221

Journal of Physiology (1990), 423, pp. 221-240 With 12 figures Printed in Great Britain

MODULATION OF Ca2+ TRANSIENTS AND CONTRACTILE PROPERTIES BY fJ-ADRENOCEPTOR STIMULATION IN FERRET VENTRICULAR MUSCLES

BY OSAMU OKAZAKI*, NORIO SUDA, KENICHI HONGO, MASATO KONISHI AND SATOSHI KURIHARAt From the Department of Physiology, The Jikei University School of Medicine. 3-25-8 Nishishinbashi, Minato-ku, Tokyo, 105 Japan

(Received 8 June 1989) SUMMARY

1. The mechanism of modulation of Ca2+ transients and contraction by /1adrenoceptor stimulation was studied in ferret ventricular muscles using aequorin to measure intracellular Ca2+. 2. Peaks of tension and light transients were increased by isoprenaline (10-9-5 X 10-7 M) which also abbreviated their time courses. 3. Time-to-peak tension was significantly shortened by 5 x 10-9 M-isoprenaline and time-to-peak light was abbreviated by 10-9 M-isoprenaline. 4. The time for the light to decay was shortened at 10-9 M-isoprenaline. However, a higher concentration of isoprenaline (10-8 M) was required for significant shortening of the half-relaxation time (TR50). 5. When isoprenaline was removed and fl-blocker (bupranolol, 1 JIM) was applied, the time course of the light transients recovered but the time course of relaxation did not recover. 6. The relationship between [Ca2+]i and tension in tetanic contraction produced in the presence of ryanodine (5 ItM) was shifted to the right by isoprenaline (10-8 M). This was recovered by the replacement of isoprenaline with bupranolol (1 JIM). 7. Isoprenaline (10-7 M) added to the solution containing 20 mm [Ca2+]o and Bay K 8644 (1 JM), which produced maximal tension, caused a large light signal and enhancement of the initial phasic tension in tetanic contraction. However, the replacement of isoprenaline with bupranolol after immersing the preparation in 20 mm [Ca2+]o solution with Bay K 8644 and isoprenaline, did not significantly change the tension level, although the light signal decreased. Similar results were obtained in the ventricular muscle of young rats. 8. These results suggest that the dose dependence of modulation of the contractile element and sarcoplasmic reticulum (SR) by fl-adrenoceptor stimulation differs, and that additional factors, other than the faster Ca2+ uptake by SR and the decrease in * Present address: Department of Internal Medicine, Fujigaoka Hospital, Faculty of Medicine, Showa University School of Medicine, 1-30 Fujigaoka, Nlidori-ku, Yokohama City, Kanagawa Prefecture, 227 Japan. t To whom all correspondence should be sent at the above address.

NIS 7737

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20. OKAZAKI AND OTHERS

Ca21 sensitivity of the contractile element, might be involved in the shortening of the half-relaxation time by /3-adrenoceptor stimulation. In addition, fi-adrenoceptor stimulation does not produce a marked change in the maximal tension level.

INTRODUCTION

/3-Adrenoceptor stimulation potentiates twitch tension and abbreviates the time course of contraction (Tsien, 1977). An increase in the peak tension is due to an increase in intracellular Ca2± concentration ([Ca2±],), which is caused by the enhancement of Ca2+ release from sarcoplasmic reticulum (SR). The increase in Ca2+ current and Ca2± content in the SR causes more Ca2+ release from the SR (Tada, Kirchberger, Repke & Katz, 1974; Tada & Katz, 1982; Reuter, Cachelin, DePeyer & Kokubun, 1983). The abbreviated time course of contraction is considered to be caused by the accelerated Ca2+ uptake by SR and the decreased Ca2+ sensitivity of the contractile element (Tsien, 1977; McClellan & Winegrad, 1978; Herzig, Kohler, Pfitzer, Riiegg & W6lffle, 1981). These changes are due to an increase in intracellular cyclic AMP concentration (Murad, Chi, Roll & Sutherland, 1962; Schumann, Endoh & Brodde, 1975), and the cyclic AMP-dependent phosphorylation of proteins (Ca21 channel protein, phospholamban, troponin I and C-protein) is considered to be the underlying mechanism of/-adrenoceptor stimulation (Ray & England, 1976; Solaro, Moir & Perry, 1976; Tada & Katz, 1982; Reuter et al. 1983; Hartzell, 1984). Simultaneous measurement of intracellular Ca2+ transients and tension with aequorin provides direct evidence of the relationships of intracellular Ca2+ movement and contraction in cardiac tissues (Allen & Blinks, 1978; Allen & Kurihara, 1980). ,8-Adrenoceptor stimulation accelerates the rate of decline of Ca2± transients and decreases the apparent Ca2+ sensitivity of the contractile element (Allen & Kurihara, 1980; Morgan & Blinks, 1982; Kurihara & Konishi, 1987; Endoh & Blinks, 1988; McIvor, Orchard & Lakatta, 1988). However, it is not clear whether the shortening of the contraction time course can be explained by the faster Ca2+ uptake by the SR and the decrease in Ca2+ sensitivity of the contractile element in ,I-adrenoceptor stimulation. In previous experiments (Endoh & Blinks, 1988; Melvor et al. 1988), the change in Ca21 sensitivity was estimated by observing the relation of the peaks of tension (or dP/dt) and Ca2+ transients in twitch responses, in which both parameters changed transiently. However, when the time courses of contraction and light transients in the twitch response are changed, the straightforward interpretation of the relationship of [Ca2+]i and tension is difficult. A decrease in Ca2+ sensitivity of the contractile element by p3-adrenoceptor stimulation in the steady state has also been demonstrated in intact cardiac preparations (Marban, Rink, Tsien & Tsien, 1980; Kurihara & Konishi, 1987). However, the relationship of [Ca2+]i and tension studied in these experiments was limited to a narrow range of [Ca2+]i. In the present study, we observed the changes in the time course of contraction and Ca2+ transients produced by 8-adrenoceptor stimulation. In particular, the dose-dependent change in both parameters was investigated. Additionally, the change in Ca2± sensitivity of the contractile element and the marked change in the maximal tension level by ,-adrenoceptor stimulation, which has been demonstrated

/i-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES 223 in hyperpermeable preparations (Winegrad, 1984), were investigated by the observation of the relationship of [Ca2±]i and tension at steady state in intact ventricular muscle using tetanic contraction (Yue, Marban & Wier, 1986). Possible mechanisms of the changes in the contractile properties induced by /?-adrenoceptor stimulation are discussed in relation to the changes in the Ca2+ sensitivity of the contractile element and Ca2± transients. The preliminary results of this study have been previously reported (Kurihara, Konishi, Okazaki & Suda, 1988). METHODS

Preparations Ferrets (body weight, 450-760 g) were anaesthetized with pentobarbitone and the hearts were quickly removed. After washing the hearts with normal Tyrode solution, the right ventricle was opened. Thin papillary muscles, with a diameter of 0 5-0-9 mm, were dissected and both ends of the preparation were tied with thin gold wires (diameter 50 ,m) or silk threads. The diameter of the preparations was 0-8+0-03 mm (mean+S.E.M., n =22) and the length 4-11 +0-17 mm (mean +S.E.M., n = 22). The papillary muscles of young rats (body weight was about 80 g) were used to observe the change in maximal tension by isoprenaline.

Tension recording and stimulation One end of the preparation was connected to a stiff hook and the other end was connected to the arm of a tension transducer (AME 801, Horten Norway or Kulite, New Jersey, USA). The preparation was mounted in a muscle chamber with a pair of platinum wires placed in parallel with the preparation for electrical stimulation. In some cases, a parabolic mirror was placed under the preparation to collect the scattered light of aequorin (Allen & Kurihara, 1982). Before the start of the experiment the muscle length was adjusted to Lmmax at which the developed tension became maximal. Generally, the preparation was stimulated by square pulses with a 5 ms duration at 0-2 Hz regularly, and the strength of the stimulation was 1-5 x threshold. In the tetanic experiment. the muscle was stimulated with square pulses with a 40 ms duration at 10 Hz (Yue et al. 1986). The intensity of the stimulation was about 3 x threshold, which produced a smooth contraction without ripples. With a lower intensity (< 3 x threshold), tension did not fuse and rippled contractions were observed.

Solutions The normal Tyrode solution used for dissecting the preparations and for the injection of aequorini had the following composition (mM): Na', 135; K+, 5; Ca2+, 2; Mg2+, 1; Cl-, 102; HC03-. 20: HPO42-, 1; So42-, 1; acetate, 20; glucose, 10; insulin, 5 units/l; pH, 7-34 at 30 TC when equilibrated with 5% C02+95% 02. In most experiments, Tyrode solution buffered with HE1PES (N-2hydroxyethyl-piperazine-N'-2-ethansulphonic acid) was employed (HEPES-Tyrode solution). which had the following composition (mM): Na+, 128; K+, 5; Ca2+, 2; Mg2+, 1; Cl-, 117; 8042-. 1 acetate, 20; glucose, 10; insulin, 5 units/l; pH was adjusted to 7-35 with NaOH at 30 'C. The solution was equilibrated with 100% 02. When [Ca2+]L was changed, the osmotic pressure of the solution was not adjusted and CaCl2 was added to or removed from the solution. The temperature of the solution was continuously monitored with a thermocouple and maintained at 30 + 0 5 'C.

Aequorin injection and measurement of light signals Aequorin, purchased from Dr J. R. Blinks, was dissolved in the 150 mM-KCl and 5 mM-HEPES at pH 7-0 solution, with a final aequorin concentration of 50- 100 JtM. A glass micropipette with a resistance of 30-80 MQ, measured after filling with aequorin solution, was used for the injection of aequorin. Aequorin was injected into about 50-100 superficial cells of each preparation by monitoring the membrane potential. Aequorin light signals were detected with a photomultiplier

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(EMT 9789A, Ruislip) which was mounted in a small housing with a cooling system. All data were stored on a tape (NFR-3515W, Sony Magnescale Inc.,Tokyo, Japan) and a computer for later analysis. In order to improve the signal-to-noise ratio, the light signals in twitch response and in tetanic contraction were recorded through 500 Hz and 1 Hz low-pass filters, respectively. Signals were averaged with the computer or a signal processor (7T07A, NEC San-ei Co., Ltd. Tokyo, Japan). The aequorin method used was essentially similar to that previously described (Allen & Kurihara, 1982). When the relationship between [Ca2+]i and tension was obtained, the light signals of aequorin were converted to [Ca2+]i using the calibration curve in a method similar to Blinks, Wier. Hess & Prendergast (1982). In the figures of the present study, the light signals were expressed as fractional luminescence (Lf, logarithm of a light signal normalized to maximal light; Allen, Blinks & Prendergast, 1977), rather than [Ca2+],, due to various unspecified factors (distribution of aequorin, validity of in vitro calibration and [Ca2+l] gradient) in the conversion of the light signals to [Ca2+]. The calibration curve was fitted using the least-squares method and three constants. KR, KTR and n, were obtained for the calculation of [Ca2i]i (Allen et al. 1977). The calibration curve was obtained by rapidly mixing the aequorin which had been pre-equilibrated with 1 mM-MgCl2 with solutions having various concentrations of Ca2+ at 30 'C. The composition of the solution used for the calibration curve was described in Kurihara & Konishi (1987). The constants used in the present study were as follows: n, 3-14; KR, 4025X106; KTR, 114-6. When tetanic contractions were observed in the solution with 20 mm [Ca2+]o with isoprenaline and Bay K 8644. the consumption of aequorin could not be ignored and correction was made for consumption in the calculation of fractional luminescence. Measured parameters For the evaluation of the effects of /J-adrenoceptor stimulation, the following parameters were measured: peaks of light and tension; time-to-peak light and time-to-peak tension, which represent the time measured from the onset of stimulus to the peak of light and tension, respectively; halfrelaxation time (TRio), which is the time required for tension to fall from the peak to 50 %; the time required for the tension to decay from 75 to 25% of the peak (TR7S 25); half-decay time of light (TL50) which is the time for the light to decay from the peak to 50% of peak; decay time of light (TL75-25) which is the time for the light signal to decay from 75 to 25 % of the peak. For analysing the relationship between [Ca2+]i and tension in the tetanized preparation, the Hill equation was used and the best-fitting curve was obtained using a computer. Two constants were calculated and compared in the presence and absence of a drug: K., [Ca2+], required for the halfmaximal tension development; n, slope of [Ca2+]1 and tension relationship. In some cases, K2, the [Ca2+]i required for producing 75 % maximal tension, was calculated.

Drugs The following drugs of analytical grade were used: L-isoprenaline D-bitartrate (Nakarai Chemicals Ltd and Sigma Chemical Co.); ryanodine (Agri Systems, Inc., Pennsvlvania); Bay K 8644 (a gift from Bayer, AG); bupranolol hydrochloride (gift from Kaken Pharmaceutical Co., Ltd). A stock solution of isoprenaline (1 mM) was prepared by dissolving in double-distilled water containing 50 ItM-EDTA and 30,uM-ascorbic acid to prevent oxidation, and was kept at low temperature. Bay K 8644 (1 mM) was dissolved in ethanol for the stock solution and then diluted in Tyrode solution when used. The effects of the drugs used on aequorin luminescence were determined in a cuvette. Two solutions with pCa 7 and pCa 6 were prepared using Ca-EGTA buffer (EGTA concentration. 15 mM). Aequorin was rapidly mixed in solutions (pCa 7 and pCa 6) with or without the drugs and the light signal was measured. In other experiments, the drugs were pre-equilibrated with aequorin and rapidly mixed in the solutions with pCa 7. The drugs used did not show significant effects on aequorin luminescence. Statistical analysis Measured values were expressed as mean+ standard error of the mean (S.E.M.) and Student's paired t test was employed for the statistical analysis. Statistical significance was verified at the 5 % level of the P value in two-sided t test unless otherwise specified.

/3-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES

B Isoprenaline (10'M)

A Control Lf -3.6

225

C Isoprenaline (5 x 10'M)

-

-4*0

/

\

/

\

\

20

mN/mm2

-4.5. 200 ms D Isoprenaline (2 x 10-7M)

E

Lf -3.6 -

-4.0

(

20

mN/mm2

4*5.

200 ms

C

c iso

(5 x 101 M) ~~~~~~~~~~~Iso

200 ms

Fig. 1. Effects of isoprenaline on light transients and tension in twitch response. Isoprenaline dose-dependently increased peaks of light signal and tension (A-D). Noisy traces with a fast time to peak are light signals. Concentrations are indicated in each panel. Small vertical marks just before both signals are stimulus markers (this is the same in the following figures). At a high concentration of isoprenaline (D), a slight increase in the light signal with a slow time course was observed during the relaxation phase (indicated by an arrow). In E, peaks of light and tension in control (A) and in isoprenaline (5 x 10-8 M) (C) were normalized and superimposed. Sixty-four signals were averaged in each record. RESULTS

Effects of isoprenaline on peaks of Ca2+ transients and tension in ferret papillary muscles Isoprenaline increased the peaks of tension and light transients accompanying the alteration in the time course of both signals in the twitch response (Fig. 1). The effects of isoprenaline on the peaks of tension and light were almost maximal at 5 x 10-8 M (Figs 1 and 2). Further increase in the concentration of isoprenaline did not significantly change the peaks of light or tension. When high concentrations of isoprenaline were used, a transient increase of light (extra-light) appeared during the relaxation phase (Fig. ID, indicated by an arrow). In some cases, this was much evident and a hump appeared on tension. The peak tension increased 36 and 78 % at 10' and 10-7 M, respectively (in controls, the peak tension was 40+5-7 mN/mm2, n = 11). [Ca21], in controls was 0-94+0-13 #M (n = 13), and increased to 1-95 + 0-20 fM (n = 5) and 2-65 + 0-27 /M (n = 13) at 10-9 and 10-7 M, respectively. However, it is known that there is a gradient of [Ca2+]i just after stimulation, and when the peak light is converted to [Ca2+]i, the [Ca2+], obtained is an overestimate.

PHY 423

226

OKAZAKI AND OTHERS

0.

Effect of isoprenaline on time courses of Ca2+ transients and tension The dose dependence of the effects of isoprenaline on the time courses of twitch tension and Ca2+ transients was compared. The main point of interest is that the relaxation of tension was affected at higher doses than the change in the time course of the light transients. 0

r-

Z2.0

ri4 2a0r >10) *> 1.° 10 (5) C

3.0

109

(8) (8) (8)

5x 10

10

~(7)

(9) (9) (9)

5x 109

10

5x 10

(11) 107

-

at 2 +0

21 .0 I, 1.0

(7)

+

+

~n+ t

me>

(13)

(5

(13)

X0

a

a

C

10-

5x 10

10-7

Isoprenaline (M) Fig. 2. Dose dependence of peaks of tension and Ca2+ transients in twitch response. Ordinate: relative tension (upper graph) and [Ca2+], (lower graph) calculated from the peak light signals. C, control. S, significant change. Vertical bars indicate S.E.M. (this is the same in the following figures). Numbers in parentheses are number of measurements.

The most significant change in the time course of contraction induced by isoprenaline was the time-to-peak tension which was significantly shortened at 5 x 10-9 M and further shortening was observed at higher concentrations (Figs 1 E and 3). The time-to-peak light was also shortened and a significant change was seen at the concentration of 10-9 M. On the other hand, the half-relaxation time (TR50; Sonnenblick, 1962) was significantly shortened at 10-8M (Fig. 4) and the change in TR50 at 10-7 M-isoprenaline was 13% (from 151-4 to 132X3 ms). However, the relaxation time (T.75-25) did not significantly change even at 10-7 M (Fig. 4; in one-sided paired t test, the change was significant at P < 005). Therefore, the significant change in the time course of contraction was the change in the time to reach the peak of contraction and to relax from the peak to 50 %. The later part of the relaxation was rather insensitive to ,-adrenoceptor stimulation. The change in the time course of light signals appeared at lower isoprenalinc concentrations than that of contraction. A significant change in the time-to-peak light was noticed at 10-9 M-isoprenaline (Fig. 3). TL7625 was significantly

/3-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES

227

shortened at 10-9 M (TL7 -25 at 10-9 M was 32 % shorter than that in control; Fig. 5). Further increase in the concentration of isoprenaline caused a slight change in TL75-25 and this effect was saturated at 5 x 10-9 M. The significant abbreviation of TL50 was also observed at 10-9 M (22 % shorter than that in control) and similar dose dependence to that in TL75-25 was recognized (Fig. 5). Therefore, the decay of the light signal (TL50 and TL75-25) was more sensitive to isoprenaline (10-fold more sensitive) than that in the half-relaxation time (TR50).

ECo

.0a200 -0 0)

-w

(11) (5)

(7)

(8) (8) (8)

(1 )

Q X100 oo

6

E

0

C 50

-

E

10-9 5 x 10-9 108 5 x 104 10-7

rS-

> (13)

- 30 .

(5)

(8)

(9) (9) (9)

(13)

CL lo Co 10

E

0

1

I

C

I

10-9 5 x 10-9

108

5 x 104

10-7

Isoprenaline (M)

Fig. 3. Dose dependence of time-to-peak tension and time-to-peak light. C, control. S, significant change. Numbers in parentheses are number of measurements.

Recovery of light transients and tension after washing out isoprenaline When isoprenaline (10-7 M) was removed from the solution and bupranolol (1 /kM), a potent fl-blocker, was applied, the time-to-peak light recovered to the control level but the falling phase was slightly prolonged. The time-to-peak tension also recovered but the relaxation did not significantly change (Fig. 6; similar phenomena were observed in three experiments). When bupranolol was added to normal HEPESTyrode solution as a control, the peak of light was slightly diminished without any change in twitch tension (three experiments). The falling phase of the light signal and the relaxation phase were slightly prolonged. Therefore, the slight prolongation of the light observed in the recovery (without isoprenaline and plus bupranolol; Fig. 6) might be partly due to the effect of bupranolol; the results in Fig. 6 suggested that 8-2

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0. OKAZAKI AND OTHERS

the falling phase of the light signal was not a critical factor in determination of the relaxation time.

Changes in Ca2+ sensitivity of contractile elements by f-adrenoceptor stimulation When the preparation was treated with ryanodine (1-5 /IM) and stimulated at 02 Hz, the peaks of light transients and tension decreased within 10 min and showed

E 80( 1 1) (5)

N rk

(7)

(8) (8) (8)

(1)

5x 10-9

10

10-7

60 E100

50

C

10O-

5x 10

Isoprenaline (M) Fig. 4. Dose dependence of relaxation time. TR75-25' time required for the decay of tension from 75 to 25% peak. TRSO, time required for the decay of tension from peak to 50%. C, control. S, significant change. Numbers in parentheses are the number of measurements.

a prolonged time course. The preparation was treated at least 40 mmn before the start of the tetanic stimulation. Tetanic stimulation was applied to the preparation every 60-75 s and the twitch responses were triggered at 0@2 Hz between tetanic stimulations. As previously shown, the tetanic stimulation with square pulses (40 ms duration, 10 Hz) produced a rather steady-state tension during the stimulation (Yue et al. 1986; Fig. 7). At the beginning of the tetanic stimulation, a twitch-like phasic contraction was frequently observed at lower [Ca2+]o, which was followed by a rather sustained tonic contraction. The phasic contraction was accompanied by a transient change in the light signal. The tonic tension was not completely steady state during the stimulation, particularly at lower [Ca2l]i. The light signal during the tetanic stimulation reached a steady level at lower LCa2+]o, but istincreased at highertCa2±]o. This is probably due to the saturation ofCa2d-binding sites in the cell as [Ca2t]i was increased by raising [Ca2+]o. When [Ca2+]o was changed from 05 to 20 mt , the

/3-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES

229

developed tension and the light signal changed depending on [Ca2+]0. The developed tension at 20 mm [Ca2+]0 was almost the same as that at 15 mm, although the light signal at 20 mm [Ca2+]o was higher than that at 15 mm. Therefore, the tension at 20 mm [Ca2+]0 was nearly maximal, with [Ca2+]i reaching 0-87 + 0-06 JM (n = 15). A-s1

60

40 - (13)

, 20

(5)

-

(9) (9) (9)

(13)

1

0

C

50

(8)

I

10-9

5 x 10-9

104

5 x 104

10-7

S]

-

(13)

+ *

(5)

30

E _LO

(8)

*

*

(9) (9) (9)

(13)

104

10-7

10 0

C

10-9

5 x 10-9

5 x 10-8

Isoprenaline (M) Fig. 5. Dose-dependent change in the decay of light signal. TL75-25' time required for the decay of light from 75 to 25 % peak. TL50, time for the decay of light from peak to 50%. C, control. S, significant change. Numbers in parentheses are the number of measurements.

Since the tension change was not parallel to the light signal change, the relationship between [Ca2+]i and tension obtained at various intervals after the start of stimulation was compared (Fig. 8A). The measured light was converted to [Ca2+]i using the calibration curve of aequorin, and both parameters were fitted by the Hill equation which can be expressed as follows: Tn

=

[Ca]2+]/(Ktp + [Ca2+]p),

where Tn is tension normalized by maximal tension, n is the Hill coefficient, [Ca2+]i is intracellular Ca2+ concentration and K, is the [Ca2+]i required for half-maximal activation of T (T = 0 5). K2 and n were both dependent on the time of measurement after the stimulation. As shown in Fig. 8A, Ki and n measured 4 and 6 s after stimulation were not greatly different. Therefore, in the following experiments, both parameters were measured 6 s after the stimulation to avoid the influence of an initial twitch-like contraction. In HEPES-Tyrode solution, K, was 0-38 + 0-02 M

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0. OKAZAKI AND OTHERS

(n = 10) but it varied from preparation to preparation. This might be due to the condition of the preparations used (endogenous catecholamine in each preparation, etc.). We tested the effects of stimulation intensity which probably changes the release of endogenous catecholamine in the [Ca2+]i-tension relationship. No

Control

Isoprenaline

(10-7 M)

- Recovery (+1

pM-bupranolol)

Control - Isoprenaline (10-7 M)

100 ms Fig. 6. Recovery of light signal and tension after removal of isoprenaline (10-7 M). In recovery, isoprenaline was replaced with bupranolol (1 gm). Peaks of light and tension were normalized and superimposed. Data in recovery were collected 90 min after removal of isoprenaline. Peaks of light transients in control and recovery were almost the same. Note the faster relaxation after the removal of isoprenaline (recovery). The falling phase of the light transients in recovery was slightly prolonged. Sixty-four signals were averaged in control; thirty-two for isoprenaline and 128 for recovery.

significant change of [Ca2+]i-tension relationship was observed even though the stimulation intensity was increased from 3 to 5 x threshold. This is probably due to the fact that endogenous catecholamine might be released at an intensity of 3 x threshold and the effects of endogenous catecholamine might be nearly maximal at that intensity. However, in the following experiments, the stimulation intensity was not changed throughout the experiment, in the absence and presence of isoprenaline. The Hill coefficient, n, was more variable than K, and thus a small change was not considered significant. Therefore, in the following experiments, the effects of isoprenaline were tested in each preparation and compared with each control using the paired t test as mentioned in the Methods section. We tested the reproducibility of the [Ca2+]i-tension relationship when [Ca2+]0 was

/3-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES

231

changed, and only a slight difference was observed between the two methods of changing [Ca2"]. (Fig. 8B). When [Ca2+]. was increased from a low to a high concentration, the slope of the [Ca2+]i-tension relationship was only slightly steeper than that observed when [Ca2+]. was reduced. However, the reproducibility of the [Ca2+]i-tension relationship within a certain range was confirmed. A Control

B Isoprenaline (10-7M)

Lf

Lf -46 r

-,4.3

20

-5.0 -

-6.0 L

20

_F

L

mN/mm2

_I

I

,

10s 10 s Fig. 7. Light signals and tension in tetanic stimulation. The preparation was treated with ryanodine (5 pM) and tetanic stimulation was applied every 65 s. Upper traces, light signals. Lower traces, tension. Tetanic stimulation was applied for 10 s after observing twitch tension (transient change before tetanus). Numbers in each panel represent [Ca2+] in solutions (mM). A, control. B, in the presence of 10-7 M-isoprenaline. In each case, four signals were averaged.

At the previously described maximal isoprenaline concentration of 10- M, the peak of twitch tension and light signal was almost fully maximal. The effects of isoprenaline at 10- M on the relationship between [Ca2+]i and tension were observed (Figs 7B and 9A). After application of isoprenaline, the transient change in the light signal, triggered just after the onset of tetanic stimulation, increased, and it accompanied a transient tension change. At lower [Ca2+]o, a larger transient contraction was followed by a tonic contraction. Upon increasing the [Ca2+]O, the tonic contraction became greater and exceeded the initial phasic contraction, which accompanied the larger light signal. Isoprenaline significantly shifted the relationship between [Ca2+]i and tension to the right, which was recognized as an increase in K, (034 + 003 /M, n = 6 in control; 056 + 007 /tM, n = 6 at 10-7 m-isoprenaline,

0. OKAZAKI AND OTHERS

232

B

A

1.05

1.05 0

0 0.5

A

6s L Down 1 -0.5 0 a 4s ~~~~~~~~~~~~~~~.Up 2 * 6s

0.5 [Ca2l; (PM)

0

*Down 2 1.0

0

1.0

2.0

[Ca2] (PM) Fig. 8. Relationship between [Ca2+]i and tension in tetanic contraction. Measured points were fitted using the Hill equation (see text). In A, [Ca2+]i and tension were measured at 2, 4 and 6 s after the start of tetanic stimulation. This was determined from the record shown in Fig. 7A. In B, reproducibility of tetanic contraction was examined. 'Up' and 'Down' represent changing [Ca2+]o from low to high and from high to low, respectively. An increase and decrease in [Ca2+]0 were repeated 2 times (labelled as 1 and 2). Note the different abscissa scale in A and B.

A

B

1.0

1.0

a 0

Control

C

o

0

Control (

='

Isoprenaline

(10/ M)

/0

0

0.50

5-

soprenaline

/

(107M)

0~~~~~~~~~~~~~~~~0 0

0.5

[Ca2]i (,uM)

1-0

0

0-5

[Ca2]i

1.0

(PM)

Fig. 9. Effects of isoprenaline on [Ca2+]i-tension relationship. In A, isoprenaline (10-7 M) shifted the relationship to the right and the slope was less steep. 0, control; Z, 10-7 Misoprenaline. In B, t0-9 M-isoprenaline did not significantly change the relationship. 0, control; O, 10- M-isoprenaline.

significant change). In addition, the slope of the relationship became less steep and n was decreased by isoprenaline (4-3 + 0-4, n = 6 in control; 2-6 + 03, n = 6 in the presence of isoprenaline, significant change). The rightward shift of the [Ca2+]i-tension relationship was more marked at higher concentrations of [Ca2+]i as the slope of the relationship became less steep in the presence of isoprenaline (Fig. 9A). Tonic tension declined more in the presence than in the absence of isoprenaline.

Ji-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES 233 However, the [Ca2+]i-tension relationship was measured at 6 s after stimulation in the presence and absence of isoprenaline as mentioned in the Methods section. The value of Ki did not significantly change at 10-' M-isoprenaline (0-24 + 0 03 /SM, n = 4 in control; 029 + 004 itM, n = 4 in the presence of 10-9 M-isoprenaline; Fig. Bay K 8644 1-0 _

1.0

o

o Control

:0

o

0-5

-

/

0 Isoprenaline (10-7M) 0 Bupranolol

0

1.5

(106M)

3.0

[Ca2]i (#M)

Fig. 10. Recovery of [Ca2+]i-tension relationship after removal of isoprenaline. 0, control; O, 10-7 M-isoprenaline. In recovery (e), isoprenaline was replaced with bupranolol (1 /zM). Bay K 8644 (1 gM) was added to 20 mm [Ca2+]o in the control.

9B). At 10-8 M-isoprenaline, the change in K, was also not significant (0-29 + 0-05 /LM, n = 4 in control; 0-53 + 0-14 /M, n = 4 at 10-8 M-isoprenaline). However, as the slope of the [Ca2+]i-tension relationship was changed, K3 ([Ca2+]i required for 75% of maximal tension) was significantly increased at 10-8 M-isoprenaline (0-42 + 0-06 /tM, n = 4 in control; 1-0+0-20/M, n = 4 at lo-8M). At 10-9 M, there was no significant change in Ki. Therefore, 10-8 M-isoprenaline might be the critical concentration which induces the rightward shift of the [Ca2+]i-tension relationship at a higher

[Ca2+]i range. The recovery of the [Ca2+]i-tension relationship after removal of isoprenaline (10-7 M) was examined (Fig. 10). After confirming the rightward shift of the relationship, isoprenaline was removed and bupranolol (1 /M) was added to the solution. The concentrations of isoprenaline and bupranolol were the same as those used in Fig. 6 in which the time course of relaxation did not recover. The relationship between [Ca2+]i and tension recovered (three experiments). Therefore, the change in Ca2+ sensitivity of the contractile element by isoprenaline is reversible.

Effects of /3-adrenoceptor stimulation on maximal tension As mentioned in the previous section, tetanic tension reached the nearly maximal level at 20 mm [Ca2+]0, and the addition of isoprenaline did not significantly change the tension level, although [Ca2+]j further increased (1I-06 + 0-14 fLM, n = 6). In some cases, the developed tension in the solution with 20 mm [Ca2+]o and isoprenaline was less than that at 20 mm [Ca2+]o. In order to confirm whether the maximal tension was

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234

changed by isoprenaline, [Ca2+], was further raised using Bay K 8644. Twitch responses were not triggered between tetanic contractions, because the tetanic tension became slightly smaller when the twitch contractions were triggered repetitively. When Bay K 8644 (1 /M) was added to 20 mm [Ca2+]0, the light signals A

B

f

-2.1

Lf -

-2.3

-

-2.5

Isoprenaline -2.5

b

~~~~-3.0 -3.5

Burnl-3.0 Buraoll-3.5

a

Bupranolol

C

Isoprenaline a

8s

25 mN/mM

Nmm2

7s

Fig. 11. Change in maximal tension in tetanized muscle. In A, isoprenaline was added to the solution with 20 mm [Ca2+]0 and Bay K 8644 (1 uM). a, light signal and tension in 20 mm [Ca2+]0. The light signal is scaled down. b, Bay K 8644 (1 /M) was added to 20 mm [Ca2+]o solution. c, 10-7 M-isoprenaline was added to the solution with 20 mm [Ca2+]0 and Bay K 8644. Note that an initial twitch-like tension appeared before the large phasic light signal but [Ca2+]i at the twitch-like tension was higher than that in a and b. In B, the preparation was immersed in the solution containing 20 mm [Ca2+]O, Bay K 8644 (1 /M) and isoprenaline (10-7 M). Then, isoprenaline was removed and bupranolol (1 uM) was applied.

increased but the increase in tension was only slight if any (Fig. 1 lA b). The [Ca2+], in the solution with 20 mm [Ca2+]o and Bay K 8644 was 2-28 + 0-43 /tM (n = 5). The addition of isoprenaline (10-7 M) to the solution containing 20 mm [Ca2+]o and Bay K 8644 induced a large phasic change in the light signals and tension (Fig. 1 lA c). The twitch-like phasic contraction with a rapid rise was triggered by an increase in the light signals which appeared just before the large phasic light signal. [Ca21], in the large phasic light signal in the presence of Bay K 8644 (1 /M) and isoprenaline (10-7 M) was 5-9 + 2-0 #m (n = 3). The phasic tension in the presence of isoprenaline and Bay K 8644 was greater (30%) than the maximal tension in the absence of isoprenaline. The tonic tension in the presence of isoprenaline and Bay K 8644 gradually decreased during tetanic stimulation; slight difference (10%) between the tension in the presence and absence of isoprenaline was recognized (thrcc experiments). The decline of tonic tension during stimulation was similar to that observed in the solutions which contained isoprenaline but not Bay K 8644. In

f-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES 235 another experiment, Bay K 8644 was applied to the preparation which had been immersed in 20 mm [Ca2+]0 solution containing isoprenaline (10-7M). The light signals increased further but tension did not significantly change. After the replacement of isoprenaline with bupranolol (1 /M), the light signals decreased but tension did not significantly change (Fig. LIB). L, B -1.8

A

cf 10 mN

2-2.0

-2.0

-2.5 L -3.0 L

I

-

I

25 -3.0

b

/

a

a C

C

[10 mN/mM2

1mNm2 8s

9s Fig. 12. Effects of isoprenaline (10-7 M) on the tetanic contraction in the papillary muscles of young rat. Upper figures, light signals. Lower figures, tension. In A, tetanus was induced in 20 mm [Ca2+]0 (a), and isoprenaline was added- (b). Bay K 8644 (1 ,M) was applied to the solution with 20 mm [Ca2+]O and isoprenaline (c). Stimulation period was 8 s. In B, Bay K 8644 (1 pM) was applied to preparation (b) after observing the tetanus in the 20 mm [Ca2+]O solution (a). Isoprenaline was added to the solution containing 20 mm [Ca2+]0 and Bay K 8644 (c). Stimulation period was 9 s.

Winegrad (1984) has suggested that /-adrenoceptor stimulation enhances the maximal tension of young rats severalfold. This was examined using tetanic contraction (three experiments). The addition of isoprenaline (0-1 ItM) to the 20 mm [Ca2+]o solution increased [Ca2+]i from 1-6 to 4 0 /M but tension increased only 10 % during the early phase of tetanic contraction. However, the tension decreased and there was no difference at as early as 8 s after the start of stimulation (Fig. 12A). Bay K 8644 added to the 20 mm [Ca2+]. solution with isoprenaline produced a large light signal (10-5 /M) at the initial phase and an increase in tension (30% greater than the maximal level of 20 mm [Ca2+]o plus isoprenaline). In the presence of both Bay K 8644 and isoprenaline, a rapid rise in the initial twitch-like tension was noticed, and this appeared prior to the large light signal. The results in young rats were qualitatively similar to those observed in ferret ventricular muscle. The tonic tension in the presence of Bay K 8644 and isoprenaline decreased, reaching a level only slightly greater than that in the solution with 20 mm [Ca2+]O and isoprenaline (16 %). In other experiments, Bay K 8644 was applied to the 20 mm [Ca2+]0 solution and then isoprenaline was added to the solution (Fig. 12B). Bay K 8644, added to

236

0. OKAZAKI AND OTHERS

20 mm [Ca2+]0, increased [Ca2+]i from 1P7 to 4 0 /tM and the tension was increased by 23 %. When isoprenaline was added to the solution with 20 mm [Ca2+]. and Bay K 8644, [Ca2+]i reached 9-7 and 4-8 ,M at the initial and later phase, respectively. The tension at the initial phase was slightly greater (16 %) than that in 20 mm [Ca2+]. plus Bay K 8644, but no significant change was observed at the later phase. DISCUSSION

Twitch potentiation by isoprenaline /-Adrenoceptor stimulation by isoprenaline potentiated the twitch tension and light transients, which accompanied the change in their time courses (Fig. 1). In addition, the [Ca2+]i-tension relationship in tetanic contraction was shifted to the right without a marked change in the maximal tension. The results suggest (1) an increase in Ca2+ release, (2) enhancement of Ca2+ removal and (3) a decrease in the Ca2+ sensitivity of the contractile element. The present results are qualitatively similar to those of previous studies (Allen & Kurihara, 1980; Morgan & Blinks, 1982; Kurihara & Konishi, 1987; Endoh & Blinks, 1988; McIvor et al. 1988). The two factors which curtail twitch tension, faster removal of Ca2+ from myoplasm and a decrease in Ca2+ sensitivity, can be overcome by a larger increase in Ca2+ release from SR; and an increase in Ca2+ release from SR is a major factor in twitch potentiation by isoprenaline. The increase in the Ca2+ release from the SR is due to an increased Ca2+ current (Fabiato, 1983; Reuter et al. 1983) and an increase in Ca2+ content in SR results from enhancement of Ca2+ uptake (Tada et al. 1974; Tada & Katz, 1982). These mechanisms occur due to the phosphorylation of Ca2+ channel protein and phospholamban in SR as suggested previously.

Changes in the time course of tension and light transients The significant changes in the time course of tension, which were induced by isoprenaline, were observed in the time-to-peak tension and relaxation time (Figs 3 and 4). The faster time-to-peak tension was partly due to the faster time-to-peak light which was probably related to the faster Ca2+ release from the SR. An increase in Ca2+ content in SR following an enhancement of Ca2+ uptake produces sufficient Ca2+ gradient across the SR membrane to induce the faster Ca2+ release from the SR. Recently, Hoh, Rossmanith, Kwan & Hamilton (1988) reported that adrenaline increases the cross-bridge cycling rate and that this is the major factor in the faster time-to-peak tension in ,-adrenoceptor stimulation in rats. The faster time-to-peak tension observed in the initial phase of tetanic contraction in the presence of both Bay K 8644 and isoprenaline is also due to the faster cross-bridge cycling rate caused by the synergistic action of cyclic AMP and the increased [Ca2+],. The faster Ca2+ release and the faster cross-bridge cycling rate might be a mechanism of the shortening of the time-to-peak tension. Relaxation is accelerated by ,3-adrenoceptor stimulation (Tsien, 1977). A significant effect on the acceleration of the relaxation was observed in TR50 (Fig. 4; Sonnenblick, 1962), although the change was small. There are at least four mechanisms which determine the time course of relaxation: (1) the rate of Ca2+

/3-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES 237 removal from myoplasm by SR, (2) Ca2+ sensitivity of the contractile element, (3) rate of cross-bridge detachment, and (4) the geometrical factor in the preparation. The result of the faster falling phase of the light signal by isoprenaline suggested that the accelerated falling phase of the light transients induced by 8-adrenoceptor stimulation reflects the faster Ca2" removal from myoplasm by SR (Tada et al. 1974; Tada & Katz, 1982; Kranias, Garvey, Srivastava & Solaro, 1985). However, the dose dependence of the falling phase of the light signals and the half-relaxation time (TR50) differed (Figs 4 and 5). In addition, the removal of isoprenaline from the solution restored the decay time course of the light but did not change the relaxation. These results suggested that the decay of Ca21 transients, which is partly related to Ca21 uptake by SR, is not the only factor in the determination of the relaxation time, as has been previously suggested (Allen & Kurihara, 1980; Kurihara & Allen, 1982; Endoh & Blinks, 1988). However, the faster Ca2+ uptake by SR might work as a back-up system to remove the Ca2+ which dissociates from the binding sites at a faster rate when Ca2+ binding to troponin C is decreased (Robertson, Johnson, Holroyde, Kranias, Potter & Solaro, 1982). The dose dependence and reversibility of the time courses of tension and light transients suggest that phosphorylation and dephosphorylation of phospholamban, troponin I and other proteins do not occur in parallel. The decrease in Ca2+ sensitivity of contractile elements by isoprenaline is due to cyclic AMP-dependent phosphorylation of troponin I as reported previously (Ray & England, 1976; Solaro et al. 1976; McClellan & Winegrad, 1978; Mope, McClellan & Winegrad, 1980; Herzig et al. 1981; Yamamoto & Ohtuki, 1982; Kranias et al. 1985). The decrease in Ca2+ sensitivity, estimated using the tetanic contraction, appeared at about 10-8 M at which significant change in TR50 was observed. This suggested that the decrease in the Ca2+ sensitivity is partially responsible for the faster halfrelaxation time. However, the faster relaxation was still observed after the removal of isoprenaline which restored the Ca2+ sensitivity (Fig. 10). A similar non-parallelism between the Ca2+ sensitivity and the relaxation has been recognized in the relationship between [Ca2+]i and tension in twitch responses in which both isoprenaline and acetylcholine were used (McIvor et al. 1988). Therefore, other factors, for example, the detachment of cross-bridges, might be important in the determination of the time course of relaxation, as has been suggested by Hoh et al. (1988). Phosphorylation of C-protein also occurs in parallel with the phosphorylation of troponin I (Kranias et al. 1985) and this is related to the faster relaxation in amphibian cardiac muscle (Hartzell, 1984). However, the role of cyclic AMPdependent C-protein phosphorylation in mammalian cardiac muscle is not clear. In addition to these factors, a geometrical factor should be considered, particularly in the Ca2+-overloaded condition. In the presence of high concentrations of isoprenaline, the light signals transiently increased in the relaxation phase (extralight) (Allen & Kurihara, 1980; Kurihara & Konishi, 1987). This was also recognized in the ferret papillary muscle, although the magnitude varied from preparation to preparation (Fig. ID). When the extra-light in the relaxation phase was remarkable, a hump was noticed on the relaxation phase. Since asynchronous contraction was produced in the Ca2+-overloaded condition, total tension was less than that in synchronous contraction (Wier, Kort, Stern, Lakatta & Marban, 1983). Therefore,

0. OKAZAKI AND OTHERS 238 when extra-light appeared at high concentrations of isoprenaline, the time course of relaxation was seriously influenced by asynchronous activation and deactivation in each cell. This probably influences TR?525 which was less sensitive to isoprenaline. Ca2± removal from myoplasm is required prior to relaxation but is not critical for determining the relaxation time.

Relationship between [Ca2+]i and tension in tetanic contraction Tetanic stimulation produces a rather steady contraction following the initial phasic tension (Yue et al. 1986). The initial twitch-like contraction might be triggered by a full-size action potential at the beginning of the tetanic stimulation, because the size of the transient tension was almost the same level as the twitch tension which was triggered before tetanic stimulation (Fig. 7) (Marban, Kusuoka, Yue, Weisfeldt & Wier, 1986). Accordingly, sustained depolarization with incomplete action potentials causes the tonic tension (S. Kurihara & K. Hongo, unpublished observation). A drastic change in the intracellular ionic environment (for example H+) which influences the tension development (Marban & Kusuoka, 1987) might not occur in the tetanic contraction for 6-10 s, although the tonic tension gradually declines. The gradual decline of the tonic tension is partly due to an internal shortening of the preparation which is induced by the sustained strong contraction. One of the disadvantages of tetanic contraction is the release of endogenous catecholamines by strong repetitive stimulation. The leftward shift of the relationship of [Ca2+]i and tetanic tension by the application of bupranolol (1 /UM) suggested that endogenous catecholamine is released by the tetanic stimulation. However, the endogenous catecholamine does not seriously influence the result, because the [Ca2+]i-tension relationship obtained by changing the strength of the tetanic stimulation did not significantly differ and the same stimulation strength was used throughout the experiment in the presence and absence of isoprenaline to avoid the change in the endogenous catecholamine release. A decrease in the Hill coefficient by isoprenaline which is not reported in the sarcolemma-free preparations is due to a change in the intracellular ionic environment produced following an increase in cyclic AMP concentration (McClellan & Winegrad, 1978; Mope et al. 1980; Herzig et al. 1981). The decline of tonic tension in tetanus in the presence of isoprenaline might be also due to the change in the intracellular ionic environment (for example H+). These results suggest that cyclic AMP changes the apparent Ca2+ sensitivity of the contractile element and the cooperativity of the contractile element as net effects in the intact preparations. Change in maximal tension by /3-adrenoceptor stimulation The present results suggested that /3-adrenoceptor stimulation does not produce a greater change in the maximal tension level, when isoprenaline was added to 20 mm [Ca2+]0 solution. However, when isoprenaline was added to the solution containing 20 mxv [Ca2+]0 and Bay K 8644, the initial twitch-like tension in the presence of isoprenaline was 30 % larger than the maximal level without isoprenaline (Fig.11 A). One possible explanation of the large twitch-like phasic tension is the synchronous contraction of each cell at a faster rate, caused by the synergistic action of the faster cross-bridge cycling induced by cyclic AMP (Hoh et al. 1988) and an increase in

/3-ADRENOCEPTOR STIMULATION IN CARDIAC MUSCLES

239

[Ca2+]i. However, the initial large tension gradually declined and the tonic tension, measured 6 s after the stimulation, was almost the same as that without isoprenaline, even though [Ca21], was higher than that in the solution with 20 mm [Ca2+]0 and Bay K 8644. In another experiment shown in Fig. 1 B, the light signals with and without isoprenaline were significantly different but the tension did not change significantly. It has been postulated that the enhancement of the maximal tension by cyclic AMP was marked in the young rat in which myosin isozyme V1 is dominant (Winegrad, 1984). However, isoprenaline showed similar results in the young rat and in the ferret (Figs 11 and 12). These results suggest that isoprenaline does not produce a large change in the maximal tension level and conflicts with the hypothesis of Winegrad (1984) that fl-adrenoceptor stimulation increases the maximal tension severalfold. Therefore, cyclic AMP does not produce a great change in the maximal tension level in mammalian cardiac muscles. These results support the finding that there is no significant change in the maximal ATPase by isoprenaline (Kranias et al. 1985). The authors thank Dr J. R. Blinks for supplying aequorin. We also thank Mrs M. Shibuya for reading the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture.

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