects m a y well account for the d e c r e a s e in the S R P t i m e constant ( Fig. 2 ),the shortening of S D P t i m e to p e a k a n d the S R P m a x i n c r e a s e ( T a b l e I) p r o d u c e d b y adrenaline. In turn, they should also p r o d u c e a fall In S D P m a x w h i c h w a s not statistically o b s e r v e d in our exp e r i m e n t s but is obvious in P u r k i n j e fibres treated with n o r e p l n e p h r i n e (32)° The reasons for this discrepancy are probably complex. Although Purklnje fibres ( 3 4 , 3 5 , 36) a n d r a t h e a r t c e l l s (37) s h o w q u i t e s i m i l a r early transient outward potassium currents there exists numerous differences between these two cardiac cell types concerning particularly other potassium channels revolved throughout the electrical cycle. Moreover in Purkinje fibres adre naline usually amplifies the action potential plateau phase, while shortening the action potential duration. In the rat atrium cell the action potential ter minal repolarization phase is lengthened by adrenahne as shown in Fig. 2 ( right part ).A prolongation of the action potential n~ay enhance the SDP max if the stin~uli excite the celt before the membrane potential has fully repolarized. Such an effect opposes the possible reduction of SDP max caused by adrenahne-induced stimulation of the electrogenlc sodium pump. As adr enahne does not change SDP in cat papillary muscle (33) t h e e v e n t u a l e f f e c ts of catecholamines on this early phase may be relative to the species. A n o t h e r w a y u s e d to a p p r o a c h adrenaline effects on the s o d i u m p u m p is illustrated by e x p e r i m e n t s carried out on regularly p a c e d left atria. H y p e r polarization of E m a x by adrenaline, partially a n t a g o n i z e d by 10-4 M ouabain a n d fully inhibited b y 10-3 M ouabain ( Fiq. 3 ), again supports the h y pothesis of a d r e n a l i n e - i n d u c e d ~timulation of the electrogenlc s o d i u m pun~po Inhibition of the N a - K pun~p in heart nluscle p r e p a r a t i o n needs doses of carglycosides c o n s i d e r a b l y n,ore i*~portant in rat than in other n.a*~malian sp ecles. In glycoside "sensitive" species insertion of sterolds to m e m b r a n e h plds m a y c h a n g e physiocherl,ical properties of the n,ellJbrane. O u a b a i n share these effects at 0. 1 raM0 T h e r e f o r e our interpretation, b a s e d on the assun~ptlon that the o b s e r v e d c h a n g e s are c a u s e d by inhibition of the s o d i u m p u m p , m i g h t be questionable. Son,e a r g u m e n t s r e m o v e thls doubt° F o r exa~nple, the con~plex f o r m e d b e t w e e n rat N a - K A T P a s e a n d o u a b a i n is far less stable than that forn,ed b e t w e e n sensitlve-species e n z y m e and cardiac glycosides (Z3). O u r experlrnents also s h o w e d that o u a b a i n effects are fully reversible in atria stimulated elther periodically (13) or continuously ( see the last part of Results ) . A c c o r d i n g to Allen and S c h w a r t z (23) the rat heart N a - K activated A T P a s e treated for 10 m i n u t e s wlth 10-4 M or 10-3 M o u a b a i n loses 4 2 ~ or 70~0 of its activity, respectively. F r o n l m o r e recent data (?.4) the rat e n z y m e activity is already r e d u c e d by 70~0 with 10-4 M ouabain° T h u s w e suggest that in p r e s e n c e of 10-4 M ouabaln, a significant nun~ber of n~en~brane pun.p sites r e m a i n s still functional. Their stimulation by adrenaline m a y then r e v e rse the depolarizing effect of ouabalno With 10-3 M ouabain the nun~ber of not inhibited sites m i g h t be too w e a k a n d unable to antagonize, w h e n stirrlulatedby adrenaline, the c o n c o m i t a n t d r o p of E Irlax a n d t r a n s n , e m b r a n e ion gradients induced by ouabaino T h e fact that d e c r e a s e in intracellular N a activity b y adrenahne (38) is inhibited by strophanthidin (39) is in a g r e e m e n t with this interpr et ation. In conclusion~the present Investigation d e m o n s t r a t e s that adrenaline is able to accelerate a n d a m p l i f y the stin,ulation repolarization p h a s e of E rrlax In rat atria s u b m i t t e d to stinlulus train a n d h y p e r p o l a r l z e s E nlax in continu ously stimulated atria. O u a b a i n r e d u c e s or abolishes both S R P a n d adrenaline
Vol. 39, No. 26, 1986
Adrenallne and Ouaba~n in Rat Atrlum
2549
i n d u c e d h y p e r p o l a r i z a t i o n of E m a x . T h e s e r e s u l t s s u g g e s t t h a t a d r e n a l i n e hyperpolarizes t h e r a t l e f t a t r i u m c e l l b y s t i m u l a t i o n of t h e e l e c t r o g e n i c sodium pump. Aknowledgen,ent s T h i s w o r k w a s s u p p o r t e d in p a r t b y g r a n t f r o m I N S E R M ( A T P 637895). T h e a u t h o r s a r e g r a t e f u l to M r s J a c q u e l i n e B e r t o n f o r h e r a s s i s t a n c e a n d to Dr. Hassan Parvez for reading the manuscript.
References I. Z. 3. 4. 50 6. 7. 8.
C.M. CONNELY, Rev. Med. Phys., 31, 475-484 (1959). R.W. STRAUB, J. Physiol. (Lond.), 1---59, 10P-Z0P (1961). E. P A G E and S.R. S T O R M , J. Gen. Physiol., 48, 957-972 (1965). HoG. G L I T S C H , PflIlgers Arch., 307, 29-46 (19-'~-9). Mo H I R A O K A a n d H . H. H E C H T , Pfltlgers Arch., 339, 25-36 (1973). H.G. G L I T S C H , J. Physiol. (Lond.~, 220, 565-582 (1972). H.G. G L I T S C H , Pfltlgers Arch., 335, 243-251 (1972). M. V A S S A L L E , F. Jo VAGNINI, A. G O U R I N and J.H. S T U C K E Y , A m . J. Physiol., 21Z, I-7 (1967)o 9. R.L. VICK, A m . J. Physiol., 271, 451-457 (1969). 10. M. V A S S A L L E , Circ. Res., 20,---~28-241 (1970). ii. H.G. G L I T S C H , Pfltlgers Arch., 344, 169-180 (1973). 12. D.L. K U N Z E , Circ. Res., 41, 12~Z~--127 (1977). 13. J. D I A C O N O , J. Mol. Cel. ~-ardiol., II, 5-30 (1979). 14. G. I S E N B E R G a n d W. T R A U T W E I N , P liT~gers A r c h . , 350,41-54(1974). 15. J. D U D E L a n d W. T R A U T W E I N , E x p e r i e n t i a , 12, 396--~'98 (1956). 16. B.]~o H O F F M A N a n d D°H. S I N G E R , A n n . N . Y . - - - A c a d . S c i . , 139, 9 1 4 939 (1967}. 17. W. T R A U T W E I N a n d R . F . S C H M I D T , P f l t l g e r s A r c h . ges. P h y s i o l . , 271, 7 1 5 - 7 2 6 (1960)o 18. A o L . W I T T a n d P . F . C R A N E F I E L D , C i r c . R e s . , 41, 4 3 5 - 4 4 5 (1977). 19. M. V A S S A L L E and O. B A R N A B E I , PflIlgers Arch., 322, 287 - 303 (1971). 20. To C L A U S E N and J.A. F L A T M A N , J. Physiol. (Lond.), 270, 383-414 (1977). 21. P.A. B O Y D E N , P.F. C R A N E F I E L D and D.C. G A D S B Y , J. Physiol. (Lond.), 339, 185-206 (1983). 22. D.C. G A D S B Y , Nature, 306, 691-693 (1983). 23. J.C. A L L E N a n d A . S C H ~ R T Z , J. Pharmacol. Exp. Ther., 168, 4Z46 (1969). 24. D. KU, T. A K E R A , T. T O B I N and T . M . B R O D Y , J. Pharmacol. Exl~ Ther., 197, 458-469 (1976). 25. R. K L I N E a n d M . M O R A D , J. Physiol. (Lond.), 280,537-558 (1978) . 26. D.J. B R O W N I N G , A.L. T I E D E M A N , A.L. S T A F F , D.G. B E N D I T T , M.M. SCHEINMANN and H.S. S T R A U S S , Circ. Res., 44___, 612 - 62Z (1979). 27. C.J. C O H E N , H.A. F O Z Z A R D and S.S. SHEU, Circ. Res., 50, 651662 (1982). 28. R. K L I N E and M. M O R A D , Biophys. J., 16, 367-372 (1976). 29. S.R. H O U S E R and A.R. F R E E M A N , A m . J. Physiol., 238, H 5 6 1 H568 (1980). 30. R. K L I N E , I. C O H E N , R. F A L K and J. K U P E R S M I T H , Nature, Z86, 68-71 (1980).
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31. S.R. H O U S E R , A. Ro F R E E I v l A N , A. Ro B R E I S C H , J . M . J A E G E R , R. Lo C O U L S O N , Ro C A R E Y and J . M . S P A N N , A m . J. Physlol., Z40 , H I 6 8 - H I 7 5 (1981). 3go R. C A R P E N T I E R and M . V A S S A L L E , in R e s e a r c h in Phvsloloov, " A tiber M e m o r l a l i s in H o n o r of Prof. C . M c C . B r o o k s " ed. by F . F . Kao, K. Koizumi, M o Vassalle. Bologna, Gaggl, pp 81-98 (1971). 33. So R. H O U S E R , V. B U R G I S , Fo M A R T I N and Do W E I N B E R G , A m . J. Physiolo, Z45, H 9 0 - H 9 7 (1983)o 34. J. D U D E L , Ko P E P E R , R. R U D E L and W. T R A U T W E I N , Pfltlsers Arch. ges. Physlolo, Z95, 197-Z12 (1967). 35° J. Lo K E N Y O N and W . R . G I B B O N S , J. Gen. Physlolo, 73, i17 - 138 (lq77). 36° E. G O R A B O E U F and Eo C A R I v I E L I E T , Pfltlgers Arch°, 392, 35Z - 359 (198Z). 37. I . R . J O S E P H S O N , J. S A N C I - I E Z - C H A P U L A a n d A. NI. BROWN, C i r c . R e s . , 54, 1 5 7 - 1 8 4 {1984~. 38. J . A . W--~TSSERSTRONf, D . J . S C H W A R T Z a n d H . A . F O Z Z A R D , A m . J. P h y s i o l . , Z43, H 6 7 0 - H 6 7 5 (1981). 39. C . O . L E E andi'~,. V A S S A L L E , A m . J. P h y s i o l . , 244, C 1 1 0 - C 114 {1983).
AND
OPPOSITE OUABAIN OF
Jacques
Pergamon Journals
EFFECTS OF ADRENALINE ON THE RESTING POTENTIAL RAT ATRIAL CELLS
D1acono, Jean Di4trich and H41~ne Lajoix
Laboratoire de Physiologie Compar4e BStiment 443, Universit6 de Parls - Sud 91404 O r s a y , F r a n c e (Recelved in final form September 17, 1986) Summary In the left rat atrium changes in diastolic potential ( E m a x ) evoked by sudden stin~ulation traln w e r e modified by adrenaline and ouabain. T h e early stimulation depolarization phase ( S D P ) of E n~ax occuring on stimulation was shortened by adrenaline , but lengthened and strongly enhanced by ouabain. The stimulation repolarization phase ( S R P ) following S D P was m a r k e d l y inhibited by ouabain, while accelerated and increased by adrenaline. In continuously stimulated atria E m a x w a s decreased by ouabain and a u g m e n t e d by adrenaline. The adrenaline-induced hyperpola rization was reduced or suppressed in the presence of 10-4 M or 10-3 M ouabain, respectively. The present data suggest that adrenaline could stinlulate the electrogenic s o d i u m p u m p in the rat atrium. C o n n e l y (I) and Straub (Z) w e r e probably the first to propose that an active transport of s o d i u m influenced the rnenlbrane potential in myelinated nerve fibres f r o m the frog. T h e participation of the s o d i u m p u m p wlth the resting potential was den~onstrated then in cardiac tissues (3, 4, 5). Activation of the s o d i u m pun~p is followed by m e m b r a n e hyperpolarlzation (6). This hyperpolarization as well as the active N a efflux are inhibited by cardiac glycosides (7).In Purkinje fibres overdriven by m o r e rapidly applied stimuli, the m a x i m u m diastolic potential ( E m a x ) increased w h e reas the subsequent spontaneous discharge w a s temporarily suppressed (8). T he stimulation hyperpolarization phase can be p r e c e d e d by transient fall of E m a x (9).A s overdrive-induced hyperpolarization was abolished by replacing s o d i u m with lithium or by adding 2-4 dinitrophenol, Vassalle (I0) suggested that the E m a x increase w a s a consequence of electrogenic sodiurn p u m p activation. This hypothesis w a s strengthened by similar results obtained f r o m guinea-pig (ll),rabbit (IZ) and rat (13)left atria submitted to stinlulus trains. Contrarily to cardiac glycosides that depolarlze m y o c a r d i u m cell (14),adrenaline increases the resting potential in atrial or Purkinje cells (15, 16). This hyperpolarization w a s also attributed to e n h a n c e m e n t of active N a - K transports (17, 18).Catecholamine-induced stimulation of the s o d i u m p u m p w a s suggested in different m u s c l e tissues such as, for example, Pur kinje fibres (19) or soleus m u s c l e (g0).However, recent studies s h o w that
0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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hyperpolarization e v o k e d by adrenaline in c o r o n a r y sinus cells (21) or P u r kinje fibres (Z2) m i g h t be attributed to an i n c r e a s e in the p e r m e a b i l i t y of the cell m e m b r a n e to p o t a s s i u m ions. T h e a i m of our e x p e r i m e n t s w a s to d e t e r m i n e w h e t h e r adrenaline h y p e r p o l a r i z e s the rat a t r i u m cell t h r o u g h stimulation of an electrogenic sod i u m p u m p or an i n c r e a s e in m e m b r a n e c o n d u c t a n c e to p o t a s s i u m ions. T h e refore, the effects of adrenaline a n d ouabain on E m a x of rat atrial cells sti m u l a t e d either periodically or continuously w e r e studied. A l t h o u g h rat r n y ocardiurn does not present a great sensitivity to c a r d i a c g l y c o s l d e s , h i g h d o ses of ouabain strongly inhibit the sarcolerrlmal N a - K ATPase involved in the active N a a n d K transport (23, 24). T h e e x p e r i m e n t a t i o n d e m o n s t r a t e s : i) opposite effects of adrenaline and o u a b a i n on the late repolarization p h a s e of E m a x that followed application of stimulus train, 2) opposite effects of adrenaline a n d ouabain on E m a x in regularly stimulated atrla a n d 3) antagonist action of o u a b a i n on the a d r e n a l i n e - i n d u c e d hyperpolarization of E m a x . T h e s e results suggest that adrenaline stimulates the electrogenic sodium p u m p in rat atrial cells.
Methods M a l e W1star rats w e i g h i n g f r o m 300 to 4 0 0 g w e r e u s e d in our exp e r i m e n t s . In brief, a n l m a l s w e r e anesthetized with s o d i u m pentobarbital (50 mg/Kg).The heart w a s r e m o v e d f r o m the chest a n d i m m e r s e d for less than one m i n u t e in n o r m a l T y r o d e ' s solution m a i n t a i n e d at r o o m t e m p e r a t u r e and saturated with 100T0 oxygen. T h e aorta w a s then canulated a n d the heart perfused for o n e m i n u t e a c c o r d i n g to Langendorff's m e t h o d . After canula r e m oval, the left a t r i u m w a s rapidly dissected free in p r e s e n c e of o x y g e n a t e d solution, t r a n s f e r r e d to a s m a l l c h a m b e r (0,5 ml) a n d s u p e r f u s e d at 6 m l / minute. The temperature w a s thermostatlcally m o n i t o r e d at 35"C. After a 30 m i n u t e equilibration p e r i o d the intracellular potential w as r e c o r d e d by m e a n s of m i c r o e l e c t r o d e s having resistance f r o m i0 to 15 M~ ( tip potential less than 5 m V ) a n d filled with 3 M K C I solution. T w o unpolarizable c a l o m e l electrodes w e r e used. T h e first one w a s inserted betwe e n the m i c r o e l e c t r o d e a n d the cathode follower input. T h e s e c o n d one m a i n tained extracellular spaces at r e f e r e n c e potential. Eventual slightasyrnn~etry of potential b e t w e e n c a l o m e l electrodes w a s electrically r e m o v e d . Atria w e r e stirnulated at 4 H z f r e q u e n c y either for periods of 4 m i n u t e s or continuously b y rectangular stimuli ( 4 m s ) 20~0 higher than the threshold. B e t w e e n two stimulus trains atria w e r e m a i n t a i n e d at rest for about ten m i n u t e s . In sonde cases action potentials w e r e triggered at f r e q u e n c y of 0.4 Hz. Electrical events w e r e displayed on a dual b e a m T e k t r o n i x oscilloscope. Isolated action potentials a n d their d V / d t w e r e r e c o r d e d on a stationary film0 D u r i n g investigations with 4 I Iz stimulus trains the horizontal s w e e p w a s stopped and vertical m o v e m e n t s of the b e a m w e r e r e c o r d e d on 5 m m / s continuously m oving film. A s vertical a m p h f i c a t i o n u s e d h e r e w a s Z m V / c m , only the Io w e r part of action potentials w a s displayed on the s c r e e n a n d r e c o r d e d o M o st of the results are p r e s e n t e d as m e a n s _4- s t a n d a r d e r r o r of m e a n s . W h e n e v e r possible the significance of m e a n difference w a s c h e c k e d by Student's t-test, T h e n o r m a l T y r o d e ' s solution contained ( in m M ) : N a C I 143, K C I 5.6, C a C I ~ .< g. 16, M g C I ~ 0.25, T r i s - H C l buffer 5, glucose I I ( p H 7.4 ) a n d w a s saturated withl00~/0 oxygen. D r u g s w e r e freshly m a d e a n d u s e d at final concentrations ( M ) of : o u a b a i n ( C a l b i o c h e m , B e h r i n g C o r p . , L a Jo-
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Adrenallne and Ouabaln in Rat Atrium
2543
lla, USA) 10-4 and 10-3 ; 1-adrenaline ( Merk, Darmstadt, Germany ) 10-6. Ascorbic acid was added to adrenaline-containing solutions to help prevent o x i d a t i o n . In o r d e r t o a v o i d a c c u m u l a t i o n phenomena the drugs were used only one time except in 4 experiments where reversibility was checked.
Results Effects tential:
of adrenaline
and ouabain
on rate-related
changes
in membrane
po -
Left atria were stimulated at 4 Hz for 4 minutes. The stimulation wa s t h e n r e m o v e d f o r a t l e a s t 10 m i n u t e s . C h a n g e s i n m e m b r a n e potential were recorded d u r i n g a n d a f t e r s t i m u l u s t r a i n a s s h o w n i n F i g . 1. E a c h t r a c e begins with the steady resting potential from quiescent atriai cell. This potentialis extrapolated to the right part of the recording by a thin horizontal l i n e . On s t i m u l a t i o n , d a r k a r e a s show the lower part of the action potentials. The oscilloscope high sensitivity selected here ( see Methods ) explains the f a c t t h a t t h e u p p e r e d g e o f t h e d a r k a r e a i s s t r a i g h t . In c o n t r o l e x p e r i m e n t
lOmV [
_
_
1 mm
B h
FIG. 1 Rate-related changesir membrane potential occurlng on stlmula~ion ( 4 H z ) f r o m q u i e s c e n c e in rat atrium. Each trace shows t h e r e s t i n g p o t e n t i a l b e f o r e , d u r i n g a n d a f t e r s t i m u l a t i o n . A: s t i mulatlon of the same atrlun, in control conditions (a) a n d i n p r esence of 10-6 M adrenahne (b). B: s t i m u l a t i o n of a n o t h e r a t r i um in control medium (a) a n d i n p r e s e n c e of 10-4 M ouabaln (b). The two controls ( A a a n d Ba) s h o w h a b i t u a l s l i g h t r e s p o n s e differences observed from one atrium to an other.
( F i g . 1 A a ), E m a x f i r s t d e c r e a s e d ( stimulation depolarization phase SDP) then increased ( stimulation repolartzation phase SRP ) to a value slightly n e g a t i v e to t h e c o n t r o l r e s t i n g potential. When stimulation was Interrupted , the resting potential augmented transiently before returning to the control steady level. Ten minutes after stimulus removal the same atrium, always
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Adrenallne and Ouabaln in Rat Atrlum
Vol. 39, No. 25, 1986
maintained at rest, was exposed to 10-6 M adrenaline f o r 10 m i n u t e s . A n e w stimulation train was then Initiated in presence of adrenaline ( F~g. 1 Ab)o It c a n b e n o t i c e d t h a t a d r e n a l i n e induced more rapid developments of SDP and SRP. Both phases were also amplified by adrenaline xn t h i s p r e p a r a t i o n . T h e sarr~e s t i m u l a t i o n protocol was apphed in an other series of atria but 1 0 - 4 M o u a b a i n w a s a d d e d to t h e p e r f u s i o n solution before the second stlniulation train. Fig. 1 B illustrates one of these experiments. The stimulation period in presence of ouabain was characterized by very large SDPfollo~¢ed by extremely weak SRP ( Fig. 1 Bb ) with respect t o c o n t r o l ( F i g . 1 Ba). Post-stimulation hyperpolarization was hardly observed in this case. T h e S R P can be fitted by single exponential c u r v e particularly after the first m i n u t e of stimulation. M e a n results f r o m 8 e x p e r i m e n t s s h o w that the S R P tin,e constant w a s r e d u c e d f r o m 48 to 30 s e c o n d s by adrenaline(Pig. 2, left part). T h e c h a n g e s in action potential induced by adrenaline w e r e also studied since they rnight explain s o m e of the features found (eog., the lack of d e c r e a s e of the initial S D P a m p l i t a d e ). in atria regularly p a c e d at
50. • Rorgllml o
E
_-1o
Il dr ol ol l l aN
\\
i
,r_,
1
I
E
~
os
E
01
o
t0mo (re,n)
FIG. 2 Left pannel: s e m i - l o g a r i t h m i c plots of S R P occuring on s t i m u lation ( 4 H z ) f r o m quiescence. T h e difference b e t w e e n E m a x value at a given time, E m a x (t),a n d its steady state value, E m a x (s),is plotted v e r s u s stimulation time. T i m e constants ( r ) are given in seconds. Points are m e a n values f r o m 8 experlrr~ents. Right pannel: action potential and dV/dt obtained without (upper part) and with ( lower part ) 10-6 M adrenaline from 0.4 Hz stimulated atrium. The horizontal line indicates 0 mV potential The square side represents 20 mV and 100 V/s vertically and 20 ms horizontally. Stimulation artefact precedes the action potential.
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Adrenaline and 0uabain in Rat Atrium
2545
0.4 H z adrenaline retarded the action potential repolarization phase, which presented m o r e evident plateau, but did not m o d i f y the m a x i m a l rate of rise ( dV/dt ) of the upstroke ( Fig. Z right part, Table I ).Estimation of the full action potential duration w a s difficult by reason of the asymptotic shape of its terminal repolarization phase. H o w e v e r , the action potential duration , at potential levels c o m p r i s e d between 10~0 and 8 5 % of the m a x i m a l ampli rude, w a s always lengthened by adrenaline.
TABLE I E f f e c t s of 1 0 - 6 M A d r e n a l i n e on R a t e - R e l a t e d C h a n g e s in E m a x and A c t i o n P o t e n t i a l U p s t r o k e
Control
SDP n=6
max
(mV)
SDP n=5
tp
( s ) 2Z. 0
SKID m a x n=5 dV/dt max n=9
(mV)
(a)
3.53 + 0.46
+ 1.8
5. Z1 -+ 0.45
(V/s) 160o33 +-11.58
Adrenaline
(b)
3.83 +- 0.75
12.6
-+ 0.4
7.80-+ 158.67
0.54
+-IZ. 14
b-a
+
(%)
8.4
P
=,0.7
-42.7
<0.01
+49.7
<0.01
1.0
>0.7
SDP max : m a x i m a l amplitude of S D P S D P tp : S D P time to peak SRP max : m a x i m a l amplitude of S R P dV/dt m a x : m a x i m a l slope of the action potential upstroke b-a : in per cent of a P - Student's t-test ~k : significantly different f r o m control M e a n s +- standard errors of m e a n s f r o m n experiments
T h e electrical effects of 10-6 M adrenaline on atria stimulated at 4 H z for 4 minutes are s u m m a r i z e d in Table I which s h o w s that : a)the m a ximal an~plitude of S D P ( S D P m a x ) r e m a i n e d statistically unchanged, b) the S D P time to peak ( S D P tp ) w a s reduced f r o m ZZ. 0 to IZ. 6 seconds and c) the m a x i m a l amplitude of S R P ( S R P m a x ), m e a s u r e d as the potential difference between the m i n i m a l and steady state values of E m a x during drive, w a s m a r k e d l y enhanced. O n the contrary, the effects of 10-4 M ouabain on changes in E m a x p r o d u c e d by identical stimulation trains w e r e roughly opposite. The amplitude and time to peak of S D P w e r e both enlarged as s h o w n in Fig. 1 B w h e r e a s S R P amplitude w a s strongly reduced in presence of the glycoside ( Table II).
2546
Adrenaline and Ouabaln
in Rat Atrlum
TABLE
Effects
of 10-4 M
Control
SRP n=4
max
(mV)
5.45
Ouabain
(a)
Same
on l~ate-l~elated C h a n g e s
(b)
b-a
2 . 2 0 - + 0.37
nomenclature
Effects of adrenallne a n d o u a b a i n continuously stimulated atria:
39, No. 26, 1986
II
Ouabain
+ 0.56
Vol.
as in Table
in E
max
(%)
P
- 59°6
~0.01
I
on the dlastolic potential
(
E
max
) of
Atria regularly p a c e d at 4 H z w e r e e x p o s e d to a d r e n a h n e ( 10-6 M ) or o u a b a i n ( 10-4 or 10-3 M ) o W h e n adrenaline alone w a s a d d e d to the perfuslc~n m e d i u m E max i n c r e a s e d in 4 m i n u t e s to a steady level in n~ean 3 . 8 2 - 0.43 m V negatlve to the control value ( F i g . 3, u p p e r part ).In an att e m p t to d e t e r m i n e w h e t h e r a d r e n a h n e - i n d u c e d h)q0erpolarlzation reflects stlmulation of the s o d i u m p u m p , the c a t e c h o l a m i n e action w a s studied in pre s e n c e of ouabain k n o w n to inhibit the m e m b r a n e Na-K ATPase activity in rat r n y o c a r d i u m . T h e Fig. 3 ( m i d d l e part) s h o w s that 10-4 M ouabaln depolarized E m a x flrst rapldly then m o r e slowly to a steady level 5.6 mV positive to the control diastohc potential. Addition of 10-6 Ivl a d r e n a h n e to ouabain w a s also followed by E m a x hyperpolarization. H o w e v e r , this effect ( about 0.9 m V ) w a s less n~arked than that o b s e r v e d in p r e s e n c e of a d r e n a h n e alone. In atrla flrst p e r f u s e d with 10-3 M ouabaln the d e c r e a s e in E m a x r e a c h e d about I0.4 n ~ V in 6 r~inutes ( F1go 3 , 1 o w e r part )oAdditlon of I0-6 M a d r e n a h n e could not n.odlfy, ln this case ,the effect of ouabain , so that a slow depolarlzation ( about 0o4 m V per m l n u t e ) perslsted even in p r e s e n c e of adrenallneo T h e s e effects w e r e totally r e v e r s i b l e o R e n ~ o v a l o f t h e two drugs c a u s e d E n~ax to h y p e r p o l a r i z e transiently b e y o n d the control value before slow return to its norn~al steady state level ,n about 30 n~inutes. In o r d e r to verify w h e t h e r adrenaline is still able to p r o d u c e hyperpolarization after previzus 10-3 M ouabaln exposure, four atria w e r e s u b m i t t e d to the perfuslon protocol of Fig. 3 ( l o w e r part ) and allowed to r e c o v e r in norn~aI medium for 30 to 40 minutes. At thls n~oment, addltlon of I0-6 M a d r e n a h n e w a s f~ollowed by E m a x hyperpolarization. This potentlal Increase reached 2.65 -' 0. 19 m V in 4 n~inutes, ioe. a value close to that obtained fror1~ atria e x p o s e d for the first t i m e to adrenaline (see a b o v e )o
Discussion In the present study c h a n g e s in dlastollc potential that con~e about during stin~ulation traln w e r e u s e d to approxlrnate the d e g r e e of N a - K pum p activation in the rat atrium. Orlginally, Vassalle (I0) d e s c r l b e d c h a n g e s in E m a x that o c c u r e d in Purklnje flbres durlng o v e r d r i v e periods a n d concluded that the S R P w a s p r o b a b l y due to enhancenlent of the s o d l u m p u m p a ctivityo S u b s e q u e n t studles have p u = s u e d in detall the basls for c h a n g e s in E ~sx o c c u r i n g on stl~ulatlon fror,~ q u l e s c e n c e {Ii, ig, 13, 25, 26, 27). S D P
Vol. 39, No. 26, 1986
Adrenaline and 0uabaln in Rat Atrium
2547
adr 10-6M ~
E i,u-4 ,el
E
w
~
w
I
[
:t--°4
-]
0
ouab 10 - 3 M adr 10-6 M ___~' +12
E÷ E
minutes
FIG.
3
Effects of 10-6 M adrenaline on E m a x of continuously 4 H z stim u l a t e d atrla in a b s e n c e ( u p p e r part ) and in p r e s e n c e o f 10-4 M ( m i d d l e part ) or 10-3 M ( l o wer part ) ouabaino E n]ax is the difference b e t w e e n E m a x value in p r e s e n c e of drugs a n d its steady state level in n o r m a l solution. M e a n values f r o m 5 to 8 e x p e r i m e n t s . B a r s represent S E M .
develops as potassium ( leaving the cell during drive ) accumulates in r e s t r icted intercellular s p a c e s ( 1 2 , 2 5 , 28). in r e t u r n , e x t r a c e l l u l a r concentration of potassium [K] decreases to a v a l u e c l o s e to t h e p r e s t i m u l a t l o n [K] during S R P ( i Z , 29, 30~. T h e s e s t u d i e s s t r e n g t h e n e d t h e i d e a t h a t c h a n g e s i n t r ° n s m e mbrane ionic gradients occuring on stimulation from quiescence ( or on overdriving spontaneously beating preparations ) activate the sodium-potassiu m p u m p w h i c h i n t u r n c a n r e s t o r e , at l e a s t i n p a r t , i n t r a - a n d e x t r a - c e l l u l a r Na and K concentrations. On t h i s p o i n t C o h e n et a l . (27) d e m o n s t r a t e d that the intracellular sodium activity was stimulation-rate dependent. When rate related changes in E max and[K]o are monitored u n d e r c o n d i t i o n s of s o d i u m p u m p i n h i b i t i o n , S D P a n d [K] are enhanced while subsequent SRP and [K]o recovery fail to develop~l~°12, 13, 3 1 ) . In o u r e x p e r i m e n t s SRP was reduced by 59.6% in the presence o f 1 0 - 4 M o u a b a i n ( T a b l e II ) . T h i s i s i n agreement with previous data indicating that the rat heart Na-K ATPase activity was diminished f r o m 4 2 % (Z3) to 70% (Z4) b y t h e s a m e d o s e o f o u a bain.
2548
Adrenaline and Ouabaln in Rat Atrlum
Vol. 39, No. 26, 1986
Stlmulation of the s o d i u m p u m p b y catecholarr.ines has long b e e n p r o posed° T h e increase of S R P m a x by adrenaline ( Table I ) confirr~s s a m e type results obtained f r o m Purkinje fibres (32) or papillary m u s c l e (33). If activation of the s o d i u m p u m p b y stimulation train is e n h a n c e d by adrenaline, two following effects can be e x p e c t e d : l) larger K influx into the cell a n d , consequently, m o r e rapid s u b s e q u e n t d e c r e a s e in [I~] L_ o , 2) larger h y p e r p o larizing p u m p current. T h e s e effects m a y well account for the d e c r e a s e in the S R P t i m e constant ( Fig. 2 ),the shortening of S D P t i m e to p e a k a n d the S R P m a x i n c r e a s e ( T a b l e I) p r o d u c e d b y adrenaline. In turn, they should also p r o d u c e a fall In S D P m a x w h i c h w a s not statistically o b s e r v e d in our exp e r i m e n t s but is obvious in P u r k i n j e fibres treated with n o r e p l n e p h r i n e (32)° The reasons for this discrepancy are probably complex. Although Purklnje fibres ( 3 4 , 3 5 , 36) a n d r a t h e a r t c e l l s (37) s h o w q u i t e s i m i l a r early transient outward potassium currents there exists numerous differences between these two cardiac cell types concerning particularly other potassium channels revolved throughout the electrical cycle. Moreover in Purkinje fibres adre naline usually amplifies the action potential plateau phase, while shortening the action potential duration. In the rat atrium cell the action potential ter minal repolarization phase is lengthened by adrenahne as shown in Fig. 2 ( right part ).A prolongation of the action potential n~ay enhance the SDP max if the stin~uli excite the celt before the membrane potential has fully repolarized. Such an effect opposes the possible reduction of SDP max caused by adrenahne-induced stimulation of the electrogenlc sodium pump. As adr enahne does not change SDP in cat papillary muscle (33) t h e e v e n t u a l e f f e c ts of catecholamines on this early phase may be relative to the species. A n o t h e r w a y u s e d to a p p r o a c h adrenaline effects on the s o d i u m p u m p is illustrated by e x p e r i m e n t s carried out on regularly p a c e d left atria. H y p e r polarization of E m a x by adrenaline, partially a n t a g o n i z e d by 10-4 M ouabain a n d fully inhibited b y 10-3 M ouabain ( Fiq. 3 ), again supports the h y pothesis of a d r e n a l i n e - i n d u c e d ~timulation of the electrogenlc s o d i u m pun~po Inhibition of the N a - K pun~p in heart nluscle p r e p a r a t i o n needs doses of carglycosides c o n s i d e r a b l y n,ore i*~portant in rat than in other n.a*~malian sp ecles. In glycoside "sensitive" species insertion of sterolds to m e m b r a n e h plds m a y c h a n g e physiocherl,ical properties of the n,ellJbrane. O u a b a i n share these effects at 0. 1 raM0 T h e r e f o r e our interpretation, b a s e d on the assun~ptlon that the o b s e r v e d c h a n g e s are c a u s e d by inhibition of the s o d i u m p u m p , m i g h t be questionable. Son,e a r g u m e n t s r e m o v e thls doubt° F o r exa~nple, the con~plex f o r m e d b e t w e e n rat N a - K A T P a s e a n d o u a b a i n is far less stable than that forn,ed b e t w e e n sensitlve-species e n z y m e and cardiac glycosides (Z3). O u r experlrnents also s h o w e d that o u a b a i n effects are fully reversible in atria stimulated elther periodically (13) or continuously ( see the last part of Results ) . A c c o r d i n g to Allen and S c h w a r t z (23) the rat heart N a - K activated A T P a s e treated for 10 m i n u t e s wlth 10-4 M or 10-3 M o u a b a i n loses 4 2 ~ or 70~0 of its activity, respectively. F r o n l m o r e recent data (?.4) the rat e n z y m e activity is already r e d u c e d by 70~0 with 10-4 M ouabain° T h u s w e suggest that in p r e s e n c e of 10-4 M ouabaln, a significant nun~ber of n~en~brane pun.p sites r e m a i n s still functional. Their stimulation by adrenaline m a y then r e v e rse the depolarizing effect of ouabalno With 10-3 M ouabain the nun~ber of not inhibited sites m i g h t be too w e a k a n d unable to antagonize, w h e n stirrlulatedby adrenaline, the c o n c o m i t a n t d r o p of E Irlax a n d t r a n s n , e m b r a n e ion gradients induced by ouabaino T h e fact that d e c r e a s e in intracellular N a activity b y adrenahne (38) is inhibited by strophanthidin (39) is in a g r e e m e n t with this interpr et ation. In conclusion~the present Investigation d e m o n s t r a t e s that adrenaline is able to accelerate a n d a m p l i f y the stin,ulation repolarization p h a s e of E rrlax In rat atria s u b m i t t e d to stinlulus train a n d h y p e r p o l a r l z e s E nlax in continu ously stimulated atria. O u a b a i n r e d u c e s or abolishes both S R P a n d adrenaline
Vol. 39, No. 26, 1986
Adrenallne and Ouaba~n in Rat Atrlum
2549
i n d u c e d h y p e r p o l a r i z a t i o n of E m a x . T h e s e r e s u l t s s u g g e s t t h a t a d r e n a l i n e hyperpolarizes t h e r a t l e f t a t r i u m c e l l b y s t i m u l a t i o n of t h e e l e c t r o g e n i c sodium pump. Aknowledgen,ent s T h i s w o r k w a s s u p p o r t e d in p a r t b y g r a n t f r o m I N S E R M ( A T P 637895). T h e a u t h o r s a r e g r a t e f u l to M r s J a c q u e l i n e B e r t o n f o r h e r a s s i s t a n c e a n d to Dr. Hassan Parvez for reading the manuscript.
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C.M. CONNELY, Rev. Med. Phys., 31, 475-484 (1959). R.W. STRAUB, J. Physiol. (Lond.), 1---59, 10P-Z0P (1961). E. P A G E and S.R. S T O R M , J. Gen. Physiol., 48, 957-972 (1965). HoG. G L I T S C H , PflIlgers Arch., 307, 29-46 (19-'~-9). Mo H I R A O K A a n d H . H. H E C H T , Pfltlgers Arch., 339, 25-36 (1973). H.G. G L I T S C H , J. Physiol. (Lond.~, 220, 565-582 (1972). H.G. G L I T S C H , Pfltlgers Arch., 335, 243-251 (1972). M. V A S S A L L E , F. Jo VAGNINI, A. G O U R I N and J.H. S T U C K E Y , A m . J. Physiol., 21Z, I-7 (1967)o 9. R.L. VICK, A m . J. Physiol., 271, 451-457 (1969). 10. M. V A S S A L L E , Circ. Res., 20,---~28-241 (1970). ii. H.G. G L I T S C H , Pfltlgers Arch., 344, 169-180 (1973). 12. D.L. K U N Z E , Circ. Res., 41, 12~Z~--127 (1977). 13. J. D I A C O N O , J. Mol. Cel. ~-ardiol., II, 5-30 (1979). 14. G. I S E N B E R G a n d W. T R A U T W E I N , P liT~gers A r c h . , 350,41-54(1974). 15. J. D U D E L a n d W. T R A U T W E I N , E x p e r i e n t i a , 12, 396--~'98 (1956). 16. B.]~o H O F F M A N a n d D°H. S I N G E R , A n n . N . Y . - - - A c a d . S c i . , 139, 9 1 4 939 (1967}. 17. W. T R A U T W E I N a n d R . F . S C H M I D T , P f l t l g e r s A r c h . ges. P h y s i o l . , 271, 7 1 5 - 7 2 6 (1960)o 18. A o L . W I T T a n d P . F . C R A N E F I E L D , C i r c . R e s . , 41, 4 3 5 - 4 4 5 (1977). 19. M. V A S S A L L E and O. B A R N A B E I , PflIlgers Arch., 322, 287 - 303 (1971). 20. To C L A U S E N and J.A. F L A T M A N , J. Physiol. (Lond.), 270, 383-414 (1977). 21. P.A. B O Y D E N , P.F. C R A N E F I E L D and D.C. G A D S B Y , J. Physiol. (Lond.), 339, 185-206 (1983). 22. D.C. G A D S B Y , Nature, 306, 691-693 (1983). 23. J.C. A L L E N a n d A . S C H ~ R T Z , J. Pharmacol. Exp. Ther., 168, 4Z46 (1969). 24. D. KU, T. A K E R A , T. T O B I N and T . M . B R O D Y , J. Pharmacol. Exl~ Ther., 197, 458-469 (1976). 25. R. K L I N E a n d M . M O R A D , J. Physiol. (Lond.), 280,537-558 (1978) . 26. D.J. B R O W N I N G , A.L. T I E D E M A N , A.L. S T A F F , D.G. B E N D I T T , M.M. SCHEINMANN and H.S. S T R A U S S , Circ. Res., 44___, 612 - 62Z (1979). 27. C.J. C O H E N , H.A. F O Z Z A R D and S.S. SHEU, Circ. Res., 50, 651662 (1982). 28. R. K L I N E and M. M O R A D , Biophys. J., 16, 367-372 (1976). 29. S.R. H O U S E R and A.R. F R E E M A N , A m . J. Physiol., 238, H 5 6 1 H568 (1980). 30. R. K L I N E , I. C O H E N , R. F A L K and J. K U P E R S M I T H , Nature, Z86, 68-71 (1980).
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Adrenallne and Ouabaln in Rat Atrlum
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31. S.R. H O U S E R , A. Ro F R E E I v l A N , A. Ro B R E I S C H , J . M . J A E G E R , R. Lo C O U L S O N , Ro C A R E Y and J . M . S P A N N , A m . J. Physlol., Z40 , H I 6 8 - H I 7 5 (1981). 3go R. C A R P E N T I E R and M . V A S S A L L E , in R e s e a r c h in Phvsloloov, " A tiber M e m o r l a l i s in H o n o r of Prof. C . M c C . B r o o k s " ed. by F . F . Kao, K. Koizumi, M o Vassalle. Bologna, Gaggl, pp 81-98 (1971). 33. So R. H O U S E R , V. B U R G I S , Fo M A R T I N and Do W E I N B E R G , A m . J. Physiolo, Z45, H 9 0 - H 9 7 (1983)o 34. J. D U D E L , Ko P E P E R , R. R U D E L and W. T R A U T W E I N , Pfltlsers Arch. ges. Physlolo, Z95, 197-Z12 (1967). 35° J. Lo K E N Y O N and W . R . G I B B O N S , J. Gen. Physlolo, 73, i17 - 138 (lq77). 36° E. G O R A B O E U F and Eo C A R I v I E L I E T , Pfltlgers Arch°, 392, 35Z - 359 (198Z). 37. I . R . J O S E P H S O N , J. S A N C I - I E Z - C H A P U L A a n d A. NI. BROWN, C i r c . R e s . , 54, 1 5 7 - 1 8 4 {1984~. 38. J . A . W--~TSSERSTRONf, D . J . S C H W A R T Z a n d H . A . F O Z Z A R D , A m . J. P h y s i o l . , Z43, H 6 7 0 - H 6 7 5 (1981). 39. C . O . L E E andi'~,. V A S S A L L E , A m . J. P h y s i o l . , 244, C 1 1 0 - C 114 {1983).