Gen. Pharmac.Vol. 23, No. 5, pp. 847-852, 1992 Printed in Great Britain. All rights reserved

0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

THE RESPONSES TO PHORBOL ESTERS WHICH STIMULATED PROTEIN KINASE C IN CANINE PURKINJE FIBERS HIROYASU SATOH,1. TOSHIKATSUNAKASHIMAI and KATSUHARUTSUCHIDA2 Department of Pharmacology, Nara Medical University, Kashihara, Nara 634, 840 Shijo-cho and 2Research Center, Talsho Pharmaceutical Co. Ltd, Ohmiya, Saitama 330, Japan

(Received 28 January 1992) Abstract--1. The effects of phorbol esters on canine Purkinje fibers were examined using conventional microelectrode techniques. 2. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (PDB), which are specific activators of protein kinase C (PKC), decreased the action potential amplitude and the maximum rate of depolarization (/;'m~) at 3 x 10-7 M or higher. These phorbol esters had little effect on the resting potential. 3. PDB (1-3 x 10-7 M) also reduced the contractile force, accompanied with initial increase (in 5 out of 8 experiments), whereas TPA did not decrease it to any significant extent. 4. An inactivate analog of phorbol esters, 4-alpha-phorbol-12,13-didecanoate (PDD), decreased the action potential amplitude and lYm~,and slightly increased the action potential duration. However, PDD failed to produce any inotropic effect. 5. Post-rest potentiation of the contractile force after a rest from stimulation for 30 sec was inhibited in the presence of 3-10 x 10-TM TPA or 3 x 10-TM PDB. 6. Isoproterenol 10 -7 M augmented the action of PDB 3 x 10-7 M. 7. These results suggest that activation of PKC may modulate myocardial Ca 2+ homeostasis and influence the excitation-contraction process.

INTRODUCTION The physiological activator of protein kinase C (PKC), diacylglycerol, is transiently produced from inositol phospholipids in response to extracellular signals. Like endogeneous diacylglycerol, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) activate P K C and increase the affinity of Ca 2+ to this enzyme (Nishizuka, 1984). Recently, the physiological role of P K C has been reported. The activation of P K C modulates several types of ionic channels and enhances the slow inward current (Baraban et al., 1985; DeRiemer et al., 1985; Strong et al., 1987). We also observed that T P A increased the slow inward current, and that in spontaneously beating rabbit sino-atrial (SA) node cells, T P A and PDB induced dysrhythmia; whereas additional application o f isoproterenol or phenylephrine exaggerated it (Satoh and Hashimoto, 1988). Furthermore, in voltage-clamped SA node cells, transient inward current also occurred. Thus, we suggest that the activation of P K C by phorbol esters may cause cellular calcium overload in rabbit SA node cells. In the present experiments, using canine Purkinje fibers, we examined whether or not the activation o f P K C causes the same effects as in rabbit SA node cells. Also, to decide whether the effects induced by phorbol esters are due to P K C activation, we

*To whom all correspondence should be addressed.

compared with other two phorbol esters (activator and non-activator of PKC). MATERIALS AND METHOI~ Preparations and recording Purkinje fibers (2-3 mm dia) obtained from dog heart were placed in an organ bath and superfused with Tyrode solution at a temperature of 36°C, as described previously (Satoh and Vassalle, 1985; Satoh and Hashimoto, 1986). One end of the preparation was fixed on the paraffin base of the bath, and the other end was connected to a force displacement transducer (Nihon Kohden SB-IT, Japan) using a fine nylon thread. Field stimulation at 1 Hz with pulses of 5 msec duration and twice threshold voltage was used. The action potential recorded using a standard glass microelectrode technique was registered on an oscilloscope (Nihon Kohden VC-I0)and photographed (Nihon Kohden RLG-6201). The contractile force was recorded with a pen-recorder (Nihon Kohden RJG-4124).

Solutions The composition of the Tyrode solution (raM) was NaCI 137, KC14.0, MgC12 1.0, CaCI2 1.8, NaH2PO 4 0.4, NaHCO3 12.0 and glucose 5.5. The solution was bubbled with 95%02 and 5%CO 2. The pH was adjusted to 7.4. The drugs used were 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma, U.S.A.), 4-beta-phorbol-12,13-dibutyrate (PDB) (Sigma, U.S.A.), and 4-alpha-phorbol-12,13-didecanoate (PDD) (Sigma, U.S.A.). Phorbol esters were dissolved in dimethyl sulfoxide (DMSO), which was diluted 7000-20,000 times in the perfusion medium. This concentration of DMSO did not cause any effects on the Purkinje fibers. Since the Tyrode solution in the bath was completely exchanged within 3 rain 847

HIROYASUSATOH el al.

848

+ISP lO'r M

3 x 1 0 "7 M

Control

-L

-L

-L\

_K

-k

-L

\

I -i\

-K5

\ j\I

TPA

ill

PDB

PDD

\

j\

\

1\

_Is

2 0 0 ms

Fig. 1. Effects on action potential in canine Purkinje fibers in the presence of TPA, PDB and PDD. The preparations were usually stimulated at 1 Hz. On right panel of PDB, the stimulation (3 Hz) was stopped to observe the occurrence of the delayed afterdepolarization. Different fibers were used for each drug. The short line before each action potential represents 0 mV. and the effects of the drugs reached a steady-state within 10min, the data were obtained approx. 10-15 min after changing to a new solution.

Statistical analysis Values are represented as mean + SEM. The differences between mean values were analyzed by the Student's t-test for paired data, and were considered significant when P values were less than 0.05.

RESULTS The action potential configuration in canine Purkinje fibers in the absence and presence o f TPA, P D B or P D D (3 x 10 -7 M ) are s h o w n in Fig. 1. A t concentrations o f 10 -7 M or lower, these p h o r b o l esters h a d no effect. T P A and P D B [specific activators for P K C (Castagna et al., 1982; K r a f t et al., 1982)]

reduced the action potential amplitude and the m a x i m u m rate o f depolarization (Vmax), and P D D , an inactivate analog (Castagna et aL, 1982; K r a f t et al., 1982), also decreased them. These data are summarized in Table 1. The agents had no effect on the resting m e m b r a n e potential. T P A and P D B tended to decrease the action potential duration at 50 and 90% repolarizations; and addition o f l0 -7 M isoproterenol ( + I S P ) decreased the durations in the presence o f P D B but had no effect in the presence o f TPA. On the other hand, P D D also decreased the action potential duration, but increased it by the addition o f 1 0 - 7 M isoproterenol. These results indicate that activation o f P K C would affect the repolarization. At 3 x 10-TM, T P A and PDB decreased the contractile force, as shown in Fig. 2(A). Since these responses were not resumed completely after

Table I. The effectsof phorbol esterson action potential parametersin canine ventricularmuscle Resting Duration (msec) potential Amplitude Vmax n (mY) (mY) (V/see) 50% 75% 90% Control TPA 10-SM 3x10 -s 10-7 3x 10-7 l0 -6 Control PDB 10-TM 3x 10-7 Control PDD 10-TM 3x 10-7

10 5 5 7 l0 3 5 5 5 5 5 5

-83+_3 -84+_2 -83+-2 -82+_3 -82+_4 -80+_2 -80+-3 -79+_2 --75+4 -80+-3 -79+_1 -78+_3

119+_4 118+_2 116+_3 109 +-4 96+_4** 98+1" 109 +-4 106+4 93+6" 105+_4 103+_3 96_+5*

213+_3 206_+7 203_+_7 202+_5 205+_3* 187 + 7" 257+-3 245+_3 242+3" 228+_3 207+_5 196+_3"

The values represent mean + SEM. *P < 0.05; **P < 0.01. n: Number of experiments.

118_+4 107+-5 120 +- 6 120_+4 114+_4 ll7_+l* 113 +-4 107_3 100+2 108+-3 110+_3 109+_4

162+_3 165+-6 164+-8 160 +- 7 151 +_4 153+9 167 +-6 164+_3 163+4 156+_4 156+_2 155+6

216 +- 5 215+6 213+-5 207+_4 207+_4 204+-6 223+_2 208+-3 207+2 220_+3 220+_7 225+_5

P h o r b o l esters o n c a r d i a c P u r k i n j e fibers

A

TPA 3 x 10.7 M

849

B

lmi._n

-I-10 %

PDD

o ~

PDB 3x 10.7 M PDD 3x 10-7M

~

,

~

TPA

1rain

1rain

-50

-log M Fig. 2. Effect o f p h o r b o l esters o n the c o n t r a c t i l e f o r c e in c a n i n e P u r k i n j e fibers. (A) D e p r e s s i o n in the

contractile force after application of phorbol esters. TPA and PDB decreased the contractile force (although PDB increased it initially), whereas PDD did not affect it. (B) Dose-response curves for the phorbol esters. At a dose of over 10-* M, PDB caused a significant negative inotropic effect. The control values were 52 + 7 nag (n = 8) in TPA, 60 + 5 mg (n = 8) in PDB and 57 + 6 mg (n = 5) in PDD. wash-out, a different preparation for each drug was used. PDB initially increased and then decreased the contractile force in 5 out of 8 experiments. The percentage changes in the contractile force are summarized in Fig. 2(B). At lower concentrations (1-3 x 10 -8 M), TPA tended to increase the contractile force. The decrease in the contractile force was stronger in the presence of PDB than TPA. PDD did not affect it. The higher frequencies of stimulation (2 or 3 Hz) induced further decrease in the contractile force in the presence of 3 x 10 - 7 M TPA or PDB, but this is not shown in Fig. 2(B). In cardiac muscle, when repetitive stimulation (1 Hz) is resumed after a rest period, the first contractile force at re-started stimulation is potentiated (post-rest potentiation). Then, the contractile force declines, and is in turn gradually increased. In the present experiments, the amplitude of the contractile force by the post-rest potentiation after rest for 30 sec became smaller in the presence of 3 x 10 -8 to 3 x 10-7M TPA (Fig. 3A). PDB also depressed it, while PDD did not have any effect (Figs 3B and C). These percentages are represented in Fig. 4. PDB was further depressed by 28% at 3 x 10 - 7 M. These results indicate that the negative inotropic effect induced by the phorbol esters might be directly due to the activation of PKC.

As shown in Fig. 5 (upper panel), the addition of 10-7M isoproterenol ( + I S P ) in the presence of 3 x 10 - 7 M PDB increased the contractile force. PDB alone decreased it. The amplitude of post-rest potentiation (rest for 20 see) was depressed by PDB, and was depressed further by the addition of isoproterenol. PDB decreased the amplitude and duration of action potentials shown in Figs 1 and 5 (lower panel). Additional application of isoproterenol potentiated these decreases and depressed the plateau of the action potential. These results suggest that activation of PKC might increase the cellular calcium level.

DISCUSSION

Tumor-promoting phorbol esters such as TPA and PDB activate Ca 2+- and phospholipid-dependent protein kinase C (PKC). PKC appears to be an important component of membrane signal transduction for a variety of biological activators of substances (Nishizuka, 1984). A stimulant specifically binds to a cell surface receptor, which in turn activates a phospholipase C for phosphatidylinositol (PI) located on the cytosolic face of plasma membrane.

Table 2. The percentage changes in the contractile force induced by phorbol esters in canine ventricular muscle Contractile force Control TPA 10-SM 3 x 10 -s 10 -7 3 x 10 -7 10 -6 Control PDB 10-TM 3 x 10-7 Control P D D 10-TM 3 x 10-7

Post-rest potentiation

n

(%)

n

(%)

15 11 8 8 10 5 8 8 8 5 5 5

52 + 7 mg 5_+4 6_+3 -4_+4 -8_+5 -7_+3 60 _+ 5 mg -43_+6*** - 5 0 + 8*** 57 _+6 mg 3_+2 5_+3

6

134 + 6 mg

5 6 6 5 4 4 4 5 5 5

-5_+4 -9_.3 -16_+5"* -18_+3"* 155 _+ 7 mg -10_+8 - 2 8 _+ 5*** 142 _+ 7 mg - ! _+5 1 _+6

The values represent means + SEM. **P < 0.01, ***P < 0.001. n: Number of experiments.

HIROYASUSATOH et al.

850

A

B T P A 3 x 10 "e M

Control

Control

PDB 3x10~

M

Control

P D D 3 x 1 0 "~ M

a

lx10

"7 M

C

b

3 x 1 0 -7 M O~

1 min

Fig. 3. Post-rest potentiation of the contractile force. (A) and (B) When stimulation was started after a rest for 30sec, the post-rest potentiation was attenuated in the presence of TPA and PDB (0.3-3 x 10-7 M). The aftercontraction did not appear when the stimulation stopped. (C) PDD did not depress the post-rest potentiation. The control values were 134 + 6 mg (n = 6) in TPA, 155 + 7 mg (n = 4) in PDB and 142 + 7 mg (n = 5) in PDD. The active enzyme phophatidylinositol- 1,4,5-bisphosphat¢ (PIP 2) into the cytosol and leaves myo-inositol1,4,5-trisphosphate (IP3) and diacylglycerol in the membrane. IP 3 mobilized Ca 2+ stored in the sarcoplasmic reticulum (Berridge, 1986; Williamson et al., 1985). Diacylglycerol or phorbol esters activate PKC by stabilizing its insertion into the membrane. Activated PKC then phosphorylates substrate proteins, thereby eliciting the physiological responses (Berridge, 1986; Streb et al., 1983). In the present experiments, both TPA and PDB, which are shown to specifically activate PKC (Castagna et al., 1982; Kraft et al., 1982), caused a negative inotropic effect. PDB decreased it more strongly than TPA. PDD, which is not an activator of PKC (Castagna et al., 1982; Kraft et aL, 1982), did not affect the contractile force. These results are consistent with those of Leatherman et aL (1987) and Yuan et aL (1987). We used H-7, an inhibitor of PKC, which caused complicated responses to cardiac muscle, and also to cardiac myocyte (Satoh, 1992). Thus, we established whether the phorbol esters-

+5 %

--30

'

/

PDB~*** M

--log Fig. 4. Dose-response curves for depression of post-rest potentiation. At 3 x 10-TM or more, TPA (n =6) depressed the amplitude of post-rest potentiation; and PDB (n = 4) did so more strongly. PDD (n = 5) did not have any effect.

induced effects were due to PKC activation by using two kinds of phorbol esters (activators and non-activator of PKC). The phorbol esters did not cause marked eleetrophysiological effects, except for the decreases in Vm~ and amplitude. Fast sodium channel blockers (class I antiarrhythmic agents) have been shown to reduce the contractile force of cardiac muscle (Satoh and Vassalle, 1985; Honerjager et al., 1986). There is the possibility that the negative inotropic effect was due to the inhibition of Na + entry and the subsequent decrease in the cytosolic free Ca 2+ level via N a - C a exchange. However, PDD, a non PKC activator, did not decrease the contractile force, despite P D D inhibited I?,ax. If the cellular free Ca 2+ level was reduced by fast Na channel inhibition, the contractile force should be also suppressed in the presence of PDD, as TPA and PDB did. But Vmax inhibition induced by the phorbol esters was less marked. Therefore, this suggests that the negative inotropic effect might not be due to the inhibition of Na + entry, but is directly due to the activation of PKC. We have already shown that in rabbit SA node cells, TPA and PDB induced dysrhythmia in the presence of isoproterenol or phenylephrine relating to afterdepolarization. The transient inward current was induced in the presence of both PDB and isoproterenol in the rabbit SA node cells, which indicates calcium overload (Satoh and Hashimoto, 1988; Tsien et al., 1982). Thus, phorbol esters was expected to induce calcium overload in canine Purkinje fibers as well. If TPA and PDB elevated cellular Ca 2+ via the activation of PKC, the contractile force should be enhanced initially before starting the negative inotropic effect. PDB caused the initial increase, but not in all experiments (see Fig. 2). TPA decreased, but at lower concentrations (1-3 x 10 -8 M) increased the contractile force. Satoh and Vassalle (1985) have demonstrated that cytosolic calcium overload depressed contractile force, and that in calcium overloaded Purkinje fibers induced by caffeine, the first

Phorbol esters on cardiac Purkinje fibers

851

mg

1 rain 2 0 0 ms 120 mV

°L I ~

k__ ,

~

I ~

~

:

~~,~=,,.~]

10 rng

Fig. 5. Responses to addition of isoproterenol during exposure to PDB. PDB (3 x 10-~ M) decreased the force, and additional application of isoproterenol (10 -7 M) increased it. After washout, the contractile force restored. The preparations were stimulated at 1 Hz. On the lower panel, the action potentials and contractile forces before stopping the stimulation were stopped are shown. The short lines represent 0 mV.

contractile force of the post-rest potentiation was depressed or abolished (Satoh and Vassalle, unpublished observation). In the present experiments, these phorbol esters had little effect. In calcium over-loaded Purkinj¢ fibers induced by caffeine, the contractile force was reduced, but the action potential configurations were unaffected (Satoh and Vassalle, 1985). Thus, the mechanical effect induced by calcium overload is not necessarily accompanied with electrical effects. The first contractile force after the onset of stimulation would be largely reflected by the amount of Ca z+ release from the sarcoplasmic reticulum (Morad and Cleemann, 1987). In this experiment, the post-rest potentiation of contractile force was depressed in the presence of TPA or PDB, but not in the presence of PDD. Additional application of isoproterenol decreased the post-rest potentiation further. It appears, therefore, that the negative inotropic effect might be due in part to cellular calcium overload. This is also supported by the evidence that TPA and PDB increased the cellular calcium concentration using fluorescent calcium indicator, fura-2 (unpublished observation). More recently, however, Leatherman et al, (1987) have shown that TPA produced concentration- and time-dependent decreases in cytosolic free Ca 2- level as well as contractile force, but that PDD did not affect them in cultured chick heart cells. In addition, they reported that a decrease in intracellular Ca '+ level may be unrelated to effects mediated through the adenylate cyclase system and to a change in intracellular pH. If so, the negative inotropic effect in canine Purkinje fibers would be responsible for the reduction in cytosolic free Ca '+. Furthermore, there are still other possibilities. In Swiss 3T3 cells, it has been shown that phorbol esters increase N a - H exchange and N a - K pump activity (Dicker and gozengurt, 1981; Moroney et ai. 1978). These mechanisms may also affect modulators of cardiac muscle contractility. Thus, PKC could play a role as a possible component of the complex mechanisms that regulate the muyocardial contractile state. These mechanisms in cardiac muscle remain unclear.

Further studies are needed to elucidate the role of PKC in regulating contractility and its precise mechanism in excitation-contraction coupling.

REFERENCES Baraban J. M., Snyder S. J. and Alger B. E. (1985) Protein kinase C regulates ionic conductance in hippocampal pyramidal neurones: electrophysiological effects of phorbol esters. Proc. hath. Acad. Sei. U.S.A. 82, 2538-2542. Berridge M. J. (1986) Intracellular signalling through inositol triphosphate and diacylglycerol. Biol. Chem. Hopp-Seyler 367, 447-456. Castagna M., Takai Y., Kalbuchi K., Sano K., Kikkawa U. and Nishizuka Y. (1982) Direct activation of calciumactivated, phospholopid-dependent protein kinase by tumor-promoting phorbol esters. J. biol. Chem. ?$7, 7847-7851. DeRiemer S. A., Strong J. A., Albert K. A., Grcengard P. and Kacmarek L. K. (1985) Enhancement of calcium current in Aplysia neurones by phorboi esters and protein kinase C. Nature 313, 313-316. Dicker P. and Rozengurt E. (1981) Phorbol esters stimulation of Nsa influx and Na-K pump activity in Swiss 3T3 ceils. Biochem. biophys. Res. Commun. 100, 433-441. Honerjager P., Loibl E., Steidl I., Schonsteiner G. and Ulm K. (1986) Negative inotropic effects of tetrodotoxin and seven class I antiarrhythmic drugs in relation to sodium channel blockade. Naunyn-Schmiedeberg's Arch. Pharmac. 332, 184-195. Kraft A. S., Anderson W. B., Cooper H. L. and Sando J. J. (1982) Decrease in cytosolic calcium/phospholipiddependent protein kinase activity following phorbol ester treatment of EtA thyroma cells. J. biol. Chem. 257, 13,193-13,196. Leatherman G. F., Kim D. and Smith T. W. (1987) Effect of phorboi esters on contractile state and calcium flux in cultured chick heart cells. Am. J. Physiol. 253, H205-H209. Morad M. and Cleemann L. (1987) Role of Ca2+ channel in development of tension in heart muscle. J. molec, cell. Cardiol. 19, 527-553. Moroney J., Smith A., Tomei L. D. and Wenner C. E. (1978) Stimulation of ~Rb + and 32pi movements in 3T3 cells by prostaglandins and phorbol esters. J. cell. Physiol. 95, 287-294.

852

H1ROYASUSATOH et aL

Nishizuka Y. (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308, 693-698. Satoh H. (1992) Inhibition in L-type Ca 2+ channel by stimulation of protein kinase C in isolated guinea-pig ventricular cardiomyocytes. Gen. Pharmac., in press. Satoh H. and Hashimoto K. (1986) An electrophysiological study of amiloride on sino-atrial node cells and ventricular muscle of rabbit and dog. Naunyn-Schmiedebergs Arch. Pharmac. 333, 8340. Satoh H. and Hashimoto K. (1988) On electrophysiological responses to phorbol esters which stimulate protein kinase C in rabbit sino-atrial node cells. Naunyn-Schmiedebergs Arch. Pharmac. 337, 308-315. Satoh H. and Vassalle M. (1985) Reversal of caffeineinduced calcium overload in cardiac Purkinje fibers. J. Pharmac. exp. Ther. 234, 172-179. Streb H., Irvine R. F., Berridge M. J. and Schulz I. (1983)

Release of Ca 2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-l,4,5-trisphosphate. Nature 306, 67-69. Strong J. A., Fox A. P., Tsien R. W. and Kaczmarek L. K. (1987) Stimulation of protein kinase C recruits over calcium channels in Aplysia bag cell neurons. Nature 325, 714-716. Tsien R. Y., Pozzan T. and Rink T. J. (1982) T-cell mitogens cause early changes in cytoplasmic free Ca ~+ and membrane potential in lymphocytes. Nature 295, 68-70. Williamson J. R., Cooper R. H., Hoseph S. K. and Thomas A. P. (1985) Inositol triphosphate and diacylglycerol as intracellular second messengers in liver. Am J. Physiol. 248, C203~2216. Yuan S., Sunahara F. A. and Sen A. K. (1987) Tumorpromoting phorbol esters inhibit cardiac functions and induce redistribution of protein kinase C in pcrfused beating rat heart. Circulation Res. 61, 372-378.