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Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: developmental changes and modulation by isoproterenol and carbachol
European Journal of Pharmacology, 240 (1993) 283-290 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
283
EJP 53249
Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: developmental changes and modulation by isoproterenol and carbachol Hiroyasu Satoh and Nicholas Sperelakis Department of Physiology and Biophysics, Unit~ersityof Cincinnati College of Medicine, Cincinnati, OH 45267, USA
Received 11 June 1993, accepted 15 June 1993
Modulation of the hyperpolarization-activated inward current (If) in embryonic chick ventricular myocytes was examined using whole-cell voltage-clamp. Long (3 s) hyperpolarizing pulses were applied from a holding potential of - 3 0 mV to steps of - 4 0 to - 120 mV. If was marked in 3-day-old cells, diminished at 10 days, and was almost completely gone at 17 days. If current density (at - 120 mV) was - 6 . 7 + 1.3 (3 days), -3.3 + 1.0 (10 days), and - 2 . 0 + 0.5 p A / p F (17 days). If reduction paralleled the decrease in spontaneous activity. In 3-day cells, the threshold potential was - 5 0 to - 6 0 mV, and the reversal potential was - 1 3 . 4 + 1.3 inV. The time course of activation was fitted by a single exponential and was temperature dependent: z was 1.3 + 0.4 s at 20°C and 0.7 _+0.4 s at 30°C (at - 120 mV). If amplitude was enhanced by 12.1 + 1.8% at 30°C compared with 20°C. Cs ÷ (3 mM) blocked If and had a negative chronotropic effect (rate decreased by 61%). Isoproterenol (1 ~zM) caused a positive chronotropic effect (17.1 _.+2.9%) and increased If by 65.2 + 5.6%. Carbachol (0.1 /xM) had a negative chronotropic effect (26.3 + 3.4%), and decreased If by 41.2 + 1.3%; it also reversed the enhancement produced by isoproterenol. Intracellular application of 100 ~M GTP-yS decreased basal If by 35.2 + 5.0%, but potentiated the stimulant effect of isoproterenol (by 37.8 + 4.7%) and the inhibitory effect of carbachol (21.2 + 4.3%). These results indicate that the If current may contribute to the pacemaker potential of young embryonic chick heart cells and decreases during development, fl-Adrenoceptor agonists stimulate If, whereas muscarinic cholinergic agonists inhibit If and reverse /3-adrenoceptor stimulation. G proteins may directly and indirectly couple autonomic receptors to If channels. Hyperpolarization-activated inward current; Pacemaker current; Automaticity; Myocytes (embryonic chick heart); Autonomic agents; Acetylcholine; Isoproterenol
1. Introduction
The hyperpolarization-activated inward current (If or I h) is one of the inward currents contributing to m e m b r a n e depolarization to threshold during diastole. Young embryonic chick ventricular myocytes exhibit spontaneous activity, which generates slow-rising action potentials (APs) from a maximum diastolic potential of around - 60 m V (Schanne et al., 1989). We have recently found that I f is present in embryonic myocytes and may be involved in generating spontaneous action potentials (Satoh and Sperelakis, 1991). The inward If current is Cs÷-sensitive, and is similar to the current which has been previously reported (DiFrancesco, 1985; Denyer and Brown, 1990).
Correspondence to: Nicholas Sperelakis, Department of Physiology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0576, USA. Tel. (513) 5585636, fax (513) 558-5738.
Some voltage-gated ionic channels are modulated by guanine nucleotide-binding (G) proteins (Brown, 1990). Automaticity of adult heart is modulated by autonomic receptors. Catecholamines accelerate heart rate, and acetylcholine slows it due to m e m b r a n e hyperpolarization and by other mechanisms that do not produce hyperpolarization (Hoffman and Cranefield, 1960; Shibata et al., 1985; DiFrancesco and Tromba, 1988). In young (3-5-day-old) embryonic chick heart, intracellular cAMP level is much higher than the c G M P level. During development, c G M P increases, whereas cAMP decreases (Thakkar and Sperelakis, 1987). The If channels in young embryonic chick heart might also be coupled to /3-adrenoceptor and muscarinic receptors through G proteins. In the present experiments, we investigated the characteristics of If, and compared its magnitude at different developmental stages in spontaneously beating embryonic chick heart cells (3-day, 10-day and 17-day). We examined whether If might contribute to
284 a u t o m a t i c i t y , a n d w h e t h e r t h e a u t o n o m i c r e c e p t o r s act through G proteins.
2. M a t e r i a l s a n d m e t h o d s
2.1. Cell culture preparation Single cell c u l t u r e s w e r e p r e p a r e d f r o m t h e l o w e r p a r t (ventricle) o f e m b r y o n i c chick h e a r t s by m e t h o d s similar to t h o s e d e s c r i b e d p r e v i o u s l y ( S a t o h a n d S p e r e lakis, 1991). T w e l v e d o z e n f e r t i l i z e d W h i t e L e g h o r n chick e m b r y o s w e r e i n c u b a t e d for 3, 10 a n d 17 days at 37.5°C a n d s t a g e d to c o n f i r m t h e i r d e g r e e o f d e v e l o p m e n t . H e a r t s w e r e r e m o v e d a n d c o l l e c t e d in a bala n c e d salt s o l u t i o n (4°C). A l t h o u g h it is difficult to d i s t i n g u i s h b e t w e e n the a t r i u m a n d t h e v e n t r i c l e in 3 - d a y - o l d h e a r t s , we u s e d t h e lower p a r t o f t h e t u b u l a r h e a r t s which, is p r i m a r i l y p r e s u m p t i v e ventricle. T h e h e a r t s w e r e m i n c e d with a scissor. T i s s u e dissociation was a c c o m p l i s h e d by g e n t l e r o t a t i o n o f t h e
tissues in a Mg2+-free a n d Ca2+-free R i n g e r s o l u t i o n c o n t a i n i n g 0 . 1 - 0 . 4 % trypsin (Sigma). T h e cell suspensions w e r e h a r v e s t e d at 5-min intervals, p o o l e d , a n d p e l l e t e d by c e n t r i f u g a t i o n (85 × g ) . T h e cells w e r e w a s h e d 3 t i m e s in tissue c u l t u r e m e d i u m (M199, G i b c o ) c o n t a i n i n g 10% fetal bovine serum. T h e cells w e r e p l a t e d on glass cover-slips p l a c e d in 35 m m plastic P e t r i d i s h e s ( F a l c o n ) at a c o n c e n t r a t i o n o f 105-106 c e l l s / m l . T h e myocyte c u l t u r e s w e r e m a i n t a i n e d at 37°C a n d p H 7.4 in a m o i s t - a i r C O 2 i n c u b a t o r until u s e d for e x p e r i m e n t a t i o n . T h e cells w e r e c u l t u r e d for 24-48 h before experimentation.
2.2. Whole-cell voltage clamp and current-clamp experimen ts W h o l e - c e l l v o l t a g e c l a m p r e c o r d i n g s w e r e m a d e using an A x o p a t c h p a t c h - c l a m p a m p l i f i e r ( A x o n Instrum e n t s ) a n d s t a n d a r d t e c h n i q u e s ( H a m i l l et al., 1981). P a t c h p i p e t t e s h a d resistances o f 3 - 5 MI2. E x p e r i m e n t s w e r e c a r r i e d o u t at r o o m t e m p e r a t u r e (20°C).
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Fig. 1. Existence oflthe hyperpolarization-activated inward current (If) in young embryonic chick ventricular cells. Test pulses were applied between - 4 0 and -120 mV, in 10 mV increments, from a holding potential (HP) of - 3 0 mV. A: Presence of If in a 3-day-old cell. Note the presence of the large inward current slowly activated by hyperpolarization. B: Smaller If in a 10-day-old cell. C: Absence of If in a 17-day-old cell D: Current/voltage relationships for If current density in the three developmental stages. Symbols used are: 3-day-old (open circles), 10-day-old (triangles) and 17-day-old cells (squares). The values are plotted as mean.+ S.E.M. The capacitances were 12.7_+2.0 pF (n = 17) in 3-d~y cells, 9.9-+ 2A pF (n = 14) in 10-day-old cells and 8.5 _+2.6 pF (n = 14) in 17-day-old cells. External solution included 3 mM BaCI 2, 10 ~M tetrodotoxin (q"rX) .and3 mM CdCI 2 to block inward rectifier K + current, fast Na + current and C a 2+ current. The short line at the left of the current records in A-C'represents the zero current level. E: Reversal potential for If in a 3-day-old cell. The If tail current reversed between - 2 0 and - 1 0 mV. Following a conditioning pulse of 3 s (to -120 mV) (from a HP of - 3 0 mV), the voltage was stepped between - 9 0 to + 10 mV, in increments of 10 inV. The dashed line indicates zero current level. External solution included 3 mM BaCI2, 10/~M TTX and 3 mM CdCI2. The experiments were performed at room temperature (20°C).
285
The amplitude of If was determined as the difference between initial baseline current and the current level at the end of the pulse. That is, the leak current at each voltage step was subtracted from the total current to give If. The current level should have approached close to steady state during each test potential of 3 s. The data were stored and analysed on an IBM-AT microcomputer using the P C L A M P analysis program (Axon Instruments). Current traces were filtered with a cut-off frequency of 1 kHz for plotting. All values are given as mean + S.E.M.
2.3. Experimental solutions The glass cover-slips containing the cells were placed in the bath-chamber and were superfused with a modified Tyrode solution. The composition of the modified Tyrode solution was (in mM): NaC1 137, KCI 5.4, CaCI 2 1.8, MgCI 2 1.0, N a H 2 P O 4 0.3, glucose 5.0, and H E P E S 5. The p H was adjusted to 7.4 with N a O H . To avoid interference from other currents, 3 m M BaC12, 10 F M tetrodotoxin and 3 m M CdCI 2 were added to the external Tyrode solution to block the fast Na ÷ current (tetrodotoxin), the Ca 2+ current (Cd2+), and the inward rectifier K ÷ current (Bae÷). l-Isoprenaline (Sigma), carbachol hydrochloride (Sigma), and guanosine 5'-O-(3-thiotriphosphate) ( G T P - y S ) (Sigma) were used. The pipette solution (intracellular) contained (in mM): K O H 110, KCI 20, MgCI z 2, E G T A 10, M g A T P 5, creatine phosphate 5, aspartic acid 100 and H E P E S 5 ( p H 7.2). The concentration of Ca z+ in the pipette solution was chosen to give a pCa of 7, according to the equation of Fabiato and Fabiato (1979), with the correction of Tsien and Rink (1980).
little or no If current was elicited in 17-day-old cells (fig. 1B and C). The c u r r e n t / v o l t a g e relationships for the three developmental stages are shown in fig. 1D. At - 120 mV, the current density for If was - 6 . 7 + 1.3 p A / p F (n = 23) in 3-day cells (open circles), -3.3_+ 1.0 p A / p F (n = 17) in 10-day cells (triangles), and - 2 . 0 + 0.5 p A / p F (n = 12) in 17-day ceils (squares). The average m e m b r a n e capacitances were 12.7 + 2.0 pF (n = 17) in 3-day cells, 10.9 + 2.1 pF (n = 14) in 10-day ceils, and 9.5 + 2.6 pF (n = 14) in 17-day cells. This indicates that the multiplying cells actually are smaller in size. The threshold potential (Vth) for If was - 5 0 to - 6 0 m V in 3-day cells, and the threshold shifted leftward to more negative potentials at the older ages (fig. 1D).
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To examine the hyperpolarization-activated inward If current at different developmental stages of embryonic chick heart cells, experiments using whole-cell voltage clamp were performed. Long duration (3 s) hyperpolarizing pulses were applied from a holding potential of - 3 0 mV; the test pulses ranged from - 4 0 to - 120 mV, in 10 mV increments. I e was activated by hyperpolarization, and it had a slow time course of activation. In 3-day cells, I e was observed consistently in all cells examined, and its variation in size was relatively small. As shown in fig. 1A, large If currents and associated tail currents were observed in a 3-day-old cell. I e current became smaller in 10-day-old cells, and
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3. Results
3.1. Changes in the hyperpolarization-actiuated current during det~elopment
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Fig. 2. Temperature dependen~ of If current in a 3-day-old emb~ onic chick cardiomyocyte. Test pulses were a~lied between -4~ and -120 mV, in 10 mV increments, from a HP of -30 m~ External solution included 3 mM BaCI~, 10 ~M ~ X and 3 mM CdCI~ A,A': Current traces at room temperature (20~C). B,B': Current traces at 30~C. C: Current-voltage cu~es for If at 20~C (circles) and 30~C(triangles). Comparison be~een time courses of I~ activation a~ 20~C (A') and 30~C (B') obtained at a test pulse to -120 mV, each from one trace fitted as a single exponential by PCLAMP/CLAMPFIT. At 20~C, the value of r was 1.2 s; at 30~C, r was reduced to 0.7 s.
286
The outward tail current was also reduced during development. For measuring the tail current in this experiment, at the end of the hyperpolarizing pulse (e.g., to - 120 mV), the voltage was stepped to various test potentials ( + 2 0 to - 9 0 mV), rather than to the holding potential ( - 30 mV). This was done to accentuate the tail currents, as described by DiFrancesco and Tromba (1988) and Hagiwara and Irisawa (1989). The average value of reversal potential (Vrev) was - 13.4 + 1.2 mV (n = 3) in 3-day cells (fig. 1D). However, this value would be affected by activation of any unblocked K + currents. The time constant of activation (~-) of If was fitted as a single exponential, and the mean value was 1.4 + 0.3 s (n = 7) in 3-day cells. These results indicate that young embryonic chick heart cells can produce a hyperpolarization-activated inward If current, and show that If becomes smaller or disappears with development.
mM) suppressed If in the 3-day-old chick cells, as previously reported (Satoh and Sperelakis, 1991). The outward tail current from - 1 2 0 to +20 mV was reduced from 84.4 to 45.7 pA by the Cs ÷ (n = 10). In the presence of Cs +, Vth for I f remained near - 50 to - 60 mW.
The If current in 10-day cells was also inhibited by 56.5 _+ 8.2% (n = 7) in the presence of Cs + (3 mM). In separate experiments, automaticity was abolished by Cs ÷ in two of three cells. In 17-day cells, If was inhibited almost completely by Cs ÷ (n = 5). In the present experiments on 18 spontaneously beating 3-day-old myocytes, the mean spontaneous firing rate was 62.3 ___2.4 beats/min, the maximum diastolic potential was - 5 7 . 3 ___2.1 mV, and the amplitude of the action potentials was 79.2 + 4.2 mV. Cs + produced a negative chronotropic effect (not illustrated) by - 3 8 . 9 + 6 . 2 % ( n = 5 ) at 0.3 mM, - 5 2 . 1 + 3 . 5 % (n = 5) at 1 mM, and - 6 8 . 7 + 4.1% (n = 10) at 3 mM Cs ÷. Cs + (3 mM) also depressed the amplitude of the spontaneous action potentials, and the maximum diastolic potential was lowered (depolarizing).
3.2. Effects of temperature on If current When the temperature of the bath solution was raised from room temperature (20°C) to 30°C, the If current had a faster time course of activation, as shown in fig. 2. The • value (at - 1 2 0 mV) was decreased from 1.3 + 0.4 s (n = 5; P < 0.05) to 0.7 +__0.4 s (n = 5; P < 0 . 0 5 ) . This corresponds to a Q10 of 1.86. The amplitude of If was only slightly enhanced (by 12.1 + 1.8% at - 120 mV; n = 5).
3.4. Modulation by isoproterenol and carbachol In 3-day-old cells, isoproterenol (1 /~M) caused a positive chronotropic effect by 17.1 + 2.9% (n = 5; P < 0.01). At - 1 2 0 mV, isoproterenol (fig. 3, filled circles) increased If by 65.2 + 5.6% (n = 7; P < 0.001) (fig. 3; table 1). r for If activation was reduced (but not significantly) to 1.0 + 0.2 s (n = 5). In 10-day cells, isoproterenol increased If by 37.2% (n = 3; P < 0.05) at - 1 2 0 mV. This agonist also restarted automaticity in Cs +-induced quiescent preparations. In 17-day cells, 1 /xM isoproterenol insignificantly increased If by only 8.2 +__2.5% (n = 8, P > 0.05),
3.3. Effects of Cs + on spontaneous action potentials The If current is known to be inhibited or abolished by Cs ÷ (Browr/ et al., 1981; Noma et al., 1983; DiFrancesco, 1985; Denyer and Brown, 1990). Cs ÷ (3
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Fig. 3. Effect of isoproterenol (ISO) on If current in a 3-day-old cell. A: Control If current in one cell. Test pulses were applied between - 4 0 and - 1 2 0 mV, in 10 mV increments, from a HP of - 3 0 mV. B: If in the presence of isoproterenol (1 /xM) for 10 min in the same cell. C: Current/voltage relationships for If with and without isoproterenol averaged for seven cells. Isoproterenol (filled circles) increased If at all potentials.
287 TABLE 1 Effects of isoproterenol, carbachol and GTP-TS on If of 3-day-old embryonic chick heart cells.
gles), to cells pretreated with 1 ~ M isoproterenol (filled circles), returned If to nearly the control value (at - 120 mV; n = 3 ) .
Numbers in parentheses represent the number of experiments. Single agent
+ GTP-TS
3.5. Effects of intracellular application of GTP-yS
Isoproterenol 1/~M +65.2_+5.6% e (7) +37.8_+4.7% b (9) a Carbachol 0 . 1 ~ M - 4 1 . 2 _ + 1 . 3 % c (7)-21.2_+4.3% (9) a GTP-yS 100 t~M -35.2_+5.0% b (17) a These values indicate further stimulation or inhibition beyond the altered new baseline (i.e., new 'control' values) indicated by the values given in the "first column ('single agent'), b p < 0.01; c p < 0.001, with respect to control value.
and ~- in 10-day cells was insignificantly changed from 2.2 + 2.1 s to 2.0 + 1.9 s (n = 3, P > 0.05). In 3-day cells, carbachol (0.1 /~M) had a negative chronotropic effect of 26.3_ 3.4% (n = 5), and produced an arrest in two of eight cells. The I f c u r r e n t was reduced by 41.2 + 1.3% (n = 7) at - 120 mV (fig. 4; table 1). Carbachol increased z to 1.5 + 0.4 s (n = 3) at - 1 2 0 mV. Carbachol was also effective in reversing the stimulatory effects of isoproterenol on I f. As shown in fig. 4D and E, addition of 0.1 /zM carbachol (trian-
A
To examine the coupling between G proteins and If channels, GTP-yS (nonhydrolysable GTP analogue) was added to the pipette solution (fig. 5). After the patch membrane was opened, If became stable at about 1 rain (Control, open circles). With GTP-yS in the pipette, GTP-yS (filled circles) diffused into the cell, and its effect reached steady state within 4 min, namely there was inhibition of If. With pretreatment by 1 /zM isoproterenol (triangles), If increased with diffusion of GTP-yS into the cell. In contrast, with 0.1 /~M carbachol pretreatment (squares), If decreased with diffusion of GTP-yS into the cell (fig. 5). Table 1 summarizes these data. GTP-yS alone depressed If by 35.2 +5.0% (n = 17). GTP-yS 100 ~zM further increased If by 37.8 + 4.7% (n = 9) beyond that produced by the 1 /zM isoproterenol pretreatment (table 1). In contrast, If was further decreased by 21.2 + 4.3% (n =
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2 Fig. 4. Effect of carbachol ( A - D ) and carbachol plus isoproterenol (E-F) on 3-day-old embryonic chick heart myocytes. A: Control If current in one cell. Test pulses were applied between - 4 0 and - 120 mV, in 10 mV increments, from a HP of - 3 0 mV. B: In the presence of carbachol (5 min) in same cell. C: Current/voltage relationships for If with and without carbachol averaged for three cells. Carbachol 0.1/xM (filled circles) decreased If at all voltages. D-E: Interaction of isoproterenol and carbachol on If current. D: Superimposed If current traces at - 120 mV, from HP of - 3 0 mV, under control condition (open circles), in the presence of 1/.tM isoproterenol (filled circles) and in the presence of 0.1/~M carbachol plus isoproterenol (triangles). E: Current/voltage relationships for If in control (open circles), 1 g M isoproterenol (filled circles) and 0.1 ~,M carbachol plus isoproterenol (1/~M) (triangles). External solution included 3 mM BaCI2, 10 ~ M "I~X and 3 mM CdCI2.
288
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Fig. 5. Effeets of GTP-yS on |~ current in the presence of isoproterenol and carbachol. Percentage cha~ge in =~ £n a ~-da~-old cell a~ a ~unction of time. ~est pulses were applied to - 120 mV from the HP of -30 mV. Control (open circles), GTP-yS 100/zM (filled circles), GTP-yS+I p~M isoproterenol (triangles) and GTP-TS+0.1 /xM carbachol (Carb) (squares). GTP-7S (100 ~M) was added into the pipette solution. External solution included 3 mM BaCI2, 10 ~M tetrodotoxin and 3 mM CdCI2.
9) beyond that produced by the 0.1 /xM carbachol pretreatment (table 1).
4. D i s c u s s i o n
4.1. Properties and developmental changes in the hyperpolarization-activated inward current In the present experiments, 3-day-old cells, from the presumptive ventricular part of the heart, exhibited spontaneous action potentials. An If current was activated in a time- and voltage-dependent manner. If required long hyperpolarizing steps of 3 s or more to reach maximum, and the -r value was 1.3 s (at - 1 2 0 mV) at 20°C. The If current was temperature sensitive. At 30°C, the r of activation became much faster (Q10 = 1.86), and the If amplitude was slightly enhanced. With development, the If pacemaker current was greatly reduced, accompanied by the disappearance of spontaneous activity in 17-day-old cells. Vth and Vrev were about - 5 0 to - 6 0 mV and about - 1 3 mV, respectively (see figs. 1C and 3C). In mammalian sino-atrial (SA) node cells and Purkinje fibers, the hyperpolarization-activated inward If current has been demonstrated in multicellular and in single-cell preparations (Brown et al., 1979; Yanagihara and Irisawa, 1980; Yanagihara et al., 1980; DiFrancesco et al., 1986; Satoh and Seyama, 1986; Hagiwara and Irisawa, 1989). Shrier and Clay (1981a,b)
showed that aggregates of embryonic chick heart ceils possessed Iv~ or If, and that this current was decreased in older ceils. In single SA node cells, Vth for If ranged between - 4 0 to - 7 0 mV (Nakayama et al., 1984; DiFrancesco et al., 1986; Satoh and Seyama, 1986), and Vrev and ~" were about - 1 5 mV and 1.3 s (Hagiwara and Irisawa, 1989). In multicellular Purkinje fibers, Vth was - 5 0 to - 6 0 mV (DiFrancesco and Ojeda, 1980). Therefore, the properties of the I f current found in young embryonic chick heart cells are the same as those found in other tissues. If is sensitive to Cs ÷, is time- and voltage-dependent, and is activated at almost the same threshold potential ( - 5 0 to - 6 0 mV). Since If became smaller and disappeared during development, it may play a role in generation of the pacemaker depolarization of young embryonic chick heart myocytes.
4.2. Contribution of I~ to the pacemaker current In cardiac Purkinje fibers, If plays an important role in the pacemaker potential (DiFrancesco and Ojeda, 1980; Noble, 1984). This is possible because the maximum diastolic potential is more negative ( - 90 to - 100 mV), thus allowing I f to be fully activated. In SA node cells, there are at least three currents known to contribute to the pacemaker potential (Noble, 1984): (a) Isi (Yanagihara and Irisawa, 1980; Yanagihara et al., 1980), (b) IK(ael) decay (Brown, 1982; Shibata and Giles, 1983), and (c) If (Maylie et al., 1981). However, | f may not play an important role in the cycle-to-cycle pacemaker potential in SA nodal cells, because (a) the time course of activation of If is too slow to account for the high frequency of the primary pacemaker, and (b) the threshold potential of activation ( - 5 0 to - 6 0 mV) is close to the maximum diastolic potential ( - 60 to - 7 0 mV) (Noma et al., 1983; Yanagihara and Irisawa, 1980; Yanagihara et al., 1980). Therefore, I f activation may make only a minor contribution to the pacemaker potential in SA node. In the present experiments, Cs ÷ blocked If, but did not completely inhibit the spontaneous action potentials (in 16 of 18 cells). Since Vth was -- 50 to -- 60 mV and the maximum diastolic potential was less than - 6 0 m V , I f may contribute to the pacemaker potential in the 3-day-old cells to a small degree. The spontaneous activity in the young embryonic chick heart could also involve other currents (e.g., I c a and IK).
4.3. Involvement of G proteins on I/ channels Isoproterenol stimulates If through stimulatory Gprotein (Gs)-mediated activation of adenylate cyclase, whereas acetylcholine inhibits I f through inhibitory G-protein (Gi)-mediated activation of guanylate cy-
289
clase (Gilman, 1987; Brown, 1990). In rabbit SA node myocytes, acetylcholine inhibits If and slows spontaneous rate at low concentrations that do not activate acetylcholine-dependent K ÷ current (IKtACh)) (DiFrancesco et al., 1989). In young (3- to 5-day-old) embryonic chick hearts, cAMP level is very high, whereas cGMP level is very low (Thakkar and Sperelakis, 1987); isoproterenol increased cAMP and acetylchoh'ne increased cGMP. With development, the change in cGMP is reciprocal to that of cAMP, namely, cAMP decreases and cGMP increases. In the present experiments, isoproterenol increased If and shifted the I / V curve in the positive direction, whereas carbachol inhibited If and shifted the I / V curve negatively (in two of eight cells, carbachol caused complete arrest). Carbachol depressed If even in the presence of isoproterenol. These results are consistent with those of DiFrancesco and Tromba (1988), who showed that acetylcholine shifted the If activation curve to more negative potentials and with the results of Chang and Cohen (1992) who showed that acetylcholine could reverse the positive voltage shift produced by fl-adrenoceptor agonists. Therefore, these effects could be due to the activation of G i and reversal of the isoproterenol-induced elevation of cAMP (Rodbell, 1980; Thakkar and Sperelakis, 1987). Acetylcholine also reduces Ica (Giles and Noble, 1976; Josephson and Sperelakis, 1982) and activates IK(ACh) by GK-protein gating of the ion channels (Pappano, 1977; Noma, 1986). The present results suggest that young embryonic cells have high sensitivity to muscarinic receptors, even though the 3-day-old embryonic chick heart is not yet innervated by parasympathetic nerves (Pappano, 1977). Acetylcholine can act directly on the heart muscle prior to the appearence of cholinergic innervation (Cullis and Lucas, 1936). Although sensitivity to acetylcholine is low in young hearts, the sensitivity to acetylcholine was found to increase with time in organ culture (Dong et al., 1986; Hatae et al., 1989). Since the 3-day cells were cultured for 1-2 days before being used for the present experiments, acetylcholine sensitivity may have developed during culture. Effects of isoproterenol and carbachol on If were potentiated further by GTP-yS. It is likely that G-protein pathways connect both muscarinic receptors and fl-adrenoceptors to If channels in embryonic chick heart cells. When intracellular application of GTP-yS was done in the absence of any agonists, If current fell somewhat, perhaps due to GTP-yS stimulation of both G proteins (G s and Gi), with G i predominating. Gilman (1987) reported that the a subunit can act on If directly or indirectly by way of adenylate cyclase, and the fly subunit may be inhibitory to the a subunit effects on adenylate cyclase. These results indicate that G proteins inhibitory to If were dominant over stimulatory G proteins in young embryonic chick heart. The
known dominance of the vagal effect may also be reflected by the dominance of the inhibitory G proteins activated by GTP-yS. Direct G-protein coupling between autonomic receptors and If channels may partly account for the ability of the autonomic nervous system to produce its effects within a single heartbeat, and an indirect coupling via second messengers can account for the more persistent effects.
Acknowledgements This study was supported by National Heart, Lung, and Blood Institute Grant HL-31942. We are grateful for Lisa WukuSick-Neumier for preparation of the cell cultures, to Anthony Sperelakis and Glenn Doerman for help with the figures, and to Rhonda'Hentz for the word processing.
References Brown, A.M., 1990, Regulation of heartbeat by G pro,tein coupled ion channels, Am. J. Physiol. 259, H1621. " Brown, H.F., D. DiFrancesco and S.J. Noble, 1979, How does adrenaline accelerate the heart, Nature 280, 235. ' Brown, H.F., D. DiFrancesco, J. Kimura, and S. Noble, 1981, Ce ~ sium: a useful tool for investigating sino-atrial (S-A) node's pacemaking, J. Physiol. 317, 54P. Brown, H.F., 1982, Electrophysiology of the sinoatrial node, Physiol. Rev. 62, 505. Chang, F. and I.S. Cohen, 1992, Mechanism of acetylcholine action on pacemaker current (i~t~) in canine Purkinje fibers, Pfliig. Arch. 420, 389. Cullis, W.C. and C.L.T. Lucas, 1936, Action of acetylcholine on the aneural chick heart, J. Physiol. 86, 53. Denyer, J.C. and H.F. Brown, 1990, Pacemaking in rabbit sino-atrial node cells during Cs ÷ block of the hyperpolarization-activated current If, J. Physiol. 429, 401. DiFrancesco, D., 1985, The cardiac hyperpolarizing-activated current, if. Origins and developments, Prog. Biophys. Mol. Biol. 46, 163. DiFrancesco, D. and C. Ojeda, 1980, Properties of the pacemaker current if in the sinoatrial node of the rabbit: a comparison with the current iK2 in Purkinje fibres, J. Physiol. 308, 331. DiFrancesco, D. and C. Tromba, 1988, Inhibition of the hyperpolarization-activated current (if) induced by acetylcholine in rabbit sino-atrial node myocytes, J. Physiol. 405, 477. DiFrancesco, D., A. Ferroni, M. Mazzanti and C. Tromba, 1986, Properties of the hyperpolarizing-activated current (If) in cells isolated from the rabbit sino-atrial node, J. Physiol. 337, 61. DiFrancesco, D., P. Ducouret and R.B. Robinson, 1989, Muscarinic modulation of cardiac rate at low acetylcholine concentrations, Science 243, 669. Dong, L., N. Sperelakis and G.M. Wahler, 1986, Development of physiological responses to acetylcholine during organ culture of young embryonic chick hearts, J. Dev. Physiol. 8, 307. Fabiato, A. and F. Fabiato, 1979, Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells, J. Physiol. 75, 463. Giles, W. and D. Noble, 1976, Changes in membrane currents in bullfrog atrium produced by acetylcholine, J. Physiol. 261, 103. Gilman, A.G., 1987, G proteins: transducers of receptor-generated signals, Annu. Rev. Biochem. 56, 615.
290 Hagiwara, N. and H. Irisawa, 1989, Modulation by intracellular Ca 2+ of the hyperpolarization-activated inward current in rabbit single sino-atrial node cells, J. Physiol. 409, 121. Hamill, O.P., A. Marty, E. Neher, B. Sakmann and J. Sigworth, 1981, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliig. Arch. 391, 85. Hatae, J., N. Sperelakis and G.M. Wahler, 1989, Development of the response to adenosine during organ culture of young embryonic chick hearts, J. Dev. Physiol. 11,342. Hoffman, B.F. and P.F. Cranefield, 1960, Electrophysiology of the Heart (McGraw-Hill, New York) p. 1. Josephson, I. and N. Sperelakis, 1982, On the ionic mechanism underlying adrenergic antagonism in ventricular muscle, J. Gen. Physiol. 79, 69. Maylie, J., M. Morad and J. Weiss, 1981, A study of pace-maker potential in the rabbit sino-atrial node. Measurement of potassium activity under voltage-clamp conditions, J. Physiol. 311, 161. Nakayama, T., Y. Kurachi, A. Noma and H. Irisawa, 1984, Action potential and membrane currents of single pacemaker cells of the rabbit heart, Pfliig. Arch. 402, 248. Noble, D., 1984, The surprising heart: a review of recent progress in cardiac electrophysiology, J. Physiol. 353, 1. Noma, A., 1986, GTP-binding proteins couple cardiac muscarinic receptors to potassium channels, Trends Neurosci. 9, 142. Noma, A., M. Morad and H. Irisawa, 1983, Does the pacemaker current generate the diastolic depolarization in rabbit SA node cells, Pfliig. Arch. 397, 190. Pappano, A.J., 1977, Ontogenetic development of autonomic neuroeffector transmission and transmitter reactivity in embryonic and fetal hearts, Pharmacol. Rev. 29, 3. Rodbell, M., 1980, The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature 284, 17.
Satoh, H. and I. Seyama, 1986, On the mechanism by which changes in extracellular pH affect the electrical activity of the sino-atrial node, J. Physiol. 381, 181. Satoh, H. and N. Sperelakis, 1991, Identification of hyperpolarization-activated inward current in young embryonic chick heart myocytes, J. Dev. Physiol. 15, 247. Schanne, O.F., L. Boutin and J. Derosiers, 1989, Effects of K-channel blockers, calcium and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells, Can. J. Physiol. Pharmacol. 67, 795. Shibata, E.F. and W.R. Giles, 1983, Ionic currents in isolated cardiac pacemaker cells from bullfrog sinus venosus, Proc. Int. Union Physiol. Sci. 15, 76. Shibata, E.F., W. Giles and G.H. Pollack, 1985, Threshold effects of acetylcholine on primary pacemaker cells of the rabbit sino-atrial node, Proc. R. Soc. London Ser. B 223, 355. Shrier, A. and J.R. Clay, 1981a, Analysis of subthreshold pace-maker currents in chick embryonic heart cells, J. Physiol. 312, 371. Shrier, A. and J.R. Clay, 1981b, Developmental changes in subthreshold pace-maker currents in chick embryonic heart cells, J. Physiol. 312, 491. Thakkar, J.K. and N. Sperelakis, 1987, Changes in cyclic nucleotide levels during embryonic development of chick hearts, J. Dev. Physiol. 9, 497. Tsien, R.Y. and T.J. Rink, 1980, Neutral carrier ion-selective microelectrodes for measurements of intracellular free calcium, Biochim. Biophys. Acta 599, 623. Yanagihara, K. and H. Irisawa, 1980, Inward current activated during hyperpolarization in rabbit sinoatrial node cell, Pfliig. Arch. 358, 11. Yanagihara, K., A. Noma and H. Irisawa, 1980, Reconstruction of sinoatrial node pacemaker potential based on the voltage clamp experiments, Jpn. J. Physiol. 30, 841.
283
EJP 53249
Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: developmental changes and modulation by isoproterenol and carbachol Hiroyasu Satoh and Nicholas Sperelakis Department of Physiology and Biophysics, Unit~ersityof Cincinnati College of Medicine, Cincinnati, OH 45267, USA
Received 11 June 1993, accepted 15 June 1993
Modulation of the hyperpolarization-activated inward current (If) in embryonic chick ventricular myocytes was examined using whole-cell voltage-clamp. Long (3 s) hyperpolarizing pulses were applied from a holding potential of - 3 0 mV to steps of - 4 0 to - 120 mV. If was marked in 3-day-old cells, diminished at 10 days, and was almost completely gone at 17 days. If current density (at - 120 mV) was - 6 . 7 + 1.3 (3 days), -3.3 + 1.0 (10 days), and - 2 . 0 + 0.5 p A / p F (17 days). If reduction paralleled the decrease in spontaneous activity. In 3-day cells, the threshold potential was - 5 0 to - 6 0 mV, and the reversal potential was - 1 3 . 4 + 1.3 inV. The time course of activation was fitted by a single exponential and was temperature dependent: z was 1.3 + 0.4 s at 20°C and 0.7 _+0.4 s at 30°C (at - 120 mV). If amplitude was enhanced by 12.1 + 1.8% at 30°C compared with 20°C. Cs ÷ (3 mM) blocked If and had a negative chronotropic effect (rate decreased by 61%). Isoproterenol (1 ~zM) caused a positive chronotropic effect (17.1 _.+2.9%) and increased If by 65.2 + 5.6%. Carbachol (0.1 /xM) had a negative chronotropic effect (26.3 + 3.4%), and decreased If by 41.2 + 1.3%; it also reversed the enhancement produced by isoproterenol. Intracellular application of 100 ~M GTP-yS decreased basal If by 35.2 + 5.0%, but potentiated the stimulant effect of isoproterenol (by 37.8 + 4.7%) and the inhibitory effect of carbachol (21.2 + 4.3%). These results indicate that the If current may contribute to the pacemaker potential of young embryonic chick heart cells and decreases during development, fl-Adrenoceptor agonists stimulate If, whereas muscarinic cholinergic agonists inhibit If and reverse /3-adrenoceptor stimulation. G proteins may directly and indirectly couple autonomic receptors to If channels. Hyperpolarization-activated inward current; Pacemaker current; Automaticity; Myocytes (embryonic chick heart); Autonomic agents; Acetylcholine; Isoproterenol
1. Introduction
The hyperpolarization-activated inward current (If or I h) is one of the inward currents contributing to m e m b r a n e depolarization to threshold during diastole. Young embryonic chick ventricular myocytes exhibit spontaneous activity, which generates slow-rising action potentials (APs) from a maximum diastolic potential of around - 60 m V (Schanne et al., 1989). We have recently found that I f is present in embryonic myocytes and may be involved in generating spontaneous action potentials (Satoh and Sperelakis, 1991). The inward If current is Cs÷-sensitive, and is similar to the current which has been previously reported (DiFrancesco, 1985; Denyer and Brown, 1990).
Correspondence to: Nicholas Sperelakis, Department of Physiology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0576, USA. Tel. (513) 5585636, fax (513) 558-5738.
Some voltage-gated ionic channels are modulated by guanine nucleotide-binding (G) proteins (Brown, 1990). Automaticity of adult heart is modulated by autonomic receptors. Catecholamines accelerate heart rate, and acetylcholine slows it due to m e m b r a n e hyperpolarization and by other mechanisms that do not produce hyperpolarization (Hoffman and Cranefield, 1960; Shibata et al., 1985; DiFrancesco and Tromba, 1988). In young (3-5-day-old) embryonic chick heart, intracellular cAMP level is much higher than the c G M P level. During development, c G M P increases, whereas cAMP decreases (Thakkar and Sperelakis, 1987). The If channels in young embryonic chick heart might also be coupled to /3-adrenoceptor and muscarinic receptors through G proteins. In the present experiments, we investigated the characteristics of If, and compared its magnitude at different developmental stages in spontaneously beating embryonic chick heart cells (3-day, 10-day and 17-day). We examined whether If might contribute to
284 a u t o m a t i c i t y , a n d w h e t h e r t h e a u t o n o m i c r e c e p t o r s act through G proteins.
2. M a t e r i a l s a n d m e t h o d s
2.1. Cell culture preparation Single cell c u l t u r e s w e r e p r e p a r e d f r o m t h e l o w e r p a r t (ventricle) o f e m b r y o n i c chick h e a r t s by m e t h o d s similar to t h o s e d e s c r i b e d p r e v i o u s l y ( S a t o h a n d S p e r e lakis, 1991). T w e l v e d o z e n f e r t i l i z e d W h i t e L e g h o r n chick e m b r y o s w e r e i n c u b a t e d for 3, 10 a n d 17 days at 37.5°C a n d s t a g e d to c o n f i r m t h e i r d e g r e e o f d e v e l o p m e n t . H e a r t s w e r e r e m o v e d a n d c o l l e c t e d in a bala n c e d salt s o l u t i o n (4°C). A l t h o u g h it is difficult to d i s t i n g u i s h b e t w e e n the a t r i u m a n d t h e v e n t r i c l e in 3 - d a y - o l d h e a r t s , we u s e d t h e lower p a r t o f t h e t u b u l a r h e a r t s which, is p r i m a r i l y p r e s u m p t i v e ventricle. T h e h e a r t s w e r e m i n c e d with a scissor. T i s s u e dissociation was a c c o m p l i s h e d by g e n t l e r o t a t i o n o f t h e
tissues in a Mg2+-free a n d Ca2+-free R i n g e r s o l u t i o n c o n t a i n i n g 0 . 1 - 0 . 4 % trypsin (Sigma). T h e cell suspensions w e r e h a r v e s t e d at 5-min intervals, p o o l e d , a n d p e l l e t e d by c e n t r i f u g a t i o n (85 × g ) . T h e cells w e r e w a s h e d 3 t i m e s in tissue c u l t u r e m e d i u m (M199, G i b c o ) c o n t a i n i n g 10% fetal bovine serum. T h e cells w e r e p l a t e d on glass cover-slips p l a c e d in 35 m m plastic P e t r i d i s h e s ( F a l c o n ) at a c o n c e n t r a t i o n o f 105-106 c e l l s / m l . T h e myocyte c u l t u r e s w e r e m a i n t a i n e d at 37°C a n d p H 7.4 in a m o i s t - a i r C O 2 i n c u b a t o r until u s e d for e x p e r i m e n t a t i o n . T h e cells w e r e c u l t u r e d for 24-48 h before experimentation.
2.2. Whole-cell voltage clamp and current-clamp experimen ts W h o l e - c e l l v o l t a g e c l a m p r e c o r d i n g s w e r e m a d e using an A x o p a t c h p a t c h - c l a m p a m p l i f i e r ( A x o n Instrum e n t s ) a n d s t a n d a r d t e c h n i q u e s ( H a m i l l et al., 1981). P a t c h p i p e t t e s h a d resistances o f 3 - 5 MI2. E x p e r i m e n t s w e r e c a r r i e d o u t at r o o m t e m p e r a t u r e (20°C).
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Fig. 1. Existence oflthe hyperpolarization-activated inward current (If) in young embryonic chick ventricular cells. Test pulses were applied between - 4 0 and -120 mV, in 10 mV increments, from a holding potential (HP) of - 3 0 mV. A: Presence of If in a 3-day-old cell. Note the presence of the large inward current slowly activated by hyperpolarization. B: Smaller If in a 10-day-old cell. C: Absence of If in a 17-day-old cell D: Current/voltage relationships for If current density in the three developmental stages. Symbols used are: 3-day-old (open circles), 10-day-old (triangles) and 17-day-old cells (squares). The values are plotted as mean.+ S.E.M. The capacitances were 12.7_+2.0 pF (n = 17) in 3-d~y cells, 9.9-+ 2A pF (n = 14) in 10-day-old cells and 8.5 _+2.6 pF (n = 14) in 17-day-old cells. External solution included 3 mM BaCI 2, 10 ~M tetrodotoxin (q"rX) .and3 mM CdCI 2 to block inward rectifier K + current, fast Na + current and C a 2+ current. The short line at the left of the current records in A-C'represents the zero current level. E: Reversal potential for If in a 3-day-old cell. The If tail current reversed between - 2 0 and - 1 0 mV. Following a conditioning pulse of 3 s (to -120 mV) (from a HP of - 3 0 mV), the voltage was stepped between - 9 0 to + 10 mV, in increments of 10 inV. The dashed line indicates zero current level. External solution included 3 mM BaCI2, 10/~M TTX and 3 mM CdCI2. The experiments were performed at room temperature (20°C).
285
The amplitude of If was determined as the difference between initial baseline current and the current level at the end of the pulse. That is, the leak current at each voltage step was subtracted from the total current to give If. The current level should have approached close to steady state during each test potential of 3 s. The data were stored and analysed on an IBM-AT microcomputer using the P C L A M P analysis program (Axon Instruments). Current traces were filtered with a cut-off frequency of 1 kHz for plotting. All values are given as mean + S.E.M.
2.3. Experimental solutions The glass cover-slips containing the cells were placed in the bath-chamber and were superfused with a modified Tyrode solution. The composition of the modified Tyrode solution was (in mM): NaC1 137, KCI 5.4, CaCI 2 1.8, MgCI 2 1.0, N a H 2 P O 4 0.3, glucose 5.0, and H E P E S 5. The p H was adjusted to 7.4 with N a O H . To avoid interference from other currents, 3 m M BaC12, 10 F M tetrodotoxin and 3 m M CdCI 2 were added to the external Tyrode solution to block the fast Na ÷ current (tetrodotoxin), the Ca 2+ current (Cd2+), and the inward rectifier K ÷ current (Bae÷). l-Isoprenaline (Sigma), carbachol hydrochloride (Sigma), and guanosine 5'-O-(3-thiotriphosphate) ( G T P - y S ) (Sigma) were used. The pipette solution (intracellular) contained (in mM): K O H 110, KCI 20, MgCI z 2, E G T A 10, M g A T P 5, creatine phosphate 5, aspartic acid 100 and H E P E S 5 ( p H 7.2). The concentration of Ca z+ in the pipette solution was chosen to give a pCa of 7, according to the equation of Fabiato and Fabiato (1979), with the correction of Tsien and Rink (1980).
little or no If current was elicited in 17-day-old cells (fig. 1B and C). The c u r r e n t / v o l t a g e relationships for the three developmental stages are shown in fig. 1D. At - 120 mV, the current density for If was - 6 . 7 + 1.3 p A / p F (n = 23) in 3-day cells (open circles), -3.3_+ 1.0 p A / p F (n = 17) in 10-day cells (triangles), and - 2 . 0 + 0.5 p A / p F (n = 12) in 17-day ceils (squares). The average m e m b r a n e capacitances were 12.7 + 2.0 pF (n = 17) in 3-day cells, 10.9 + 2.1 pF (n = 14) in 10-day ceils, and 9.5 + 2.6 pF (n = 14) in 17-day cells. This indicates that the multiplying cells actually are smaller in size. The threshold potential (Vth) for If was - 5 0 to - 6 0 m V in 3-day cells, and the threshold shifted leftward to more negative potentials at the older ages (fig. 1D).
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To examine the hyperpolarization-activated inward If current at different developmental stages of embryonic chick heart cells, experiments using whole-cell voltage clamp were performed. Long duration (3 s) hyperpolarizing pulses were applied from a holding potential of - 3 0 mV; the test pulses ranged from - 4 0 to - 120 mV, in 10 mV increments. I e was activated by hyperpolarization, and it had a slow time course of activation. In 3-day cells, I e was observed consistently in all cells examined, and its variation in size was relatively small. As shown in fig. 1A, large If currents and associated tail currents were observed in a 3-day-old cell. I e current became smaller in 10-day-old cells, and
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3. Results
3.1. Changes in the hyperpolarization-actiuated current during det~elopment
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Fig. 2. Temperature dependen~ of If current in a 3-day-old emb~ onic chick cardiomyocyte. Test pulses were a~lied between -4~ and -120 mV, in 10 mV increments, from a HP of -30 m~ External solution included 3 mM BaCI~, 10 ~M ~ X and 3 mM CdCI~ A,A': Current traces at room temperature (20~C). B,B': Current traces at 30~C. C: Current-voltage cu~es for If at 20~C (circles) and 30~C(triangles). Comparison be~een time courses of I~ activation a~ 20~C (A') and 30~C (B') obtained at a test pulse to -120 mV, each from one trace fitted as a single exponential by PCLAMP/CLAMPFIT. At 20~C, the value of r was 1.2 s; at 30~C, r was reduced to 0.7 s.
286
The outward tail current was also reduced during development. For measuring the tail current in this experiment, at the end of the hyperpolarizing pulse (e.g., to - 120 mV), the voltage was stepped to various test potentials ( + 2 0 to - 9 0 mV), rather than to the holding potential ( - 30 mV). This was done to accentuate the tail currents, as described by DiFrancesco and Tromba (1988) and Hagiwara and Irisawa (1989). The average value of reversal potential (Vrev) was - 13.4 + 1.2 mV (n = 3) in 3-day cells (fig. 1D). However, this value would be affected by activation of any unblocked K + currents. The time constant of activation (~-) of If was fitted as a single exponential, and the mean value was 1.4 + 0.3 s (n = 7) in 3-day cells. These results indicate that young embryonic chick heart cells can produce a hyperpolarization-activated inward If current, and show that If becomes smaller or disappears with development.
mM) suppressed If in the 3-day-old chick cells, as previously reported (Satoh and Sperelakis, 1991). The outward tail current from - 1 2 0 to +20 mV was reduced from 84.4 to 45.7 pA by the Cs ÷ (n = 10). In the presence of Cs +, Vth for I f remained near - 50 to - 60 mW.
The If current in 10-day cells was also inhibited by 56.5 _+ 8.2% (n = 7) in the presence of Cs + (3 mM). In separate experiments, automaticity was abolished by Cs ÷ in two of three cells. In 17-day cells, If was inhibited almost completely by Cs ÷ (n = 5). In the present experiments on 18 spontaneously beating 3-day-old myocytes, the mean spontaneous firing rate was 62.3 ___2.4 beats/min, the maximum diastolic potential was - 5 7 . 3 ___2.1 mV, and the amplitude of the action potentials was 79.2 + 4.2 mV. Cs + produced a negative chronotropic effect (not illustrated) by - 3 8 . 9 + 6 . 2 % ( n = 5 ) at 0.3 mM, - 5 2 . 1 + 3 . 5 % (n = 5) at 1 mM, and - 6 8 . 7 + 4.1% (n = 10) at 3 mM Cs ÷. Cs + (3 mM) also depressed the amplitude of the spontaneous action potentials, and the maximum diastolic potential was lowered (depolarizing).
3.2. Effects of temperature on If current When the temperature of the bath solution was raised from room temperature (20°C) to 30°C, the If current had a faster time course of activation, as shown in fig. 2. The • value (at - 1 2 0 mV) was decreased from 1.3 + 0.4 s (n = 5; P < 0.05) to 0.7 +__0.4 s (n = 5; P < 0 . 0 5 ) . This corresponds to a Q10 of 1.86. The amplitude of If was only slightly enhanced (by 12.1 + 1.8% at - 120 mV; n = 5).
3.4. Modulation by isoproterenol and carbachol In 3-day-old cells, isoproterenol (1 /~M) caused a positive chronotropic effect by 17.1 + 2.9% (n = 5; P < 0.01). At - 1 2 0 mV, isoproterenol (fig. 3, filled circles) increased If by 65.2 + 5.6% (n = 7; P < 0.001) (fig. 3; table 1). r for If activation was reduced (but not significantly) to 1.0 + 0.2 s (n = 5). In 10-day cells, isoproterenol increased If by 37.2% (n = 3; P < 0.05) at - 1 2 0 mV. This agonist also restarted automaticity in Cs +-induced quiescent preparations. In 17-day cells, 1 /xM isoproterenol insignificantly increased If by only 8.2 +__2.5% (n = 8, P > 0.05),
3.3. Effects of Cs + on spontaneous action potentials The If current is known to be inhibited or abolished by Cs ÷ (Browr/ et al., 1981; Noma et al., 1983; DiFrancesco, 1985; Denyer and Brown, 1990). Cs ÷ (3
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Fig. 3. Effect of isoproterenol (ISO) on If current in a 3-day-old cell. A: Control If current in one cell. Test pulses were applied between - 4 0 and - 1 2 0 mV, in 10 mV increments, from a HP of - 3 0 mV. B: If in the presence of isoproterenol (1 /xM) for 10 min in the same cell. C: Current/voltage relationships for If with and without isoproterenol averaged for seven cells. Isoproterenol (filled circles) increased If at all potentials.
287 TABLE 1 Effects of isoproterenol, carbachol and GTP-TS on If of 3-day-old embryonic chick heart cells.
gles), to cells pretreated with 1 ~ M isoproterenol (filled circles), returned If to nearly the control value (at - 120 mV; n = 3 ) .
Numbers in parentheses represent the number of experiments. Single agent
+ GTP-TS
3.5. Effects of intracellular application of GTP-yS
Isoproterenol 1/~M +65.2_+5.6% e (7) +37.8_+4.7% b (9) a Carbachol 0 . 1 ~ M - 4 1 . 2 _ + 1 . 3 % c (7)-21.2_+4.3% (9) a GTP-yS 100 t~M -35.2_+5.0% b (17) a These values indicate further stimulation or inhibition beyond the altered new baseline (i.e., new 'control' values) indicated by the values given in the "first column ('single agent'), b p < 0.01; c p < 0.001, with respect to control value.
and ~- in 10-day cells was insignificantly changed from 2.2 + 2.1 s to 2.0 + 1.9 s (n = 3, P > 0.05). In 3-day cells, carbachol (0.1 /~M) had a negative chronotropic effect of 26.3_ 3.4% (n = 5), and produced an arrest in two of eight cells. The I f c u r r e n t was reduced by 41.2 + 1.3% (n = 7) at - 120 mV (fig. 4; table 1). Carbachol increased z to 1.5 + 0.4 s (n = 3) at - 1 2 0 mV. Carbachol was also effective in reversing the stimulatory effects of isoproterenol on I f. As shown in fig. 4D and E, addition of 0.1 /zM carbachol (trian-
A
To examine the coupling between G proteins and If channels, GTP-yS (nonhydrolysable GTP analogue) was added to the pipette solution (fig. 5). After the patch membrane was opened, If became stable at about 1 rain (Control, open circles). With GTP-yS in the pipette, GTP-yS (filled circles) diffused into the cell, and its effect reached steady state within 4 min, namely there was inhibition of If. With pretreatment by 1 /zM isoproterenol (triangles), If increased with diffusion of GTP-yS into the cell. In contrast, with 0.1 /~M carbachol pretreatment (squares), If decreased with diffusion of GTP-yS into the cell (fig. 5). Table 1 summarizes these data. GTP-yS alone depressed If by 35.2 +5.0% (n = 17). GTP-yS 100 ~zM further increased If by 37.8 + 4.7% (n = 9) beyond that produced by the 1 /zM isoproterenol pretreatment (table 1). In contrast, If was further decreased by 21.2 + 4.3% (n =
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9) beyond that produced by the 0.1 /xM carbachol pretreatment (table 1).
4. D i s c u s s i o n
4.1. Properties and developmental changes in the hyperpolarization-activated inward current In the present experiments, 3-day-old cells, from the presumptive ventricular part of the heart, exhibited spontaneous action potentials. An If current was activated in a time- and voltage-dependent manner. If required long hyperpolarizing steps of 3 s or more to reach maximum, and the -r value was 1.3 s (at - 1 2 0 mV) at 20°C. The If current was temperature sensitive. At 30°C, the r of activation became much faster (Q10 = 1.86), and the If amplitude was slightly enhanced. With development, the If pacemaker current was greatly reduced, accompanied by the disappearance of spontaneous activity in 17-day-old cells. Vth and Vrev were about - 5 0 to - 6 0 mV and about - 1 3 mV, respectively (see figs. 1C and 3C). In mammalian sino-atrial (SA) node cells and Purkinje fibers, the hyperpolarization-activated inward If current has been demonstrated in multicellular and in single-cell preparations (Brown et al., 1979; Yanagihara and Irisawa, 1980; Yanagihara et al., 1980; DiFrancesco et al., 1986; Satoh and Seyama, 1986; Hagiwara and Irisawa, 1989). Shrier and Clay (1981a,b)
showed that aggregates of embryonic chick heart ceils possessed Iv~ or If, and that this current was decreased in older ceils. In single SA node cells, Vth for If ranged between - 4 0 to - 7 0 mV (Nakayama et al., 1984; DiFrancesco et al., 1986; Satoh and Seyama, 1986), and Vrev and ~" were about - 1 5 mV and 1.3 s (Hagiwara and Irisawa, 1989). In multicellular Purkinje fibers, Vth was - 5 0 to - 6 0 mV (DiFrancesco and Ojeda, 1980). Therefore, the properties of the I f current found in young embryonic chick heart cells are the same as those found in other tissues. If is sensitive to Cs ÷, is time- and voltage-dependent, and is activated at almost the same threshold potential ( - 5 0 to - 6 0 mV). Since If became smaller and disappeared during development, it may play a role in generation of the pacemaker depolarization of young embryonic chick heart myocytes.
4.2. Contribution of I~ to the pacemaker current In cardiac Purkinje fibers, If plays an important role in the pacemaker potential (DiFrancesco and Ojeda, 1980; Noble, 1984). This is possible because the maximum diastolic potential is more negative ( - 90 to - 100 mV), thus allowing I f to be fully activated. In SA node cells, there are at least three currents known to contribute to the pacemaker potential (Noble, 1984): (a) Isi (Yanagihara and Irisawa, 1980; Yanagihara et al., 1980), (b) IK(ael) decay (Brown, 1982; Shibata and Giles, 1983), and (c) If (Maylie et al., 1981). However, | f may not play an important role in the cycle-to-cycle pacemaker potential in SA nodal cells, because (a) the time course of activation of If is too slow to account for the high frequency of the primary pacemaker, and (b) the threshold potential of activation ( - 5 0 to - 6 0 mV) is close to the maximum diastolic potential ( - 60 to - 7 0 mV) (Noma et al., 1983; Yanagihara and Irisawa, 1980; Yanagihara et al., 1980). Therefore, I f activation may make only a minor contribution to the pacemaker potential in SA node. In the present experiments, Cs ÷ blocked If, but did not completely inhibit the spontaneous action potentials (in 16 of 18 cells). Since Vth was -- 50 to -- 60 mV and the maximum diastolic potential was less than - 6 0 m V , I f may contribute to the pacemaker potential in the 3-day-old cells to a small degree. The spontaneous activity in the young embryonic chick heart could also involve other currents (e.g., I c a and IK).
4.3. Involvement of G proteins on I/ channels Isoproterenol stimulates If through stimulatory Gprotein (Gs)-mediated activation of adenylate cyclase, whereas acetylcholine inhibits I f through inhibitory G-protein (Gi)-mediated activation of guanylate cy-
289
clase (Gilman, 1987; Brown, 1990). In rabbit SA node myocytes, acetylcholine inhibits If and slows spontaneous rate at low concentrations that do not activate acetylcholine-dependent K ÷ current (IKtACh)) (DiFrancesco et al., 1989). In young (3- to 5-day-old) embryonic chick hearts, cAMP level is very high, whereas cGMP level is very low (Thakkar and Sperelakis, 1987); isoproterenol increased cAMP and acetylchoh'ne increased cGMP. With development, the change in cGMP is reciprocal to that of cAMP, namely, cAMP decreases and cGMP increases. In the present experiments, isoproterenol increased If and shifted the I / V curve in the positive direction, whereas carbachol inhibited If and shifted the I / V curve negatively (in two of eight cells, carbachol caused complete arrest). Carbachol depressed If even in the presence of isoproterenol. These results are consistent with those of DiFrancesco and Tromba (1988), who showed that acetylcholine shifted the If activation curve to more negative potentials and with the results of Chang and Cohen (1992) who showed that acetylcholine could reverse the positive voltage shift produced by fl-adrenoceptor agonists. Therefore, these effects could be due to the activation of G i and reversal of the isoproterenol-induced elevation of cAMP (Rodbell, 1980; Thakkar and Sperelakis, 1987). Acetylcholine also reduces Ica (Giles and Noble, 1976; Josephson and Sperelakis, 1982) and activates IK(ACh) by GK-protein gating of the ion channels (Pappano, 1977; Noma, 1986). The present results suggest that young embryonic cells have high sensitivity to muscarinic receptors, even though the 3-day-old embryonic chick heart is not yet innervated by parasympathetic nerves (Pappano, 1977). Acetylcholine can act directly on the heart muscle prior to the appearence of cholinergic innervation (Cullis and Lucas, 1936). Although sensitivity to acetylcholine is low in young hearts, the sensitivity to acetylcholine was found to increase with time in organ culture (Dong et al., 1986; Hatae et al., 1989). Since the 3-day cells were cultured for 1-2 days before being used for the present experiments, acetylcholine sensitivity may have developed during culture. Effects of isoproterenol and carbachol on If were potentiated further by GTP-yS. It is likely that G-protein pathways connect both muscarinic receptors and fl-adrenoceptors to If channels in embryonic chick heart cells. When intracellular application of GTP-yS was done in the absence of any agonists, If current fell somewhat, perhaps due to GTP-yS stimulation of both G proteins (G s and Gi), with G i predominating. Gilman (1987) reported that the a subunit can act on If directly or indirectly by way of adenylate cyclase, and the fly subunit may be inhibitory to the a subunit effects on adenylate cyclase. These results indicate that G proteins inhibitory to If were dominant over stimulatory G proteins in young embryonic chick heart. The
known dominance of the vagal effect may also be reflected by the dominance of the inhibitory G proteins activated by GTP-yS. Direct G-protein coupling between autonomic receptors and If channels may partly account for the ability of the autonomic nervous system to produce its effects within a single heartbeat, and an indirect coupling via second messengers can account for the more persistent effects.
Acknowledgements This study was supported by National Heart, Lung, and Blood Institute Grant HL-31942. We are grateful for Lisa WukuSick-Neumier for preparation of the cell cultures, to Anthony Sperelakis and Glenn Doerman for help with the figures, and to Rhonda'Hentz for the word processing.
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Satoh, H. and I. Seyama, 1986, On the mechanism by which changes in extracellular pH affect the electrical activity of the sino-atrial node, J. Physiol. 381, 181. Satoh, H. and N. Sperelakis, 1991, Identification of hyperpolarization-activated inward current in young embryonic chick heart myocytes, J. Dev. Physiol. 15, 247. Schanne, O.F., L. Boutin and J. Derosiers, 1989, Effects of K-channel blockers, calcium and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells, Can. J. Physiol. Pharmacol. 67, 795. Shibata, E.F. and W.R. Giles, 1983, Ionic currents in isolated cardiac pacemaker cells from bullfrog sinus venosus, Proc. Int. Union Physiol. Sci. 15, 76. Shibata, E.F., W. Giles and G.H. Pollack, 1985, Threshold effects of acetylcholine on primary pacemaker cells of the rabbit sino-atrial node, Proc. R. Soc. London Ser. B 223, 355. Shrier, A. and J.R. Clay, 1981a, Analysis of subthreshold pace-maker currents in chick embryonic heart cells, J. Physiol. 312, 371. Shrier, A. and J.R. Clay, 1981b, Developmental changes in subthreshold pace-maker currents in chick embryonic heart cells, J. Physiol. 312, 491. Thakkar, J.K. and N. Sperelakis, 1987, Changes in cyclic nucleotide levels during embryonic development of chick hearts, J. Dev. Physiol. 9, 497. Tsien, R.Y. and T.J. Rink, 1980, Neutral carrier ion-selective microelectrodes for measurements of intracellular free calcium, Biochim. Biophys. Acta 599, 623. Yanagihara, K. and H. Irisawa, 1980, Inward current activated during hyperpolarization in rabbit sinoatrial node cell, Pfliig. Arch. 358, 11. Yanagihara, K., A. Noma and H. Irisawa, 1980, Reconstruction of sinoatrial node pacemaker potential based on the voltage clamp experiments, Jpn. J. Physiol. 30, 841.