European Journal of Pharmacology, 229 (1992) 163- 169
163
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52799
Caffeine inhibits depolarization-activated outward currents in rat ventricular myocytes Jose Sanchez-Chapula Centro de lnt~estigaciones Biomedicas de la Unit~ersidad de Colima, Apartado Postal 199, C.P. 28000, Colima, Col., Mdxico Received 1 September 1992, accepted 15 September 1992
The effects of caffeine (10 mM) on depolarization-activated, calcium-independent outward K + currents were investigated in isolated rat ventricular myocytes, using whole-cell clamping. The external solution contained CoC1z 2mM and the internal solution contained ethylene glycol-bis(-aminoethyl ether) N,N,N',N'-tetraacetic acid 10 mM. Caffeine decreased the peak amplitude of the total current and the sustained plateau current. Caffeine did not modify the steady state inactivation curve, which was fitted by two Boltzmann functions. Caffeine blocked the tetraethylammonium-sensitive slowly activating and inactivating outward current by 32% and the 4-aminopyridine-sensitive rapidly activating and inactivating transient outward current by 19%. Caffeine did not modify the inactivation rate or the time course of the recovery from inactivation of the transient current. Ryanodine 10 /zM did not modify any of the current components and the effect of caffeine was not modified by ryanodine pretreatment. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxantine 100 /zM, did not modify thc depolarization-activated calcium-independent outward currents. Myocytes (rat ventricular); Whole cell clamping; Caffeine; Depolarization-activated, CaZ+-independent outward currents
1. Introduction Caffeine exerts complex effects on the mechanical and electrophysiological properties of cardiac muscle. Caffeine influences the inotropic state of cardiac muscle at least partially by altering the uptake and release of Ca 2+ by the sarcoplasmic reticulum (Bodem and Sonnenblick, 1975; C h a p m a n and Leoty, 1976; Konishi et al., 1984). Caffeine has been found to affect different m e m b r a n e currents in myocardial cells. Various authors (except Kisner et al., 1979) have found that caffeine enhances the L-type Ca 2+ current in cardiac cells (Goto et al., 1979; Yatani et al., 1984). This effect has been attributed to an increase in intracellular adenosine 3'5'-cyclic monophosphate ( c A M P ) v i a suppression of phosphodiesterase activity (Beavo et al., 1970). Recently, Habuchi et al. (1991) have found that caffeine produces direct blocking effects on the sodium current (INa) in guinea-pig ventricular myocytes. This effect was independent of the internal calcium concentration or intracellular signaling system. Two transient outward currents have been identified in different cardiac preparations (Coraboeuf and
Correspondence to: J. Sanchez-Chapula, Apartado Postal 199, C.P. 28000, Colima, Col., M~xico. Tel. 52 (331) 40573.
Carmeliet, 1982; Escande et al., 1987; Giles and Imaizumi, 1988; Tseng and Hoffman, 1989; Kimura et al., 1990). One current (I t o r It01) is sensitive to 4aminopyridine (4-AP); the primary charge carrier appears to be K + (Isenberg, 1978; Kenyon and Gibbons, 1979a, b). This first current, It, is voltage-activated and is independent of intracellular calcium (Kenyon and Sutko, 1987). The second transient outward current (It02) is briefer and usually smaller than It0 p This second transient outward current is not sensitive to 4-AP and appears to be calcium-activated (Sigelbaum and Tsien, 1980; Coraboeuf and Carmeliet, 1982). The fact that It02 is sensitive to externally applied caffeine and raynodine at concentrations higher than 3 mM suggests that the transient outward current is activated by Ca 2+ released by the sarcoplasmic reticulum (Coraboeuf and Carmeliet, 1982; Escande et al., 1987; Tseng and Hoffman, 1989; Hiraoka and Kawano, 1989). Caffeine has been found to reduce to a large extent the systolic increase of Ca 2+ by inhibiting calcium accumulation in the sarcoplasmic reticulum isolated from skeletal (Weber and Herz, 1968) and cardiac muscle by making vesicle membranes more permeable to Ca 2+ (Blayney et al., 1978). The 4-AP-resistant Ca 2+activated transient outward current had been assumed to be carried by K + ions (Kenyon and Sutko, 1987). Zygmunt and Gibbons (1991), however, have recently
1~4 presented evidence that this current may be a CI current. Since caffeine has been widely used as a pharmacological tool to study different mechanical and electrophysiological properties of cardiac muscle, more information is needed about direct and indirect actions of this drug. In the present work. we report on the effects of caffeine on depolarization-induced calcium-independent outward currents of rat ventricular myocytcs, under experimental conditions that permitted examination of the direct actions of caffeine.
2. Materials and methods
2.1. Isolation of cardiac myoo'tes Cardiac myocytes were isolated according to an enzymatic procedure (Mitra and Morad, 1986). Briefly, rats weighing 200-300 g, were anesthetized with pentobarbital sodium (35 m g / k g ) . The hearts were rapidly excised and mounted via the aorta to a Langendorff perfusion system for retrograde coronary perfusion. The initial perfusion was nominally zero Ca 2* Tyrode solution. After 5 min of perfusion, the perfusate was switched to a zero Ca 2" solution containing collagenase (Sigma, type l, 2 m g / m l ) and protease (Sigma, tycp XIV, (I.2 m g / m l ) for 20 min. After 5 min of perfusion with a 0.1 mM CaCI, Tyrode solution, the heart was removed, the ventricle was dissected out and the cells were dispersed by gentle mechanical agitation.
2. 2. Solutions The Tyrode solution for cell isolation contained (mM): NaC1 112, NaHCO~ 24, KCI 5.4, CaCI 2 1.8, MgC12 1.0, N a H z P O 4 0.42, glucose 11, taurine 10 (pH 7.4). Nominally calcium-free Tyrode solution was prepared by omitting CaC12 from the solution. The 'control' external solution contained (in mM): NaCI 140, KCI 4, MgC12 1, CaCI~ 1.8, H E P E S 10. glucose 11 (pH 7.4 by NaOH). To measure total calcium-independent, depolarization-activated outward K + currents, the 'Ca2+-Co e~' external solution was used which contained (in mM): NaCI 140, KCI 4, MgCI: 1, CaCI: 0.1, H E P E S - N a 10, glucose 11, CoCI 2 2 (to suppress Ca 2 + currents), tetrodotoxin (TTX) 0.02 (to suppress Na + currents), pH 7.4, and was bubbled with 100% 0 2. The depolarization-induced outward K + current under these conditions showed that the activated outward current wave form results from the activation of two kinetically distinct K + currents, one that activates and inactivates rapidly and is sensitive to 4-AP (I~) and a second one that activates and inactivates slowly (IK). Tetraethylammonium (TEA) pre-
dominantly suppresses 1K (Apkon and Nerbonne. 1991 ). In order to study the effects of caffeine on l,, we used the T E A external solution composed as follows (in raM): NaCI 90, TEA-CI 50, KCI 4, MgCI: I, ('aCl~ 0.1, H E P E S - N a 10, CaCI~ 2, T T X 0.02 (pH 7.4). In some experiments, in order to study 1K with minimal interference of I t , we used the 'Ca-~+-Co e+" external solution plus 4-aminopyridinc 5 raM. The potassium internal solution used was (in mM): KCI 120, KftxPO4 IlL H E P E S 10, E G T A lt), ATP-Mg 3 (pH 7.2-7.3, by KOH). In experiments in which the L-type Ca 2~ current was studied, the external solution contained (in raM): TEA-CI 140, CsC1 4, MgCl 2 1, CaCI~ 1.8, H E P E S IlL glucose 11 (pH 7.4 by CsOH). The cesium internal solution was (in raM): CsCI t30, H E P E S 1(I, E G T A 10, ATP-Mg 3 (pH 7.2-7.3 by CsOH).
2.3. Electrophysiological recording4 Cells were transferred to a perfusion chamber (0.3 ml) mounted on the stage of an inverted microscope (Nikon Diaphot, Nikon Co.). The cells were pcrfuscd at a rate of 2 - 3 m l / m i n . The experiments were performed at room temperature (22-23°C). Macroscopic current recordings were obtained with the whole-cell voltage-clamp method as described by Hamill el al. (1981) using a patch-clamp amplifier ( A X O P A T C H l-C, Axon Instruments). Glass pipettes had tip resistances of 1-3 M,(,) when filled with internal solution. The resistance in series with the cell membrane was compensated to provide the fastest possibe capacity transient without ringing. Neither capacitivc current nor leakage current were compensated. Currents werc filtered with a four-pole Bessel filter at 5 kHz, digitized at 4 kHz and stored on an AST 286/16 computer ( p C L A M P software, Axon Instruments).
2.4. Data attalysis When possible, data are presented as arithmetic means + S.D. mean. The statistical significancc of differences in calculated mean values was evaluated using Student's t-test. Differences were considered significant when P values were < 0.05.
2.5. Drugs used Caffeine, tetrodotoxin (TTX) and 4-aminopyridine (4-AP) were dissolved in the external solution: 3-isobutyl-l-methyl-xantine (IBMX) (Sigma Chemical) was dissolved in dimethyl sulfoxide; ryanodine (S.B. Pennick) was added to the external solution from an aqueous stock solution.
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3. Results In a first series of experiments we studied the effect of caffeine (10 mM) on action potentials in the presence of 'normal' external solution and potassium internal solution (fig. 1A). Caffeine increased the plateau level and prolonged the duration of the action potential. The duration measured at 50% repolarization (APDs,) was significantly increased, by 142 _+ 12%, from the control (n = 4). The effect of caffeine was completely reversed after 5 - 7 min of washout. The effect of caffeine on the L-type Ca -,+ current (lc,,_ L) was studied in four cells, under experimental conditions designed to record lc,,_L) with minimal interference of sodium and K + currents (see Materials and methods). From a holding potential (HP) of - 4 0 mV, depolarizing pulses of 500 ms duration were applied to potentials between - 3 0 and + 5 0 mV in steps of 10 inV. Records of currents induced by depolarizing pulses to +10, + 3 0 and + 5 0 mV are shown in fig. lB. Records made under control conditions are shown in (a) and 7 rain after the addition of caffeine in (b). The
A
\
.•J2omV 20ms B
Q
1
C
lOOms O control /x caffeine E'm (mV)
,,
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(pA) 2000 1500 1000
~-e~-m-0~-~-~-m"l -50 - 5 0 _ 5 0 0
0
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I
I
30
50
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-1500
Fig. 2. A. Effect of caffeine (10 # M ) on action potential in the presence of 'Ca2+-Co 2+' external solution. Stimulation frequency 0.1 Hz. Control conditions (a), in the presence of caffeine (b). B. Effect of caffeine on depolarization-activated outward currents. From a holding potential of - 7 0 mV pulses of 500 ms duration were applied to - 3 0 , - 1 0 , +10, + 3 0 and + 5 0 mV, under control conditions (a) and in the presence of caffeine (b). C. I-V curve of peak current for potentials between - 9 0 to + 50 mV under control conditions ( o ) and in the presence of caffeine ( ,5 ).
._•J20mV
20ms
B
b
a
lOOms C
I(pA)
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-50 -10 T 600 20 J,_ ,-, . e , , a ~ L'~.~, ~ _ _ [5~~ i~ . . . . .
k~ ~
.
40 f"j ~ .
50 L7 ]
o
0 [ 3 control ~ , ~ - 1 8 0 0 \ ~ /O/A A O caffeine x ~ " ~ o ~ J O / A ~
_58oo y ~ Fig. 1. A. Effect of caffeine (10 mM) on action potential in a rat ventricular cell. The cell was perfused with control external solution and the pipette was filled with potassium internal solution. Stimulation frequency 0.1 Hz. Control conditions (a), in the presence of caffeine (b). B. Effect of caffeine on Ic,_u. in a cell other than that in Fig. 1A. The external and internal solutions were designed lo decrease interference of Na + and K + currents (see Materials and methods section). From a holding potential (HP) and - 4 0 mV depolarizing pulses of 500 ms duration to + 10, + 3 0 and + 5 0 mV were applied under control conditions (a) and in the presence of caffeine 10 mM (b). C. I-V curves of the peak inward current (~ , zx ) and current measured at the end of the 500 ms pulse ([i], o), in the presence and absence of caffeine 10 raM.
drug increased the amplitude of Ic,_ L without affecting the current at the end of the 500 ms pulse. The I - V curves for peak inward current and current measured at the end of the pulse under control conditions and after addition of caffeine are shown in fig. 1C. Caffeine increased the amplitude of the peak inward current, but did not modify the current at the end of the pulse. These data confirm earlier results obtained by Yatani et al. (1984) under conditions that did not permit the internal buffering of Ca 2+ (single high-resistance microelectrode voltage-clamp). In another series of experiments, we studied the effect of caffeine (10 raM) on action potentials of cells perfused with a 'Ca 2+-Co 2 +, external solution to block Ic, ~ and a potassium internal solution. Action potentials under control conditions and after addition of caffeine are shown in fig. 2A. Under these conditions, with I(,~, blocked, caffeine reversibly increased action potential duration. In four cells, caffeine significantly increased APDs, by 48 +_ 7% from control conditions. This result shows that even after blockade of lc~,, caffeine can still increase action potential duration. This result could be explained by an increase in the inward current (IN,) or a decrease in the outward current. Recently, Habuchi et al. (1991) have found
166
that caffeine decreased INa, making it unlikely that the increase in action potential duration caused by caffeine is due to an increase in inward current. In order to explore the possibility that caffeine can modify calcium-independent outward currents, experiments were performed under voltage-clamp conditions. We used the C a 2 + - C o 2÷ external solution and the potassium internal solution. From a HP of - 70 mV, voltage-clamp steps to potentials between - 9 0 and + 5 0 mV were applied at a frequency of 0.1 Hz. Records of currents in the absence and presence of caffeine to - 3 0 , -1(}, -Fl0, + 3 0 and + 5 0 mV are shown in fig. 2B. The depolarization-evoked outward currents rose rapidly to a peak, then decayed slowly to an apparent plateau. Caffeine decreased outward peak current amplitude and the current at the end of the pulse without apparent changes in the time course of inactivation of the current. The I - V curve for peak current is shown in fig. 2C. Caffeine did not significantly alter the current for voltages negative to -2(1 mV. This results suggests that caffeine did not modify the inward rectifying K + current. Caffeine decreased the peak amplitude of outward currents for voltages positive to - 2 0 mV. In six experiments 10 mM caffeine decreased peak current amplitude measured at + 50 mV by 27 ± 6% from the control values. These effects of caffeine were completely reversed after 5 - 7 min of washout. The voltage dependence of steady-state inactivation of the peak current in rat ventricular cells can be fitted by the sum of two Boltzmann distributions (Apkon and Nerbonne, 19911. The interpretation of that result is that it reflects the steady state inactivation of two channel populations, most likely to be two distinct channel types (Apkon and Nerbonne, 1991). To examine the voltage dependence of steady state inactivation, peak currents evoked by test depolarizing pulses to + 5(1 mV from conditioning pulses of 10 s duration at potentials between 100 and 0 mV were measured. The steady state inactivation curves under control conditkms and in the presence of caffeine are shown in fig. 3, The data were fitted by using the sum of two Boltzmann functions, according to the expression: itV)
A,*{I/l+exp[(V
V, e ( , , ) / k i ] ) }
10 I : ~ : : ~ #
~
, EJ~::~ ~
08 t
-'~,
100
[2 CAFFEINE
80 -60 40 20 HOLDING POTENTIAL (n,V)
0
Fig. 3. Effect of caffeine on voltage dependence of steady state inactivation of the peak outward current. Outward currents were evoked during depolarizations to +50 mV after l0 s conditkming pulses to potentials between - 100 and 0 mV. The amplitudes of the peak currents were normalized to the amplitude of the currenl evoked from - 100 inV. The data were fitted using the sum of two Boltzmann distributions. (©) Under control conditions : (r_J) in the presence of caffeine l0 ,aM.
and were: A~ = (/.23 ± 0.4; V]/2(])= - 6 3 ± 2 mV; k~ = 14.1 ± 1.3 mV; A , = 0.76 ± 0.06; V~., zc_~= - 4 5 ± 3 mV; k, = 3.9 +_ 0.4 mV. In all the present experiments we used a high (10 mM) concentration of E G T A in the internal solution. However, it is possible that caffeine could producc its effects on the depolarization-induced outward currents by blocking an EGTA-resistant sarcoplasmic reticulum calcium release. Ryanodine is a potent alkaloid that also decreases sarcoplasmic reticulum release of Ca2 + during excitation-contraction coupling (Sutko et al., 1979). We then studied the effect of ryanodine at a high concentration (10 p,M) on the depolarization-induced outward currents. In fig. 4A we show records of
A
a
b
F'"-
"-t.
B a
XA 2 * { 1 / I + exp[(V
,:) CONTR,:)t
b
Vl. 2(21)/k2])}
where i is the normalized current as a function of the conditioning potential, V; A~ and A z are amplitude factors of both distributions: Vl/2(l t and V1/,(2 ) are the voltages at which 50% of each channel population is inactivated; k I and k 2 a r e the Boltzmann factors. Under control conditions we found that following value from four cells: A~ = (I.25 ± 0.03: Vl/2(l)= --65 ~ 3 mV; k ~ = 13.6± 1.1 mV; A , = 0 . 7 4 _ + 0 . 0 4 ; V~/e(e)= - 4 4 + _ 2 mV; k , - 4 . 1 +_0.5 mV. These values were not significantly changed, in the presence of caffeine
rt-
__ nA ' lOOms
Fig. 4. A. Effects of ryanodine (10 `aM) on depolarization-activated outward currents. From a holding potential of - 70 mV, depolarizing pulses to - 3 0 , -10, +10, +30 and +50 mV were applied under control conditions (a) and in the presence of ryanodine (b). 13. Effect of IBMX 100 ,aM on outward currents activated by depolarization. From a holding potential of 70 mV, depolarizing pulses to 3(1, - 10, + 10, + 3{) and + 50 mV were applied under control conditions (a) and in the presence of II3MX (bL
167
in the presence of 4-AP alone are shown in fig. 5Aa. Substantial suppression of the peak current is evident and only a slowly activating and very slowly inactivating current is apparent, termed I K by Apkon and Nerbonne (1991). Caffeine significantly decreased I K (fig. 5Ab). The I - V curve for the maximum outward current is shown in fig. 5Ac. Caffeine decreased the outward current at potentials positive to - 2 0 inV. Caffeine decreased I K measured at + 5 0 mV by 32 _+ 5% of the control values (n = 5). Records of currents induced by voltage steps to - 2 0 , 0, + 20 and + 40 mV, in the presence of T E A 50 mM are shown in fig. 5Ba. In the presence of TEA, the current wave forms show only a rapidly activating transient outward current (It) with substantial attenuation of the sustained component. Caffeine decreased the peak amplitude of the current at potentials positive to - 2 0 mV (fig. 5Bb, 5Bc). Caffeine decreased the peak amplitude of the current measured at + 50 mV by 19 _+ 5% of the control values (n = 5). The drug did not significantly modify the rate of inactivation of the current. Under control conditions, the rate of inactivation of the currents induced by a depolarizing pulse to + 50 mV was fitted by a monoexponential process with a time constant of 34 _+ 5 ms (n = 5). In the presence of caffeine, the time constant of inactivation was 36 _+ 6 ms (n = 5). The effect of caffeine on the time course of reactivation of I t was assessed at a HP of - 7 0 mV, using a standard paired pulse (100 ms of duration, to + 50 mV) protocol in the presence of a T E A external solution.
current induced by depolarizing pulses to - 3 0 , - 1 0 , +10, + 3 0 and + 5 0 mV from a HP of - 7 0 mV. Ryanodine did not modify the currents at any of the voltage studied. Similar results were found for two other cells. In five cells which were exposed to ryanodine for 30 rain before seal formation and internal dialysis, the response to caffeine (10 mM) was similar to that in cells without p r e t r e a t m e n t with ryanodine (data not shown). Caffeine has been found to increase the cAMP level by inhibiting phosphodiesterase activity (Beavo et al., 1970). In order to test the possibility that this mechanism of action could explain the effect of the drug on outward currents, we tested the effect of another phosphodiesterase inhibitor, I B M X 100 ~ M , on the depolarization-induced outward currents. Records of currents induced by depolarizing pulses to - 3 0 , - 1 0 , +10, + 3 0 and + 5 0 mV from a HP of - 7 0 mV under control conditions (a) and in the presence of IBMX (b) are shown in fig. 4B. I B M X did not modify either the peak amplitude or the sustained component at the end of the pulse of the outward currents. Similar results were found for two other cells. The K + channel blocker; 4-AP, preferentially blocks the fast transient outward K + current (It), and T E A preferentially blocks the sustained component of the current (1 K) (Apkon and Narbonne, 1991). In another series of experiments, we studied the effect of caffeine in the presence of 4-AP 5 raM. Records of currents induced by depolarization between - 3 0 and + 50 mV
B
A
a
a
b
r;
lnA
lOOms
50ms
900
0
3500
/ 0 4-AP /O A 4-AP + coffeine /O /0 /A / /O . /.z~" 0 A/A
600
300
~_0.~°~
0
o 2500 A
n
O~ A
TEA + coffeine v
1500
/ o J/A /
500
--
-3o
0 TEA
0
so
o
Em (mY)
60
-30
-lo
lo Em (mY)
30
50
Fig. 5. A. Effect of caffeine on depolarization-activated outward currents in a cell perfuscd with the 'Ca 2 "-Co 2+' external solution plus 4-AP (5 mM). Currents evoked by depolarizing pulses to - 3 0 , - 1 0 , + 10, +30 and +50 mV from a holding potential of - 7 0 mV, in the presence of 4-AP alone (a) and in the presence of 4-AP plus caffeine (b). (C) I - I V curve of maximum outward current for potential between - 30 and + 50 mV, under control conditions ( o ) and in the presence of caffeine (A). B. Effect of caffeine on depolarization-activated transient outward current in the presence of the TEA external solution. Currents evoked by depolarizing pulses to - 2 0 , 0, + 20 and +40 mV from a holding potential of - 70 mV, under control conditions (a) and in the presence of caffeine (b). I-V current of peak outward current for poentials between - 30 and + 50 mV, under control conditions ( o ) and in the presence of caffeine ( n ).
168
,oo 80,o .~ 60~--4020 O'--
0
,60
e6o
36o
4&0
~6o
T (ms) Fig. 6. Effect of caffeine on the recovery, from inactivation of I t. The cells were p e r f u s e d with T E A e x t e r n a l solution. From a h o l d i n g p o t e n t i a l of 70 mV, p a i r e d pulses of 100 ms d u r a t k m to + 5 0 mV were a p p l i e d cvery 30 s. The interval b e t w e e n the pulses was c h a n g e d from 20 to 500 ms. T h e n o r m a l i z e d current (lt t / l ~ < , w h e r e l i t is the t r a n s i e n t o u t w a r d c u r r e n l e v o k e d by the second or 'test' pulse, and I t~ is the t r a n s i e n t o u t w a r d current ew)ked by the first or 'conditioning" pulse) was p l o n e d against the interval b e t w e e n the pulses. The data were fitted by single e x p o n e n t i a l s with a lime constan! of 37 ms u n d e r control c o n d i t k m s C ) ) and 40 ms in the p r e s e n c e of caffeine ( ~ }.
Under control conditions the time course of the recovely from inactivation was fitted by an exponential process with a time constant of 39 ± 7 ms (fig. 6). Caffeine did not modify the time course of the recovery from inactivation of I t. The time constant obtained in the presence of the drug was 42 ± 6 ms (n = 5).
4. Discussion In the present work, we have found that caffeine inhibits the intracellular calcium-independent, depolarization-induced outward K + currents in rat ventricular myocytes reversibly. We also confirmed that caffeine increased I t > L in the presence of 10 mM of E G T A in the internal solution. Caffeine has numerous effects on the clectrophysiological and mechanical properties of the myocardium. Most of these effects result from caffeine actions on the sarcoplasmic reticulum (Konishi et al., 1984) and on phosphodiesterase (Beavo et al. 1970). However, Hughes et al. (1990) have found that caffeine inhibits directly I t , in rabbit vascular cells. Moreover, Habuchi et al. (1991) have found that caffeine also blocks IN:, directly and shifts the steady state inactivation curve toward more hyperpolarized potentials. Two distinct depolarization-activated K + currents have been characterized in rat ventricular myocytes: a 4-AP-sensitive current that activates and inactivates rapidly (I t) and a TEA-sensitive current that activates ten times slower and inactivates thirty times slower than 1, (1 k) (Apkon and Nerbonne, 1991). We have
now found that caffeine inhibits both currents. In experiments on the efefects of caffeine on the total depolarization-activated outward current, the drug decreased both the peak amplitude and the sustained component of the current. This effect of caffeine was not accompanied by a shift in the steady-state inactiw> tion curve. In experiments in which I t and I k were dissected out pharmacologically, the blocking effect of caffeine was more pronounced on the slowly activating and inactivating current It; (32q4) than on 1~ (19~/c,). Most of the blockers of I t accelerate the rate of inactivation of the current in addition to decreasing its peak amplitude. Among these drugs are quinidine (lmaizumi and Giles, 1988), tedisamil (Dukes et al., 1991 ), bupiwtcaine (Castle, 1990), clofilium (Castle, 1991) and D601) (Lefevre et al., 1991). Caffeine did not modify either the rate of inactivation of or the recovery from inactivation of I t. The acceleration of the rate of inactivation with some drugs like quinidine (lmaizumi and Giles, 1987), bupivacaine (Castle, 1990) and clofilium (Castle, 1991) has been explained by a preferential binding of the drug to the open configuration of thc I~ channel, leading to an apparent acceleration of current inactiwttion (Castle, 1990, 1991). Depending on the unblocking rate and the interval between successive depolarizations, some block may persist, thus inducing a delay in the recovery from inactivation and use-dependent effects, Quinidine has been shown to produce a delay in the recovery from inactivation of I t in rabbit atrial ceils (lmiazumi and Giles, 1987) and clofilium has been shown to induce a delay in the recovery from inactiwltion and usc-dependent block of I t in rat vcntricular myocytes (Castle, 1991). The results obtained in thc prcsent work suggest thal caffeine should preferentially bind to the I t channel in the rested state configuration, producing only a tonic effect. The effect of caffeine on 1, and IK in rat ventricular myocytes secms to bc independent of voltagc-depcndent Ca 2÷ influx, since the experiments wcrc performed in thc presence of the Ca 2+ channel blocker, Co e+. Moreover, this effect of caffeine also seems to be independent from the internal Ca 2 ~ concentration, since the experiments were performed in the prcscncc of an internal solution with a high intraccllular capacity for buffering Ca 3~ ( E G T A 10 mM). Moreovcr, a high concentration (10 /a,M) of ryanodinc suppresses sarcoplasmic reticulum function in cardiac tissues by a mechanism different from that of caffeine (Sutko ct al., 1979; Nieman e t a [ . , 1985). Ryanodinc did not affect the depolarization-induced outward currents and the effects of caffeine on the outward currents were similar in cells pretreated with ryanodine. The possibility that caffeine could be decreasing I t and 1K by increasing cAMP via non-selective phosphodiesterase inhibition was tested indirectly. Wc tk)und that IBMX, another non-selective phosphodieslerase
169
inhibitor, did not modify either peak amplitude or sustained outward current. We can conclude from these results that caffeine can increase action potential duration in rat ventricular myocytes by mechanisms that do not involve an effect on sarcoplasmic reticulum. These mechanisms are: an increase in Ic~_L, mediated by phosphodiesterase inhibition, which results in an increase in cAMP level and a direct inhibition of the depolarization-induced outward currents, I t and IK.
Acknowledgements This work was supported by C O N A C y T (Mexico) Grant 0432M9108. The author wishes to thank Gusti Gould de Pineda for editorial assistance, Juan Fernando Fernandez for technical asistance and Alfredo Schulte for preparation of the figures.
References Apkon, M. and J.M. Nerbonne, 1991, Characterization of two distinct depolarization-activated K + currents in isolated adult rat ventricular myocytes, J. Gen. Physiol. 97, 973. Beavo, J.A., N.L. Rogers, O.P. Crofford, J.G. Hardman, E.W. Sutherland and E.V. Newman, 1970, Effects of xantine derivatives on lipolysis and on adenosine 3 ' 5 ' - m o n o p h o s p h a t e phosphodiesterase activity, Mol. Pharmacol. 6, 597. Blayney, L., H. Thomas, J. Muir and A. Henderson, 1978, Actions of caffeine on calcium transport by isolated fractions of myofibrils, mitochondria and sarcoplasmic reticulum from rabbit heart, Circ. Res. 43, 520. Bodem, R. and E.H. Sonnenblick, 1975, Mechanical activity of mammalian heart muscle: variable onset, species differences, and the effects of caffeine, Am. J. Physiol. 228, 250. Castle, N.A., 1990, Bupivacaine inhibits the transient outward K ~ current but not the inward rectifier in rat ventricular myocytes. J. Pharmacol. Exp. Ther. 255, 1038. Castle, N.A., 1991, Selective inhibition of potassium currents in rat ventricle by clofilium and its tertiary homolog, J. Pharmacol. Exp. Ther. 257, 342. C h a p m a n , R.A. and C. Leoty, 1976, The time-dependent and dosed e p e n d e n t effects of caffeine on the contraction of the ferret heart, J. Physiol. (London) 256, 287. Coraboeuf, E. and E. Carmeliet, 1982, Existence of two transient outward currents in sheep cardiac Purkinje fibers, Pfluegers Arch. 392, 352. Dukes, I.D., L. Cleemann and M. Morad, 1990, Tedisamil blocks the transient and delayed rectifier K + currents in mammalian cardiac and glial cells, J. Pbarmacol. Exp. Ther. 254, 560. Eisner, D.A., W. J. Lederer and D. Noble, 1979, Caffeine and tetracaine abolish the slow inward calcium current in sheep cardiac Purkinje fibres, J. Physiol. (London) 293, 76P (abstract). Escande, D., A. Coulombe, J-F. Faivre, E. Deroubaix and E. Coraboeuf, 1985, Two types of transient outward currents in adult h u m a n atrial cells, Am. J. Physiol. 252, H142. Giles, W.R. and Y. Imaizumi, 1988, Comparison of potassium currents in rabbit atrial and ventricular cells, J. Physiol. (London) 405, 123. Goto, M., A. Yatani and T. Ehara, 1979, Interaction between caffeine and adenosine on the m e m b r a n e current and tension component in the bullfrog atrial muscle, Jap. J. Physiol. 29, 393.
Habuchi, Y., H. Tanaka, T. Furukawa and Y. Tsujimura, 1991, Caffeine-induced block of Na + current in guinea pig ventricular cells, Am. J. Physiol. 261, H1855. Hamill, O.P., A. Marry, E. Neher, B. Sakmann and F.J. Sigworth, 198l, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfluegers Arch. 391, 85. Hiraoka, M. and S. Kawano, 1989, Calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes, J. Physiol. (London) 410, 187. Hughes, A.D., S. Hering and T.B. Bolton, 1990, The action of caffeine on inward barium current through voltage-dependen't calcium channels in single rabbit ear artery cells, Pfluegers Arch. 416, 462. Imaizumi, Y. and W.R. Giles, 1987, Quinidine-induced inhibition of transient outward current in cardiac muscle, Am. J. Physiol. 253, H704. Isenberg, G., 1978, The positive dynamic current of the cardiac Purkinje fiber is not a chloride but a potassium current, Pfluegers Arch. 377, R4 (abstract). Kenyon, J.L. and W.R. Gibbons, 1979a, Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers, J. Gen. Physiol. 73, 117. Kenyon, J.k. and W.R. Gibbons, 1979b, 4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers, J. Gen. Physiol. 73, 139. Kenyon, J.L. and J.L. Sutko, 1987, Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers, J. Gen. Physiol. 89, 921. Kimura, S., A.L. Basset, T. Furukawa, J. Cuevas and R.J. Myerburg, 1990, E[ectrophysiological properties in response to simulated ischemia in cat ventricular myocytes of endocardial and epicardial origin, Circ. Res. 66, 469. Konishi, M., S. Kurihara and T. Sakai, 1984, The effects of caffeine on tension development and intracellular calcium transients in rat ventricular muscle, J. Physiol. (London) 355, 605. Lefevre, I.A., A. Coulombe and E. Coraboeuf, 1991, The calcium antagonist D600 inhibits calcium-independent transient outward current in isolated rat ventricular myocytes, J. Physiol. (London) 432, 65. Mitra, R. and M. Morad, 1986, a uniform enzymatic method for the dissociation of myocytes from heart and stomach of vertebrates, Am. J. Physiol. 249, HI056. Nieman, D. and D.A. Eisner, 1985, Effects of caffeine, tetracaine and ryanodine on calcium-dependent oscillations in sheep cardiac Purkinje fibers, J. Gen. Physiol. 86, 877. Siegelbaum, S.A. and R.W. Tsien, 1980, Calcium-activated transient outward current in calf cardiac Purkinje fibers, J. Physiol. (London) 299, 485. Sutko, J.K., J.T. Willerson, G.H. Templeton, J.R. Jones and H.R. Besch Jr., 1979, Ryanodine: its alterations of cat papillary muscle contractile state and responsiveness to inotropic interventions and a suggested mechanism of action, J. Pharmacol. Exp. Ther. 209, 37. Tseng, G.-N. and B.F. Hoffman, 1989, Two components of transient outward current in canine ventricular myocytes, Circ. Res. 64, 633. Weber, A. and R. Herz, 1968, The relationship between caffeine contracture on intact muscle and effet of caffeine on reticulum, J. Gen. Physiol. 52, 750. Yatani, A., Y. Imoto and M. Goto, 1984, The effects of caffeine on the electrical properties of isolated, single rat ventricular cells, Jap. J. Physiol. 34, 337. Zygmunt, A.C. and W.R. Gibbons, 1991, Calcium-activated chloride current in rabbit ventricular myocytes, Circ. Res. 68, 424.
163
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52799
Caffeine inhibits depolarization-activated outward currents in rat ventricular myocytes Jose Sanchez-Chapula Centro de lnt~estigaciones Biomedicas de la Unit~ersidad de Colima, Apartado Postal 199, C.P. 28000, Colima, Col., Mdxico Received 1 September 1992, accepted 15 September 1992
The effects of caffeine (10 mM) on depolarization-activated, calcium-independent outward K + currents were investigated in isolated rat ventricular myocytes, using whole-cell clamping. The external solution contained CoC1z 2mM and the internal solution contained ethylene glycol-bis(-aminoethyl ether) N,N,N',N'-tetraacetic acid 10 mM. Caffeine decreased the peak amplitude of the total current and the sustained plateau current. Caffeine did not modify the steady state inactivation curve, which was fitted by two Boltzmann functions. Caffeine blocked the tetraethylammonium-sensitive slowly activating and inactivating outward current by 32% and the 4-aminopyridine-sensitive rapidly activating and inactivating transient outward current by 19%. Caffeine did not modify the inactivation rate or the time course of the recovery from inactivation of the transient current. Ryanodine 10 /zM did not modify any of the current components and the effect of caffeine was not modified by ryanodine pretreatment. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxantine 100 /zM, did not modify thc depolarization-activated calcium-independent outward currents. Myocytes (rat ventricular); Whole cell clamping; Caffeine; Depolarization-activated, CaZ+-independent outward currents
1. Introduction Caffeine exerts complex effects on the mechanical and electrophysiological properties of cardiac muscle. Caffeine influences the inotropic state of cardiac muscle at least partially by altering the uptake and release of Ca 2+ by the sarcoplasmic reticulum (Bodem and Sonnenblick, 1975; C h a p m a n and Leoty, 1976; Konishi et al., 1984). Caffeine has been found to affect different m e m b r a n e currents in myocardial cells. Various authors (except Kisner et al., 1979) have found that caffeine enhances the L-type Ca 2+ current in cardiac cells (Goto et al., 1979; Yatani et al., 1984). This effect has been attributed to an increase in intracellular adenosine 3'5'-cyclic monophosphate ( c A M P ) v i a suppression of phosphodiesterase activity (Beavo et al., 1970). Recently, Habuchi et al. (1991) have found that caffeine produces direct blocking effects on the sodium current (INa) in guinea-pig ventricular myocytes. This effect was independent of the internal calcium concentration or intracellular signaling system. Two transient outward currents have been identified in different cardiac preparations (Coraboeuf and
Correspondence to: J. Sanchez-Chapula, Apartado Postal 199, C.P. 28000, Colima, Col., M~xico. Tel. 52 (331) 40573.
Carmeliet, 1982; Escande et al., 1987; Giles and Imaizumi, 1988; Tseng and Hoffman, 1989; Kimura et al., 1990). One current (I t o r It01) is sensitive to 4aminopyridine (4-AP); the primary charge carrier appears to be K + (Isenberg, 1978; Kenyon and Gibbons, 1979a, b). This first current, It, is voltage-activated and is independent of intracellular calcium (Kenyon and Sutko, 1987). The second transient outward current (It02) is briefer and usually smaller than It0 p This second transient outward current is not sensitive to 4-AP and appears to be calcium-activated (Sigelbaum and Tsien, 1980; Coraboeuf and Carmeliet, 1982). The fact that It02 is sensitive to externally applied caffeine and raynodine at concentrations higher than 3 mM suggests that the transient outward current is activated by Ca 2+ released by the sarcoplasmic reticulum (Coraboeuf and Carmeliet, 1982; Escande et al., 1987; Tseng and Hoffman, 1989; Hiraoka and Kawano, 1989). Caffeine has been found to reduce to a large extent the systolic increase of Ca 2+ by inhibiting calcium accumulation in the sarcoplasmic reticulum isolated from skeletal (Weber and Herz, 1968) and cardiac muscle by making vesicle membranes more permeable to Ca 2+ (Blayney et al., 1978). The 4-AP-resistant Ca 2+activated transient outward current had been assumed to be carried by K + ions (Kenyon and Sutko, 1987). Zygmunt and Gibbons (1991), however, have recently
1~4 presented evidence that this current may be a CI current. Since caffeine has been widely used as a pharmacological tool to study different mechanical and electrophysiological properties of cardiac muscle, more information is needed about direct and indirect actions of this drug. In the present work. we report on the effects of caffeine on depolarization-induced calcium-independent outward currents of rat ventricular myocytcs, under experimental conditions that permitted examination of the direct actions of caffeine.
2. Materials and methods
2.1. Isolation of cardiac myoo'tes Cardiac myocytes were isolated according to an enzymatic procedure (Mitra and Morad, 1986). Briefly, rats weighing 200-300 g, were anesthetized with pentobarbital sodium (35 m g / k g ) . The hearts were rapidly excised and mounted via the aorta to a Langendorff perfusion system for retrograde coronary perfusion. The initial perfusion was nominally zero Ca 2* Tyrode solution. After 5 min of perfusion, the perfusate was switched to a zero Ca 2" solution containing collagenase (Sigma, type l, 2 m g / m l ) and protease (Sigma, tycp XIV, (I.2 m g / m l ) for 20 min. After 5 min of perfusion with a 0.1 mM CaCI, Tyrode solution, the heart was removed, the ventricle was dissected out and the cells were dispersed by gentle mechanical agitation.
2. 2. Solutions The Tyrode solution for cell isolation contained (mM): NaC1 112, NaHCO~ 24, KCI 5.4, CaCI 2 1.8, MgC12 1.0, N a H z P O 4 0.42, glucose 11, taurine 10 (pH 7.4). Nominally calcium-free Tyrode solution was prepared by omitting CaC12 from the solution. The 'control' external solution contained (in mM): NaCI 140, KCI 4, MgC12 1, CaCI~ 1.8, H E P E S 10. glucose 11 (pH 7.4 by NaOH). To measure total calcium-independent, depolarization-activated outward K + currents, the 'Ca2+-Co e~' external solution was used which contained (in mM): NaCI 140, KCI 4, MgCI: 1, CaCI: 0.1, H E P E S - N a 10, glucose 11, CoCI 2 2 (to suppress Ca 2 + currents), tetrodotoxin (TTX) 0.02 (to suppress Na + currents), pH 7.4, and was bubbled with 100% 0 2. The depolarization-induced outward K + current under these conditions showed that the activated outward current wave form results from the activation of two kinetically distinct K + currents, one that activates and inactivates rapidly and is sensitive to 4-AP (I~) and a second one that activates and inactivates slowly (IK). Tetraethylammonium (TEA) pre-
dominantly suppresses 1K (Apkon and Nerbonne. 1991 ). In order to study the effects of caffeine on l,, we used the T E A external solution composed as follows (in raM): NaCI 90, TEA-CI 50, KCI 4, MgCI: I, ('aCl~ 0.1, H E P E S - N a 10, CaCI~ 2, T T X 0.02 (pH 7.4). In some experiments, in order to study 1K with minimal interference of I t , we used the 'Ca-~+-Co e+" external solution plus 4-aminopyridinc 5 raM. The potassium internal solution used was (in mM): KCI 120, KftxPO4 IlL H E P E S 10, E G T A lt), ATP-Mg 3 (pH 7.2-7.3, by KOH). In experiments in which the L-type Ca 2~ current was studied, the external solution contained (in raM): TEA-CI 140, CsC1 4, MgCl 2 1, CaCI~ 1.8, H E P E S IlL glucose 11 (pH 7.4 by CsOH). The cesium internal solution was (in raM): CsCI t30, H E P E S 1(I, E G T A 10, ATP-Mg 3 (pH 7.2-7.3 by CsOH).
2.3. Electrophysiological recording4 Cells were transferred to a perfusion chamber (0.3 ml) mounted on the stage of an inverted microscope (Nikon Diaphot, Nikon Co.). The cells were pcrfuscd at a rate of 2 - 3 m l / m i n . The experiments were performed at room temperature (22-23°C). Macroscopic current recordings were obtained with the whole-cell voltage-clamp method as described by Hamill el al. (1981) using a patch-clamp amplifier ( A X O P A T C H l-C, Axon Instruments). Glass pipettes had tip resistances of 1-3 M,(,) when filled with internal solution. The resistance in series with the cell membrane was compensated to provide the fastest possibe capacity transient without ringing. Neither capacitivc current nor leakage current were compensated. Currents werc filtered with a four-pole Bessel filter at 5 kHz, digitized at 4 kHz and stored on an AST 286/16 computer ( p C L A M P software, Axon Instruments).
2.4. Data attalysis When possible, data are presented as arithmetic means + S.D. mean. The statistical significancc of differences in calculated mean values was evaluated using Student's t-test. Differences were considered significant when P values were < 0.05.
2.5. Drugs used Caffeine, tetrodotoxin (TTX) and 4-aminopyridine (4-AP) were dissolved in the external solution: 3-isobutyl-l-methyl-xantine (IBMX) (Sigma Chemical) was dissolved in dimethyl sulfoxide; ryanodine (S.B. Pennick) was added to the external solution from an aqueous stock solution.
165
3. Results In a first series of experiments we studied the effect of caffeine (10 mM) on action potentials in the presence of 'normal' external solution and potassium internal solution (fig. 1A). Caffeine increased the plateau level and prolonged the duration of the action potential. The duration measured at 50% repolarization (APDs,) was significantly increased, by 142 _+ 12%, from the control (n = 4). The effect of caffeine was completely reversed after 5 - 7 min of washout. The effect of caffeine on the L-type Ca -,+ current (lc,,_ L) was studied in four cells, under experimental conditions designed to record lc,,_L) with minimal interference of sodium and K + currents (see Materials and methods). From a holding potential (HP) of - 4 0 mV, depolarizing pulses of 500 ms duration were applied to potentials between - 3 0 and + 5 0 mV in steps of 10 inV. Records of currents induced by depolarizing pulses to +10, + 3 0 and + 5 0 mV are shown in fig. lB. Records made under control conditions are shown in (a) and 7 rain after the addition of caffeine in (b). The
A
\
.•J2omV 20ms B
Q
1
C
lOOms O control /x caffeine E'm (mV)
,,
90 0 /
(pA) 2000 1500 1000
~-e~-m-0~-~-~-m"l -50 - 5 0 _ 5 0 0
0
d.2:< l
I
I
30
50
looo O
-1500
Fig. 2. A. Effect of caffeine (10 # M ) on action potential in the presence of 'Ca2+-Co 2+' external solution. Stimulation frequency 0.1 Hz. Control conditions (a), in the presence of caffeine (b). B. Effect of caffeine on depolarization-activated outward currents. From a holding potential of - 7 0 mV pulses of 500 ms duration were applied to - 3 0 , - 1 0 , +10, + 3 0 and + 5 0 mV, under control conditions (a) and in the presence of caffeine (b). C. I-V curve of peak current for potentials between - 9 0 to + 50 mV under control conditions ( o ) and in the presence of caffeine ( ,5 ).
._•J20mV
20ms
B
b
a
lOOms C
I(pA)
'l
-50 -10 T 600 20 J,_ ,-, . e , , a ~ L'~.~, ~ _ _ [5~~ i~ . . . . .
k~ ~
.
40 f"j ~ .
50 L7 ]
o
0 [ 3 control ~ , ~ - 1 8 0 0 \ ~ /O/A A O caffeine x ~ " ~ o ~ J O / A ~
_58oo y ~ Fig. 1. A. Effect of caffeine (10 mM) on action potential in a rat ventricular cell. The cell was perfused with control external solution and the pipette was filled with potassium internal solution. Stimulation frequency 0.1 Hz. Control conditions (a), in the presence of caffeine (b). B. Effect of caffeine on Ic,_u. in a cell other than that in Fig. 1A. The external and internal solutions were designed lo decrease interference of Na + and K + currents (see Materials and methods section). From a holding potential (HP) and - 4 0 mV depolarizing pulses of 500 ms duration to + 10, + 3 0 and + 5 0 mV were applied under control conditions (a) and in the presence of caffeine 10 mM (b). C. I-V curves of the peak inward current (~ , zx ) and current measured at the end of the 500 ms pulse ([i], o), in the presence and absence of caffeine 10 raM.
drug increased the amplitude of Ic,_ L without affecting the current at the end of the 500 ms pulse. The I - V curves for peak inward current and current measured at the end of the pulse under control conditions and after addition of caffeine are shown in fig. 1C. Caffeine increased the amplitude of the peak inward current, but did not modify the current at the end of the pulse. These data confirm earlier results obtained by Yatani et al. (1984) under conditions that did not permit the internal buffering of Ca 2+ (single high-resistance microelectrode voltage-clamp). In another series of experiments, we studied the effect of caffeine (10 raM) on action potentials of cells perfused with a 'Ca 2+-Co 2 +, external solution to block Ic, ~ and a potassium internal solution. Action potentials under control conditions and after addition of caffeine are shown in fig. 2A. Under these conditions, with I(,~, blocked, caffeine reversibly increased action potential duration. In four cells, caffeine significantly increased APDs, by 48 +_ 7% from control conditions. This result shows that even after blockade of lc~,, caffeine can still increase action potential duration. This result could be explained by an increase in the inward current (IN,) or a decrease in the outward current. Recently, Habuchi et al. (1991) have found
166
that caffeine decreased INa, making it unlikely that the increase in action potential duration caused by caffeine is due to an increase in inward current. In order to explore the possibility that caffeine can modify calcium-independent outward currents, experiments were performed under voltage-clamp conditions. We used the C a 2 + - C o 2÷ external solution and the potassium internal solution. From a HP of - 70 mV, voltage-clamp steps to potentials between - 9 0 and + 5 0 mV were applied at a frequency of 0.1 Hz. Records of currents in the absence and presence of caffeine to - 3 0 , -1(}, -Fl0, + 3 0 and + 5 0 mV are shown in fig. 2B. The depolarization-evoked outward currents rose rapidly to a peak, then decayed slowly to an apparent plateau. Caffeine decreased outward peak current amplitude and the current at the end of the pulse without apparent changes in the time course of inactivation of the current. The I - V curve for peak current is shown in fig. 2C. Caffeine did not significantly alter the current for voltages negative to -2(1 mV. This results suggests that caffeine did not modify the inward rectifying K + current. Caffeine decreased the peak amplitude of outward currents for voltages positive to - 2 0 mV. In six experiments 10 mM caffeine decreased peak current amplitude measured at + 50 mV by 27 ± 6% from the control values. These effects of caffeine were completely reversed after 5 - 7 min of washout. The voltage dependence of steady-state inactivation of the peak current in rat ventricular cells can be fitted by the sum of two Boltzmann distributions (Apkon and Nerbonne, 19911. The interpretation of that result is that it reflects the steady state inactivation of two channel populations, most likely to be two distinct channel types (Apkon and Nerbonne, 1991). To examine the voltage dependence of steady state inactivation, peak currents evoked by test depolarizing pulses to + 5(1 mV from conditioning pulses of 10 s duration at potentials between 100 and 0 mV were measured. The steady state inactivation curves under control conditkms and in the presence of caffeine are shown in fig. 3, The data were fitted by using the sum of two Boltzmann functions, according to the expression: itV)
A,*{I/l+exp[(V
V, e ( , , ) / k i ] ) }
10 I : ~ : : ~ #
~
, EJ~::~ ~
08 t
-'~,
100
[2 CAFFEINE
80 -60 40 20 HOLDING POTENTIAL (n,V)
0
Fig. 3. Effect of caffeine on voltage dependence of steady state inactivation of the peak outward current. Outward currents were evoked during depolarizations to +50 mV after l0 s conditkming pulses to potentials between - 100 and 0 mV. The amplitudes of the peak currents were normalized to the amplitude of the currenl evoked from - 100 inV. The data were fitted using the sum of two Boltzmann distributions. (©) Under control conditions : (r_J) in the presence of caffeine l0 ,aM.
and were: A~ = (/.23 ± 0.4; V]/2(])= - 6 3 ± 2 mV; k~ = 14.1 ± 1.3 mV; A , = 0.76 ± 0.06; V~., zc_~= - 4 5 ± 3 mV; k, = 3.9 +_ 0.4 mV. In all the present experiments we used a high (10 mM) concentration of E G T A in the internal solution. However, it is possible that caffeine could producc its effects on the depolarization-induced outward currents by blocking an EGTA-resistant sarcoplasmic reticulum calcium release. Ryanodine is a potent alkaloid that also decreases sarcoplasmic reticulum release of Ca2 + during excitation-contraction coupling (Sutko et al., 1979). We then studied the effect of ryanodine at a high concentration (10 p,M) on the depolarization-induced outward currents. In fig. 4A we show records of
A
a
b
F'"-
"-t.
B a
XA 2 * { 1 / I + exp[(V
,:) CONTR,:)t
b
Vl. 2(21)/k2])}
where i is the normalized current as a function of the conditioning potential, V; A~ and A z are amplitude factors of both distributions: Vl/2(l t and V1/,(2 ) are the voltages at which 50% of each channel population is inactivated; k I and k 2 a r e the Boltzmann factors. Under control conditions we found that following value from four cells: A~ = (I.25 ± 0.03: Vl/2(l)= --65 ~ 3 mV; k ~ = 13.6± 1.1 mV; A , = 0 . 7 4 _ + 0 . 0 4 ; V~/e(e)= - 4 4 + _ 2 mV; k , - 4 . 1 +_0.5 mV. These values were not significantly changed, in the presence of caffeine
rt-
__ nA ' lOOms
Fig. 4. A. Effects of ryanodine (10 `aM) on depolarization-activated outward currents. From a holding potential of - 70 mV, depolarizing pulses to - 3 0 , -10, +10, +30 and +50 mV were applied under control conditions (a) and in the presence of ryanodine (b). 13. Effect of IBMX 100 ,aM on outward currents activated by depolarization. From a holding potential of 70 mV, depolarizing pulses to 3(1, - 10, + 10, + 3{) and + 50 mV were applied under control conditions (a) and in the presence of II3MX (bL
167
in the presence of 4-AP alone are shown in fig. 5Aa. Substantial suppression of the peak current is evident and only a slowly activating and very slowly inactivating current is apparent, termed I K by Apkon and Nerbonne (1991). Caffeine significantly decreased I K (fig. 5Ab). The I - V curve for the maximum outward current is shown in fig. 5Ac. Caffeine decreased the outward current at potentials positive to - 2 0 inV. Caffeine decreased I K measured at + 5 0 mV by 32 _+ 5% of the control values (n = 5). Records of currents induced by voltage steps to - 2 0 , 0, + 20 and + 40 mV, in the presence of T E A 50 mM are shown in fig. 5Ba. In the presence of TEA, the current wave forms show only a rapidly activating transient outward current (It) with substantial attenuation of the sustained component. Caffeine decreased the peak amplitude of the current at potentials positive to - 2 0 mV (fig. 5Bb, 5Bc). Caffeine decreased the peak amplitude of the current measured at + 50 mV by 19 _+ 5% of the control values (n = 5). The drug did not significantly modify the rate of inactivation of the current. Under control conditions, the rate of inactivation of the currents induced by a depolarizing pulse to + 50 mV was fitted by a monoexponential process with a time constant of 34 _+ 5 ms (n = 5). In the presence of caffeine, the time constant of inactivation was 36 _+ 6 ms (n = 5). The effect of caffeine on the time course of reactivation of I t was assessed at a HP of - 7 0 mV, using a standard paired pulse (100 ms of duration, to + 50 mV) protocol in the presence of a T E A external solution.
current induced by depolarizing pulses to - 3 0 , - 1 0 , +10, + 3 0 and + 5 0 mV from a HP of - 7 0 mV. Ryanodine did not modify the currents at any of the voltage studied. Similar results were found for two other cells. In five cells which were exposed to ryanodine for 30 rain before seal formation and internal dialysis, the response to caffeine (10 mM) was similar to that in cells without p r e t r e a t m e n t with ryanodine (data not shown). Caffeine has been found to increase the cAMP level by inhibiting phosphodiesterase activity (Beavo et al., 1970). In order to test the possibility that this mechanism of action could explain the effect of the drug on outward currents, we tested the effect of another phosphodiesterase inhibitor, I B M X 100 ~ M , on the depolarization-induced outward currents. Records of currents induced by depolarizing pulses to - 3 0 , - 1 0 , +10, + 3 0 and + 5 0 mV from a HP of - 7 0 mV under control conditions (a) and in the presence of IBMX (b) are shown in fig. 4B. I B M X did not modify either the peak amplitude or the sustained component at the end of the pulse of the outward currents. Similar results were found for two other cells. The K + channel blocker; 4-AP, preferentially blocks the fast transient outward K + current (It), and T E A preferentially blocks the sustained component of the current (1 K) (Apkon and Narbonne, 1991). In another series of experiments, we studied the effect of caffeine in the presence of 4-AP 5 raM. Records of currents induced by depolarization between - 3 0 and + 50 mV
B
A
a
a
b
r;
lnA
lOOms
50ms
900
0
3500
/ 0 4-AP /O A 4-AP + coffeine /O /0 /A / /O . /.z~" 0 A/A
600
300
~_0.~°~
0
o 2500 A
n
O~ A
TEA + coffeine v
1500
/ o J/A /
500
--
-3o
0 TEA
0
so
o
Em (mY)
60
-30
-lo
lo Em (mY)
30
50
Fig. 5. A. Effect of caffeine on depolarization-activated outward currents in a cell perfuscd with the 'Ca 2 "-Co 2+' external solution plus 4-AP (5 mM). Currents evoked by depolarizing pulses to - 3 0 , - 1 0 , + 10, +30 and +50 mV from a holding potential of - 7 0 mV, in the presence of 4-AP alone (a) and in the presence of 4-AP plus caffeine (b). (C) I - I V curve of maximum outward current for potential between - 30 and + 50 mV, under control conditions ( o ) and in the presence of caffeine (A). B. Effect of caffeine on depolarization-activated transient outward current in the presence of the TEA external solution. Currents evoked by depolarizing pulses to - 2 0 , 0, + 20 and +40 mV from a holding potential of - 70 mV, under control conditions (a) and in the presence of caffeine (b). I-V current of peak outward current for poentials between - 30 and + 50 mV, under control conditions ( o ) and in the presence of caffeine ( n ).
168
,oo 80,o .~ 60~--4020 O'--
0
,60
e6o
36o
4&0
~6o
T (ms) Fig. 6. Effect of caffeine on the recovery, from inactivation of I t. The cells were p e r f u s e d with T E A e x t e r n a l solution. From a h o l d i n g p o t e n t i a l of 70 mV, p a i r e d pulses of 100 ms d u r a t k m to + 5 0 mV were a p p l i e d cvery 30 s. The interval b e t w e e n the pulses was c h a n g e d from 20 to 500 ms. T h e n o r m a l i z e d current (lt t / l ~ < , w h e r e l i t is the t r a n s i e n t o u t w a r d c u r r e n l e v o k e d by the second or 'test' pulse, and I t~ is the t r a n s i e n t o u t w a r d current ew)ked by the first or 'conditioning" pulse) was p l o n e d against the interval b e t w e e n the pulses. The data were fitted by single e x p o n e n t i a l s with a lime constan! of 37 ms u n d e r control c o n d i t k m s C ) ) and 40 ms in the p r e s e n c e of caffeine ( ~ }.
Under control conditions the time course of the recovely from inactivation was fitted by an exponential process with a time constant of 39 ± 7 ms (fig. 6). Caffeine did not modify the time course of the recovery from inactivation of I t. The time constant obtained in the presence of the drug was 42 ± 6 ms (n = 5).
4. Discussion In the present work, we have found that caffeine inhibits the intracellular calcium-independent, depolarization-induced outward K + currents in rat ventricular myocytes reversibly. We also confirmed that caffeine increased I t > L in the presence of 10 mM of E G T A in the internal solution. Caffeine has numerous effects on the clectrophysiological and mechanical properties of the myocardium. Most of these effects result from caffeine actions on the sarcoplasmic reticulum (Konishi et al., 1984) and on phosphodiesterase (Beavo et al. 1970). However, Hughes et al. (1990) have found that caffeine inhibits directly I t , in rabbit vascular cells. Moreover, Habuchi et al. (1991) have found that caffeine also blocks IN:, directly and shifts the steady state inactivation curve toward more hyperpolarized potentials. Two distinct depolarization-activated K + currents have been characterized in rat ventricular myocytes: a 4-AP-sensitive current that activates and inactivates rapidly (I t) and a TEA-sensitive current that activates ten times slower and inactivates thirty times slower than 1, (1 k) (Apkon and Nerbonne, 1991). We have
now found that caffeine inhibits both currents. In experiments on the efefects of caffeine on the total depolarization-activated outward current, the drug decreased both the peak amplitude and the sustained component of the current. This effect of caffeine was not accompanied by a shift in the steady-state inactiw> tion curve. In experiments in which I t and I k were dissected out pharmacologically, the blocking effect of caffeine was more pronounced on the slowly activating and inactivating current It; (32q4) than on 1~ (19~/c,). Most of the blockers of I t accelerate the rate of inactivation of the current in addition to decreasing its peak amplitude. Among these drugs are quinidine (lmaizumi and Giles, 1988), tedisamil (Dukes et al., 1991 ), bupiwtcaine (Castle, 1990), clofilium (Castle, 1991) and D601) (Lefevre et al., 1991). Caffeine did not modify either the rate of inactivation of or the recovery from inactivation of I t. The acceleration of the rate of inactivation with some drugs like quinidine (lmaizumi and Giles, 1987), bupivacaine (Castle, 1990) and clofilium (Castle, 1991) has been explained by a preferential binding of the drug to the open configuration of thc I~ channel, leading to an apparent acceleration of current inactiwttion (Castle, 1990, 1991). Depending on the unblocking rate and the interval between successive depolarizations, some block may persist, thus inducing a delay in the recovery from inactivation and use-dependent effects, Quinidine has been shown to produce a delay in the recovery from inactivation of I t in rabbit atrial ceils (lmiazumi and Giles, 1987) and clofilium has been shown to induce a delay in the recovery from inactiwltion and usc-dependent block of I t in rat vcntricular myocytes (Castle, 1991). The results obtained in thc prcsent work suggest thal caffeine should preferentially bind to the I t channel in the rested state configuration, producing only a tonic effect. The effect of caffeine on 1, and IK in rat ventricular myocytes secms to bc independent of voltagc-depcndent Ca 2÷ influx, since the experiments wcrc performed in thc presence of the Ca 2+ channel blocker, Co e+. Moreover, this effect of caffeine also seems to be independent from the internal Ca 2 ~ concentration, since the experiments were performed in the prcscncc of an internal solution with a high intraccllular capacity for buffering Ca 3~ ( E G T A 10 mM). Moreovcr, a high concentration (10 /a,M) of ryanodinc suppresses sarcoplasmic reticulum function in cardiac tissues by a mechanism different from that of caffeine (Sutko ct al., 1979; Nieman e t a [ . , 1985). Ryanodinc did not affect the depolarization-induced outward currents and the effects of caffeine on the outward currents were similar in cells pretreated with ryanodine. The possibility that caffeine could be decreasing I t and 1K by increasing cAMP via non-selective phosphodiesterase inhibition was tested indirectly. Wc tk)und that IBMX, another non-selective phosphodieslerase
169
inhibitor, did not modify either peak amplitude or sustained outward current. We can conclude from these results that caffeine can increase action potential duration in rat ventricular myocytes by mechanisms that do not involve an effect on sarcoplasmic reticulum. These mechanisms are: an increase in Ic~_L, mediated by phosphodiesterase inhibition, which results in an increase in cAMP level and a direct inhibition of the depolarization-induced outward currents, I t and IK.
Acknowledgements This work was supported by C O N A C y T (Mexico) Grant 0432M9108. The author wishes to thank Gusti Gould de Pineda for editorial assistance, Juan Fernando Fernandez for technical asistance and Alfredo Schulte for preparation of the figures.
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