J Mol
Cell
Cardiol22,
Protein
Noritsugu
725-734
Kinase Potassium Tohse,
(1990)
C Activation Current
Masaki
Enhances in Guinea-pig
the Delayed Rectifier Heart Cells
Kameyamal, Kazuo Sekigucki and Mario Ksnno
*, Mark
S. Shearman*
Department of Pharmacology, Hokkaido University School of Medicine, Sapporo 060, Japan, INational Institute for Physiological Sciences, Okazaki 444, Japan, and ‘Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan (Received 5 May 1989, accepted in revisedform
16 February 1990)
M. KAMEYAMA, K. SEKIGUCHI, M. S. SHEARMAN, AND M. KANNO. Protein Kinase C Activation Enhances the Delayed Rectifier Potassium Current in Guinea-pig Heart Cells. jounzal of Molecular and Cellular Cardiology (1990) 22, 725-734. The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na+- and K+-free external solution, the delayed rectifier K+ cument (ZK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol13-acetate (TPA). The amplitude of the ZK tail elicited by a return from a depolarizing pulse for 3 s at +50 mV to a holding potential of -30 mV was increased by 32 + 4% (mean + s.E., n = 6) after the external application of 1 nM TPA, and by 60 + 17% (n = 5) after 10 11~. The increase in ZK produced by 1 nM TPA was abolished by the inhibitor of PKC, I-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 FM). In addition, the synthetic diacylglycerol I-oleoyl-2-acetylglycerol (OAG, 125 PM) also increased ZK (58 + 9%, n = 3). PKC purified from bovine brain remarkably increased ZK ( 15 1 f 101 %, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve ofZx for the intracellular concentration action of PKC and/or altered Ca’+of Ca2+ was shifted to the left by 1 m TPA, suggesting a Ca ‘+-dependent sensitivity of ZK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 & 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 + 5% in outward component, and 3 f 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase ZK without affecting the other two currents. N. TOHSE,
KEY
WORDS:
Protein
kinase C; Delayed
rectifier
K+ current;
rntroduction Protein kinase C (PKC) is now widely accepted to be one of the key enzymes in intracellular signal transduction for various biologically active substances (Nishizuka, 1984). PKC, when activated, translocates from the cytosol to the plasma membrane (Dunphy et al., 1981; Yuan et al., 1987, and see review Hirasawa and Nishizuka, 1985), and thereby becomes eligible to modulate ion channels therein. Recent studies have indicated that purified PKC and phorbol esters that activate PKC, are capable of modulating ion channel activities in various types of cells (Baraban et al., 1985; DeRiemer et al., 1985; Farley and Auerbach, 1986; Madison et al., 1986; Llano and Marty, 1987; Strong et al., 1987; Brown and Higashida, 1988). We have previously reported that a phorbol ester 12-O-tetradecanoylphorbol13-acetate 0022-2828/90/060725
+ 10 $03.00/O
Heart;
Single
cell; TPA,
H-7;
OAG.
(TPA) increases the delayed rectifier K+ current (1~) in guinea-pig ventricular cells, through an activation of PKC, with little influence on the other currents in these cells (Tohse et al., 1987). However, some recent reports argued that phorbol esters modulate the Ca*+ current (1,-J in cardiac cells (Leatherman et al., 1987; Dosemeci et al., 1988; Lacerda et al., 1988; Satoh and Hashimoto, 1988). Therefore, we designed the present study in order to examine in detail whether the activation of PKC modulates cardia.c membrane currents, including Z,. We applied PKC intracellularly by a cell dialysis method (Mastuda and Noma, 1984), in addition to extracellular application of TPA and 1 -oleoyl-2-acetylglycerol (OAG), in isolated ventricular cells of guinea-pigs, and investigated their influences on ionic currents. From the results obtained, we conclude that 0 1990 Academic
Press Limitetd
726
N. Tohse et al.
PKC, when activated, increases IK in guineapig ventricular cells without affecting other ionic currents. Materials
and Methods
Isolation of cardiac cells Single ventricular cells of guinea-pig heart were obtained essentially by the same technique as described previously (Taniguchi et al., 1981; Isenberg and Klockner, 1982). Briefly, collagenase (0.04% w/v, Sigma type I) in a low-Cazf Tyrode solution (Ca2+ concentration: c. 60 ,UM) was perfused for 12 to 20 min through the coronary artery using a LangendorfI apparatus. The collagenase solution was washed out by high-K+, low-Clsolution (KB-solution, cf Isenberg and Klockner, 1982). The ventricular tissue was cut into small pieces, agitated gently in a small beaker with KB-solution, and filtered through a lopm stainless-steel mesh. The cell suspension was stored in a refrigerator (4°C) for later use. Solutions and materials The composition of normal Tyrode solution was (in mM): NaCl 143, KC1 5.4, CaCl2 1.8, MgClz 0.5, NaH2P04 0.33, glucose 5.5 and HEPES-NaOH buffer (pH 7.4) 5.0. A Na+and K+-free external solution was prepared by replacing NaCl and KC1 with equimolar choline Cl. A Na+-free internal solution in the recording pipette contained (in mM): KOH 110, KC1 20, MgC12 1.0, ATP-K2 5.0, creatine-phosphate-K2 5.0, EGTA 10, HEPES 5.0, and the pH (= 7.4) of the internal solution was adjusted by 90 to 100 mM aspartic acid. Concentrations of free Ca2+ (PCs 11 to PCs 6) in the internal solution were calculated according to Fabiato and Fabiato (1979) with the correction of Tsien and Rink (1980). The temperature of the external solutions was 34 to 36°C. TPA was dissolved in dimethyl sulfoxide as a stock solution (1 mhr). l-(5-Isoquinolinesulfonyl)-2methylpiperazine (H-7; Seikagaku Kogyo, Tokyo) was dissolved in water to make a 10 mM stock solution; 1-oleoyl-2-acetylglycerol (OAG, Sigma) was dissolved in 10% dimethyl sulfoxide to make a 5 mM stock solution. The stock solutions were diluted by the perfiusate to a given concentration. Nifedipine (3 PM;
Bayer, FRG) was used to block the Ca2+ current and was dissolved in ethanol to make a 10 mM stock solution. Purified PKC [type III(a); Kikkawa et al., 19871 was obtained from bovine whole brain as described by Shearman et al. (1989). For internal cellular dialysis, a solution of the enzyme was included in the pipette internal solution. The final concentration of the enzyme was 10 pg/ml (8.1 nmol Pi/min/ml for incorporation into H 1 histone). Recording techniques The whole-cell membrane currents were recorded by the patch clamp method (Hamill et al., 1981), using glass patch pipettes with a diameter of 3 to 4 pm. The resistance of the pipette was 1.8 to 2 MQ when filled with normal Tyrode solution. The pipette solution was connected through a Ag/AgCl wire to the input stage of a current-voltage converter with a feedback register of 100 Ma. Current and voltage signals were filtered at 2 kHz, digitalized by an AD converter (ADX-98, Canopus Electronics, Japan) at 100 Hz for a pulse duration of 3 s or at 2 kHz for a pulse duration of 300 ms, and stored on a 20 MByte hard disk of a computer (PC-98XA, NEC, Japan). The signals were simuhaneously fed to a data recorder (R-2 10, TEAC, Japan) as a back-up. The current density of the Ix tail was obtained by dividing the amplitude of the IK tail by the surface area of cell. The width and length of cells were measured using a scale on eyepieces of a microscope. The thickness of cells was assumed as 10 pm (Taniguchi et al., 1981). We calculated the surface area in assumption that the cells were a rectangular solid. Intracellular dialysis was performed by the same method as that of Matsuda and Noma (1984). Briefly, the test internal solution was sucked into the pipette from a plastic container through fine polyethylene tubing, with a negative pressure of 40 to 50 cm HzO. The flow rate was approximately 0.06 ml/min, counted as drops in the waste chamber. Three drops (about 0.3 ml) of the fluid were sufficient to exchange the intra-pipette fluid completely. The pipette potential was adjusted to zero with normal Tyrode solution both in the pip-
Enhancement ette and the recording chamber. When a gigaseal was established, the pipette solution was changed to the control internal solution and then the cell membrane was ruptured to make the whole-cell recording mode. Therefore, the junction potential was 0 mV. All data are presented as mean + standard errors (S.E.).
of Zg by PKC
7!!7
external solution. The membrane potential was held at - 30 mV, clamped to + 50 mV for 3 s, and then repolarized to - 30 mV. A small1 outward current at the holding potential of -30 mV was observed. The calculated COIIcentration of intracellular calcium ([Ca’+]i) was 0.1 nM (PCs 10). The Ca’+ current was blocked by 3 PM nifedipine. Application of TPA at 1 nM increased both the activation and deactivation of Ix at 2 min after the start of its Results administration [Fig. l(a)]. The amplitude of Eflectsof TPA andPKC on IK the tail current of Ix, defined as the difference IK can be isolated from other membrane currbetween the peak current of the tail and the ents by using a Na+- and K+-free external holding current, was 0.53nA and 0.69 nA, solution, a Na+-free internal solution and a before and after the application of 1 nM TPA, Ca2+ channel blocker such as nifedipine respectively. In six cells, 1 nM TPA increased (Tohse et al., 1987; Tohse, 1990). Figure 1 the amplitude of the tail current bly shows the concentration-related effect of TPA 31.9 + 3.5% over the control value. When 10 on the isolated IK in the Na+- and Kf-free nM TPA was applied to another single cell, thie increase in Iz became more marked [Fig. Is 1 (b)]. The amplitude of the tail current was 0.71 nA and 1.33 nA, before and after the application of 10 nM TPA, respectively. In five (a) cells, the increase in the amplitude of ta.il current was 60.0 + 16.6% over the control value. On the other hand, 0.1 rm TPA produced no change in Ix, the tail current being 95.5 + 3.1 y. (n = 4) of the control value. The effective concentration range of TPA was comparable to the dissociation constant of TPA binding to PKC (Dunphy et al., 1980; Driedger and Blumberg, 1980; Ashendel et al., 1983), implying that the effect of TPA was mediated by PKC. A suppressing effect of H-7, an inhibitor of PKC (Hidaka et al., 1984), on the increase in IK induced by TPA supports this assertion. Application of 10~~ H-7 to single cells decreased Zk and its tail current gradually, with the decrease reaching a steady state within 5 to 7 min after application.. When 1 nM TPA was added to the external solutian in the presence of 10 pM H-7, TPA did not exert its stimulatory effect on 1, as shown in Figure 2. Figure 2(a) shows families of 1,; traces elicited by depolarizing pulses for 3 s up to +70 mV. In the presence of H-7, TPA ____-_ - -------____------___----- _____---_________---_ -_. failed to increase Ix (compare the current FIGURE 1. Effects ofTPA at 1 no (a) and 10 IIM (b) on traces depicted in panels 2 and 3). In five cells, the isolated Z,. Current traces were elicited by depolarizthe tail current of Ix in response to a deing pulses for 3 s to + 50 mV from the holding potential of polarizing pulse to +50 mV did not increase,, -30 mV. The [Ca’+]i was kept at Z’Ca 10. Each current but even slightly decreased by 9.1 + 6.4%, inI trace was obtained in the absence (unmarked) and at 5 the presence of H-7. Figure 2(b) shows the min after the application of TPA (marked). The broken lines indicate zero current level. current-voltage relation oflx. Treatment with1
N. Tohse et 41.
728
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(b)
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-40
+A
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(mV)
FIGURE 2. (a) Effect of TPA on Ik in the presence of 10 PM H-7. Current traces of Zk were recorded by depolarizing pulses to 10 different potentials by 10 mV steps between -20 and + 70 mV. Duration of the pulse was 3 s, and holding potential was -30 mV. The horizontal bar in left corner indicates zero current level. The [Caz+li was kept at PCs 10. 1, Control; 2, at 30 min after the application of 10 mr H-7; 3, at 5 min after the application of 1 tm TPA in the presence of 10 PM H-7. (b) Current-voltage relations ofthe I, tail current obtained in each condition in (a). The amplitude of the tail current was defined as the difference between a peak of the tail current and a steady-state holding current. Tail current shown from 1 (0), 2 (0) and 3 (0) of (a). H.P., holding potential.
H-7 alone, or the combined treatment of H-7 with TPA produced no change in the relation. The synthetic diacylglycerol OAG is known to activate PKC by extracellular application (Kaibuchi et al., 1983; Nishizuka, 1984). Application of OAG 125 PM produced an increase in 1x [Fig. 3(a)]. In three cells, OAG increased the amplitude of the tail current of Ix, in response to a depolarizing pulse to +50 mV, by .58.4 + 8.8% over the control value. The OAG produced little change in the current-voltage relation of Ix [Fig. 3 (b)]. The increase of Ix by OAG, without affecting the voltage-dependency of I,, is qualitatively the same as that produced by TPA (Tohse et al., 1987). These findings, using activators and an inhibitor of PKC, provide pharmacological
evidence to support the contention that PKC mediates the increase in I,. This approach, however, remains indirect. To overcome this drawback, PKC [type III(a)], purified from bovine brain, was directly applied to single cells using the intracellular dialysis method. Merits of the dialysis method for applying large molecules such as enzymes to the intracellular space have already been reported by Kameyama et al. (1985). The PKC used in the present study was a non-activated form. Therefore, in order to transform the non-activated PKC into the active form, 1 nM TPA was perfused externally before and during the intracellular application of protein kinase C. Figure 4 shows a typical example of the effect of protein kinase
Enhancement
729
of & by PKC
InA
fnA)
20 CmV)
FIGURE 3. (a) Effects of OAG different potentials by lo-mV steps - 30 mV. The horizontal bar in left at 5 min after the application ofOAG. presence (0 ) of OAG. The amplitude and a steady-state holding current.
(125 PM) on ZK. Current traces of 2, were recorded by depolarizing pulses to nine between -20 and +60 mV. Duration of pulse was 3 s, and holding potential was corner indicates zero current level. The [Ca*+]. was kept at PCs 10. 1, Control; 2, (b) Current-voltage relations of the Z, tail c&rent obtained in absence (0) and of the tail current was defined as the difference between a peak of the tail current H.P., holding potential.
C on 1~. The single cell was clamped at - 30 mV and ZK was elicited by depolarizing pulses for 3 s to +50 mV. The application of 1 nM TPA also increased IK by 24.6% as measured in the amplitude of tail current. When the IK increase by TPA reached a steady state, protein kinase C (10 pg/ml) was intracellularly applied to the single cell through the inner tube of the patch pipette. The amplitude of the tail current of 1~ was gradually increased until 13 min after the start of application, and reached an almost stable level, being increased 5.5-fold compared with the level of IK obtained in the presence of TPA only. In five cells, PKC increased the amplitude of the tail
current by 150.9 f lOl.lo/o over the value obtained in the presence of TPA. These findings provide direct evidence that activation of PKC increases IK. The relationship between the action of PKC and the Ca’+ -sensitivity of IK We have reported that IK is increased by [Ca*+]i elevation (Tohse et al., 1987; Tohse, 1990) and it is known that the activation olf PKC is dependent upon Ca2+ (Takai et al., 1979). These findings together with the present results, suggest the possibility that the Ca2+-sensitivity of IK may be influenced by
730
,N. Tohse et al. (0)
IS
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TPA
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FIGURE 4. Time course oflx increase produced by an internal application of 10 pg/ml type III (a) protein kinase C. The amplitude of 2~ tail current was measured with a holding potential of -30 mV and a test potential of - 50 mV. The [Ca’+]i was kept at PCs 9. At time 0, external perfusion of the cell with 1 no TPA was started. After 7 min, dialysis of the cell with protein kinase C was started. Examples of the current record are shown above the graph (A-D) for the time indicated in the graph. The broken lines indicate zero current level.
the activation of PKC. Therefore, we examined the effects of PKC on the Ca’+sensitivity of Zk. In the previous study, Ix increased at [Ca’+]i of PCs 8 and higher concentrations in the absence of TPA. In the present study, the concentration-response curve of [Ca’+]i for the increase in Zk was constructed in the presence of 1 nM TPA (Fig. 5). Since continued measurement of the current was not possible using a range of different calcium within a single cell, a few concentrations of [CaZ+li were examined in one single cell at a time. To avoid the cell-to-cell deviation of data, the current density of the Ix tail elicited by repolarization from +50 mV to - 30 mV was used as an index of Ix activation by PKC. The current density of, the Ix tail obtained in the presence of TPA at PCs 11 was 5.67 + 1.07 PA/cm’ (a = 5), which is almost the same as that in the absence of TPA. At PCs 10, 9, and 8, TPA produced 1.53-, 1.66-, and 1.34-fold increases, respectively. At PCs 7, the current density of the 1~ tail in the absence and presence of TPA reached a similar level of about 15 PA/cm’. Effects of [Ca2+]i higher than PCs 7 on Zk were not
investigated because at those [Ca2+]i the cells developed a contracture. These data indicate that TPA apparently shifts the concentration-response curve of the Zk tail in the direction of lower [Ca’+]i. The effect of protein kinase C on the Ca2’ current and the inward-rectifier X’ current Recently, it has been suggested that activation of PKC might increase Zc, in cardiac cells (Dosemeci et al., 1988; Lacerda et al., 1988). Therefore, we examined effects of PKC on currents other than Ix. Figure 6(a) shows Zca elicited by depolarizing pulses for 300 ms from -37 mV to + 3 mV in normal Tyrode solution at a [Ca’+]i of PCs 10. The amplitude of Zca was defined as the difference between the peak inward current and the current at the end of depolarizing pulse. The application of 1 nM TPA decreased the amplitude of Zc, from 0.89 to 0.80 nA. In five cells, however, this decrease was only 6.5 +_ 4.3%, and thus statistically insignificant. Figure 6(b) shows the inward-rectifier K+ current (ZKrec,) in normal Tyrode solution. An outward component of
Enhancement T
of IK by PKC
73 1
fecting other membrane currents. In the present study, we used three different procedures in order to activate protein kinase C; external application of TPA and OAG, and intracellular application of PKC [type III (a)] purified from bovine brain. Phorbol esters provide a useful pharmacological tool for activating PKC (for references, see the review by Ashendel, 1985). Among them, TPA is reported to be the most potent and a concentration of 10 rig/ml (16 nM) is sufficient to obtain full activation of PKC in vitro (Castagna et al., 1982). In the intact cell, however, TPA at concentrations higher than 50 rig/ml often perturbs the membrane structure and results in the degradation of membrane phospholipids (Yamanishi et al., 1983; Nishizuka, 1984). Hockberger et al. (1989) reported that 8 8 7 II IO 9 micromolar levels of phorbol esters decreased Intracellular calcium (pco) Ca’ + current in chick sensory neuron independently of their effect as activation of PKC. FIGURE 5. Concentration-response curve for the increasing effect of [Ca’+J on ZK in presence of TPA. Therefore, when TPA is used to activate PKC, Ordinate gives current density of the tail current of ZK its concentration should be chosen carefully in elicited by return to the holding potential of -30 mV order to avoid this nonspecific membrane acfrom a depolarizing pulse of +50 mV for 3 s. Squares tion. The TPA-induced increase in 1, deindicate the mean values and numerals in parentheses give the number ofcells used in the presence of 1 nM TPA. scribed in the present study was obtained with Circles are the current density of the Z, tail obtained in the external application of TPA at concentthe same condition except in absence of TPA, taken from rations of 1 to 10 nM. This concentration range a previous study (Tohse, 1990). Vertical bars give S.E. is a very reasonable one for TPA to act as a Curves were fitted by eye. selective activator of PKG. Another phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10 I Krect was elicited by hyperpolarizing pulses for nM) has been reported to enhance IK in ven300 ms to - 47 mV from a holding potential of tricular cells (Walsh and Kass, 1988). Fur-37 mV, and its inward component was thermore, H-7, possessing an inhibitory effect elicited by hyperpolarizing pulses to -87 mV. on PKC (Hidaka et al., 1984), clearly blocked It is evident that neither component of IKrect the TPA-induced increase in 1~. Physiological was affected by 1 nM TPA. In six cells, TPA activators of PKC are 1,2-diacylglycerols produced a 5.3 + 5.2% decrease in the outwhich are generated by phospholipase C from ward component and a 3.5 + 8.0% decrease polyphosphoinositides in the plasma memin the inward component, respectively. In brane. The membrane-permeable OAG normal Tyrode solution, however, TPA con(Kaibuchi et al., 1983), at a concentration of sistently enhanced the time-dependent out125 PM, markedly increased tK in guinea-pig ward current elicited by depolarizing pulses to ventricular myocytes. These lines of evidence +43 mV (figure not shown). These findings strongly support the proposal that activation indicate that activation of PKC does not affect of PYC modulates 1~ channels. Further conI& and IKrect. firmation of this is provided by the effect on Ib; of PKC applied internally by cell dialysis. Type III (LX) PKC, which is the major subspecies expressed in cardiac tissue (Kosaka et Discussion al., 1988), markedly increased & in the pre,The major finding in the present study is that sence of TPA. An activity of type II (/?) PKC the activation of PKC increases IK in isolated was also observed in cardiac tissue (Kosaka et guinea-pig ventricular myocytes without af- al., 1988). We have yet to examine the effect of (61
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M 100
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FIGURE 6. (a) Effect of TPA on Za in normal Tyrode solution. Current traces were obtained by depolarizing pulse for 300 ms to + 3 mV from a holding potential of - 37 mV. The [Ca’+]i was PCs 10. Left, control; right, at 5 min after the application of 1 IIM TPA. (b) Effect of TPA on I,,,,, in normal Tyrode solution. Current traces were obtained by hyperpolarizing pulses for 300 ms to -47 and -87 mV from a holding potential of -37 mV. The [Ca2+]i was PCs 10. Left, control; right, at 5 min after the application of 1 nM TPA. The broken lines in both (a) and (b) indicate zero current level. -
this subspecies on ZK, but this is an important experiment to determine if there is a functional difference between the two enzymes. To our knowledge, this is the first demonstration of ion channel modulation by application of a resolved PKC subspecies to intact mammalian cardiac cells. Based on these findings we conclude that the activation of PKC increases Zx in ventricular cells of guinea-pig. Recently, Apkon and Nerbonne (1988) showed that TPA decreased the delayed outward current in rat ventricular cells. However, they did not isolate the current from the transient outward current. The discrepancy between these findings may be caused by the difference of experimental conditions or species differences. Hirasawa and Nishizuka (1985) have proreceptor hypothesis”, in posed the “mobile which PKC may phosphorylate functional proteins bound to the plasma membrane. Thus, it is quite possible that PKC phosphorylates
IK channels
in the
membrane
and
modu-
lates its function. A number of reports have shown that phorbol esters and purified PKC modulate ion channels in various types of cells
(Baraban et al., 1985; DeRiemer et al., 1985; Farley and Auerbach, 1986; Madison et al., 1986; Llano and Marty, 1987; Strong et al., 1987; Brown and Higashida, 1988). In cardiac cells, TPA has been suggested to modulate ionic currents including Ix (Tohse et al., 1987; Apkin and Nerbonne, 1988; Dosemeci et al., 1988; Lacerda et al., 1988; Satoh and Hashimoto, 1988). Dosemeci et al. (1988) and Lacerda et al. ( 1988) reported that TPA increased Zca or produced a dual effect of increasing and decreasing on Zca in cultured ventricular cells of neonatal rats. Satoh and Hashimoto (1988) described that TPA increased Zc, and decreased ZK in sino-atria1 nodal cells of rabbits. These results, however, were obtained by application of TPA at relatively high concentrations ( > 100 nM), at which TPA is reported to perturb the membrane structure (see above). In the present study, we used TPA at low concentrations (< 100 nM) and found that TPA increases Zx but does not affect Zca or ZKrect in ventricular cells. From the present study, we conclude that the activation of PKC selectively increases ZK in guinea-pig ventri-
Enhancement cular cells. Recently, Hartmann et al. (1988) reported that TPA had no significant effect on Ca2+ current in feline ventricular myocytes. Walsh and Kass (1988) reported results consistently with the present study using another phorbol ester, PDBu. We have previously reported that Zk in guinea-pig ventricular cells is sensitive to Ca2+ (Tohse et al., 1987; Tohse, 1990). Intracellular CaZf may increase Ix, not only by activating PKC in the presence of TPA or second messengers, but also by a direct action on the channels. The present study revealed that TPA apparently shifts the concentration-response curve of [Ca’+]i for Ix, in the direction of lower [ Ca2 ‘Ii, although effects of high [Ca’+]t on Ix remain obscured. This result raises the possibility that the activation of PKC might enhance the [Ca2+]isensitivity of Zx. Thus, an intriguing future study would be to investigate the possible modulation of the Ca’+-sensitivity of Zx by PKC-mediated channel phosphorylation. The role of PKC in cardiac function remains unknown, although PKC activity has been observed in cardiac tissues (Wise et al., 1982; Yuan et al., 1987; Kosaka et al., 1988).
of ZK by PKC
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Phorbol esters have been reported to produlce negative inotropic effects on cardiac muscle cells (Leatherman et al., 1987; Yuan et al., 1987; Dosemeci et al., 1988). According to our results, PKC increases ZK without afecting Ic, thereby shortens the or 4heet and probably action potential duration (APD). This shortening in APD would restrict Ca’+-influx during the plateau phase (Morad and Trautwein, 1968). Therefore, the negative inotropic effect produced by activation of PKC may be related to the APD-shortening. Further study is required to resolve the physiological funlction of cardiac PKC. Acknowledgements We thank Professors Y. Nishizuka and l-l. Irisawa for their encouragement, Professor N. Sperelakis for critical reading of the manuscript, Mrs M. Ohara, 0. Nagata and K. Komeichi for their technical assistance and Miss M. Kobayashi for providing excellelnt secretarial assistance. N.T. is a fellow of the Japan Society for the Promotion of Science for Japanese Junior Scientist, M.S.S. is a recipient of a Research Fellowship from the JSPS.
References APKON M, NERBONNE JM (1988) al-adrenergic agonists selectively suppress voltage-dependent K+ currents in rat ventricular myocytes. F’roc Natl Acad Sci USA 85: 8756-8760. AWENDEL CL (1985) The phorbol ester receptor: a phospholipid-regulated protein kinase. Biochem Biophys Acta 82!2: 219-242. ASHELDEL CL, STALLER JM, BOUTWELL RK (1983) Identification of a calcium- and phospholipid-dependent phorbol
ester binding activity in the soluble fraction of mouse tissues.Biochem Biophys Res Commun 111: 340-345. BARABAN JM, SNYDER SH, ALGAR BE (1985) Protein kinase C replates ionic conductance in hippocampal pyramidal neurons: electrophysiological effectsof phorbol esters. Proc Nat1 Acad Sci USA 82: 25382542. BROWN DA, HIGA~HIDA H (1988) Inositol 1,4,5-t&phosphate and diacylglycerol mimic bradykinin effectson mouse neuroblastoma x rat glioma hybrid cells. 3 Physiol397: 185-207. CASTAGNA M, TAKAI Y, KAIBUCHI K, SANO K, KIKKAWA U, NISHIZUKA Y (1982) Direct activation ofcalcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.J Biol Chem 257: 7847-7851. DERIEMER SA, STRONG JA, ALBERT KA (1985) Enhancement of calcium current in Aplysa neurones by phorbol ester and protein kinase C. Nature (Lond) 313: 313-316. DOSEMECI A, DHALLAN RS, COHEN NM, LEDERER WJ, ROGERS TB (1988) Phorbol ester increases calcium current arld simulates the effectsof angiotensin II on cultured neonatal rat heart myocytes. Circ Res 62: 347-357. DRIEDGER PE, BLUMBERG PM (1980) Specific binding of phorbol ester tumor promoters. Proc Natl Acad Sci USA 77: 567-571. DUNPHY WG, DELCLOS KM, BLUMBERG PM (1980) Characterization of specific binding of PH] phorbol 12,13dibutyrate and PH] phorbol 12-myristate 13-acetate to mouse brain. Cancer Res 40: 3635-3641. DUNPHY WG, KOCHENBURGER RJ, CASTAGNA M, BLUMBERG PM (1981) Kinetics and subcellular localization ofspecific PH] phorbol 12,13-dibutyrate binding by mouse brain. Cancer Res 41: 2640-2647. FAB~ATO A, FABLATO F (1979) Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 75: 463-505. FARLEY J, AUERBACH S (1986) Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning. Nature (Lond) 319: 22@223. HAMILL OP, MARTY A, NEHER E, SAKMANN B, SIGWORTH FJ (1981) Improved patch-clamp techniques for highresolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391: 85-100.
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Enhancement
of ZR by PKC
HA, MAZZOCCA NJ, KLE~MAN RB, HOUSER SR (1988) Effects of phenylephrine on calcium current and contractility of feline ventricular myocytes. Am Physio1255: H1173-H1180. HIRASAWA K, NISHIZUKA Y (1985) Phosphatidylinositol turnover in receptor mechanism and signal transduction. Ann Rev Pharmacol Toxic01 25: 147-170. HIDAKA H, INAGAKI M, KAWAMOTO S, SAKAIR Y (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036-5041. HOCKBERGER P, TOSELLI M, SWANDULLA D, Lux HP (1989) A diacylglycerol analogue reduces neuronal calcium carrents independently of protein kinase activation. Nature (Lond) 338: 34@342. ISENBERC G, KLOCKNER IJ (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”. Plhigers Arch 395: 618. KA~BUCHI K, TAKAI Y, SAWAMURA M, HOSHIJIMA M, FUJIWARA T, NISHIZUKA Y (1983) Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. J Biol Chem 258: 67016704. KAMEYAMA M, HOFFMANN F, TRAUTWEIN W (1985) On the mechanism of/l-adrenergic regulation of the Ca channel in the guinea-pig heart. Pfliigers Arch 405: 285-293. KIKKAWA U, ONO Y, OGITA K, FUJII T, ASAOKA Y, SEIUGLJCHI K, KOSAKA Y, IGARASHI K, NISHIZUKA Y (1987) Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain. FEBS Lett 217: 227-231. KOSAKA Y, OGITA K, ASE K, NOMURA H, KIKKAWA U, NISHIZUKA Y (1988) The heterogeneity of protein kinase C in various rat tissues. Biochem Biophys Res Commun 151: 973-981. LACERDA AE, RAMPE D, BROWN AM (1988) Effects of protein kinase C activators on cardiac Ca’+ channels. Nature (Lond) 355: 249-25 1. LEATHERMAN GF, KIM D, SMITH TW (1987) Effects of phorbol esters on contractile state and calcium flux in cultured chick heart cells. Am J Physiol253: H205-H209. LJ..ANO I, MARTY A (1987) Protein kinase C activators inhibit the inositol’triphosphate-mediated muscarinic current responses in rat lacrimal cells. J Physiol394: 239-248. MADISON DV, MALENKA RC, NICOLL RA (1986) Phorbol esters block a voltage-sensitive chloride current in hippocampal pyramidal cells. Nature (Lond) 321: 695697. MATSUDA H, NOMA A (1984) Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea-pig. J PhysioI357: 553-573. MORAD M, TRAUTWEIN W (1968) The effect of the duration of the action potential on contraction in the mammalian heart muscle. Pfliigers Arch 299: 6682. NISHIZUKA Y (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (Lond) SO& 6933698. SATOH H, HASHIMOTO K (1988) On electrophysiological responses to phorbol esters which stimulate protein kinase C in rabbit sino-atria1 node cells. Naunyn Schmiedeberg’s Arch Pharmacol337: 308-315. SHEARMAN MS, NAOR Z, SEKIGUCHI K, KISHIMOTO A, NISHIZUKA Y (1989) Selective activation of the y-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites. FEBS Lett 243: 177-182. STRONG JA, Fox AP, TSIEN RW, KACZMAREK LK (1987) Stimulation of protein kinase C recruits covert calcium channels in AprySia bag cell neurons. Nature (Lond) 325: 7 14-7 17. TAKAI Y, KISHIMOTO A, ISAWA Y, KAWAHARA Y, MORI T, NISHIZUKA Y (1979) Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J Biol Chem 2% 3692-3695. TANIGUCHI J, KOKOBUN S, NOMA A, IRISAWA H (1981) Spontaneously active cells isolated sino-atria1 and atrioventricular nodes of the rabbit heart. Jpn J Physio131: 547-558. TOHSE N (1990) Calcium sensitive delayed rectifier potassium current in guinea-pig ventricular cells. Am J Physiol, 257: H1200-H1206. TOHSE N, KAMEYAMA M, IR~SAWA H (1987) Intracellular Gas+ and protein kinase C modulate K2+ current in guineapig heart cells. Am J Physiol253: H1321-H1324. TSIEN RY, RINK T (1980) Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium. Biochim Biophys Acta 599: 623-638. WALSH KB, KASS RS (1988) Regulation of a heart potassium channel by protein kinase A and C. Science 242: 67-69. protein kinase from heart. J Biol Chem WISE BC, RAYNOR RL, Kvo JF (1982) Phospholipid-sensitive Ca ‘+-dependent 257: 84818488. YAMANISHI J, TAKAI Y, KAIEXJC~I K, SANO K, CASTAGNA M, NISHIZUKA Y (1983) Synergistic functions of phorbol ester and calcium in serotonin release from human platelets. B&hem Biophys Res Commun 112: 778-786. YUAN S, SUNAHARA EA, SEN AK (1987) T umor-promoting phorbol esters inhibit cardiac functions and induce redistribugion of protein kinase C in perfused beating rat heart. Circ Res 61: 372-378. HARTMANN
Cell
Cardiol22,
Protein
Noritsugu
725-734
Kinase Potassium Tohse,
(1990)
C Activation Current
Masaki
Enhances in Guinea-pig
the Delayed Rectifier Heart Cells
Kameyamal, Kazuo Sekigucki and Mario Ksnno
*, Mark
S. Shearman*
Department of Pharmacology, Hokkaido University School of Medicine, Sapporo 060, Japan, INational Institute for Physiological Sciences, Okazaki 444, Japan, and ‘Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan (Received 5 May 1989, accepted in revisedform
16 February 1990)
M. KAMEYAMA, K. SEKIGUCHI, M. S. SHEARMAN, AND M. KANNO. Protein Kinase C Activation Enhances the Delayed Rectifier Potassium Current in Guinea-pig Heart Cells. jounzal of Molecular and Cellular Cardiology (1990) 22, 725-734. The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na+- and K+-free external solution, the delayed rectifier K+ cument (ZK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol13-acetate (TPA). The amplitude of the ZK tail elicited by a return from a depolarizing pulse for 3 s at +50 mV to a holding potential of -30 mV was increased by 32 + 4% (mean + s.E., n = 6) after the external application of 1 nM TPA, and by 60 + 17% (n = 5) after 10 11~. The increase in ZK produced by 1 nM TPA was abolished by the inhibitor of PKC, I-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 FM). In addition, the synthetic diacylglycerol I-oleoyl-2-acetylglycerol (OAG, 125 PM) also increased ZK (58 + 9%, n = 3). PKC purified from bovine brain remarkably increased ZK ( 15 1 f 101 %, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve ofZx for the intracellular concentration action of PKC and/or altered Ca’+of Ca2+ was shifted to the left by 1 m TPA, suggesting a Ca ‘+-dependent sensitivity of ZK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 & 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 + 5% in outward component, and 3 f 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase ZK without affecting the other two currents. N. TOHSE,
KEY
WORDS:
Protein
kinase C; Delayed
rectifier
K+ current;
rntroduction Protein kinase C (PKC) is now widely accepted to be one of the key enzymes in intracellular signal transduction for various biologically active substances (Nishizuka, 1984). PKC, when activated, translocates from the cytosol to the plasma membrane (Dunphy et al., 1981; Yuan et al., 1987, and see review Hirasawa and Nishizuka, 1985), and thereby becomes eligible to modulate ion channels therein. Recent studies have indicated that purified PKC and phorbol esters that activate PKC, are capable of modulating ion channel activities in various types of cells (Baraban et al., 1985; DeRiemer et al., 1985; Farley and Auerbach, 1986; Madison et al., 1986; Llano and Marty, 1987; Strong et al., 1987; Brown and Higashida, 1988). We have previously reported that a phorbol ester 12-O-tetradecanoylphorbol13-acetate 0022-2828/90/060725
+ 10 $03.00/O
Heart;
Single
cell; TPA,
H-7;
OAG.
(TPA) increases the delayed rectifier K+ current (1~) in guinea-pig ventricular cells, through an activation of PKC, with little influence on the other currents in these cells (Tohse et al., 1987). However, some recent reports argued that phorbol esters modulate the Ca*+ current (1,-J in cardiac cells (Leatherman et al., 1987; Dosemeci et al., 1988; Lacerda et al., 1988; Satoh and Hashimoto, 1988). Therefore, we designed the present study in order to examine in detail whether the activation of PKC modulates cardia.c membrane currents, including Z,. We applied PKC intracellularly by a cell dialysis method (Mastuda and Noma, 1984), in addition to extracellular application of TPA and 1 -oleoyl-2-acetylglycerol (OAG), in isolated ventricular cells of guinea-pigs, and investigated their influences on ionic currents. From the results obtained, we conclude that 0 1990 Academic
Press Limitetd
726
N. Tohse et al.
PKC, when activated, increases IK in guineapig ventricular cells without affecting other ionic currents. Materials
and Methods
Isolation of cardiac cells Single ventricular cells of guinea-pig heart were obtained essentially by the same technique as described previously (Taniguchi et al., 1981; Isenberg and Klockner, 1982). Briefly, collagenase (0.04% w/v, Sigma type I) in a low-Cazf Tyrode solution (Ca2+ concentration: c. 60 ,UM) was perfused for 12 to 20 min through the coronary artery using a LangendorfI apparatus. The collagenase solution was washed out by high-K+, low-Clsolution (KB-solution, cf Isenberg and Klockner, 1982). The ventricular tissue was cut into small pieces, agitated gently in a small beaker with KB-solution, and filtered through a lopm stainless-steel mesh. The cell suspension was stored in a refrigerator (4°C) for later use. Solutions and materials The composition of normal Tyrode solution was (in mM): NaCl 143, KC1 5.4, CaCl2 1.8, MgClz 0.5, NaH2P04 0.33, glucose 5.5 and HEPES-NaOH buffer (pH 7.4) 5.0. A Na+and K+-free external solution was prepared by replacing NaCl and KC1 with equimolar choline Cl. A Na+-free internal solution in the recording pipette contained (in mM): KOH 110, KC1 20, MgC12 1.0, ATP-K2 5.0, creatine-phosphate-K2 5.0, EGTA 10, HEPES 5.0, and the pH (= 7.4) of the internal solution was adjusted by 90 to 100 mM aspartic acid. Concentrations of free Ca2+ (PCs 11 to PCs 6) in the internal solution were calculated according to Fabiato and Fabiato (1979) with the correction of Tsien and Rink (1980). The temperature of the external solutions was 34 to 36°C. TPA was dissolved in dimethyl sulfoxide as a stock solution (1 mhr). l-(5-Isoquinolinesulfonyl)-2methylpiperazine (H-7; Seikagaku Kogyo, Tokyo) was dissolved in water to make a 10 mM stock solution; 1-oleoyl-2-acetylglycerol (OAG, Sigma) was dissolved in 10% dimethyl sulfoxide to make a 5 mM stock solution. The stock solutions were diluted by the perfiusate to a given concentration. Nifedipine (3 PM;
Bayer, FRG) was used to block the Ca2+ current and was dissolved in ethanol to make a 10 mM stock solution. Purified PKC [type III(a); Kikkawa et al., 19871 was obtained from bovine whole brain as described by Shearman et al. (1989). For internal cellular dialysis, a solution of the enzyme was included in the pipette internal solution. The final concentration of the enzyme was 10 pg/ml (8.1 nmol Pi/min/ml for incorporation into H 1 histone). Recording techniques The whole-cell membrane currents were recorded by the patch clamp method (Hamill et al., 1981), using glass patch pipettes with a diameter of 3 to 4 pm. The resistance of the pipette was 1.8 to 2 MQ when filled with normal Tyrode solution. The pipette solution was connected through a Ag/AgCl wire to the input stage of a current-voltage converter with a feedback register of 100 Ma. Current and voltage signals were filtered at 2 kHz, digitalized by an AD converter (ADX-98, Canopus Electronics, Japan) at 100 Hz for a pulse duration of 3 s or at 2 kHz for a pulse duration of 300 ms, and stored on a 20 MByte hard disk of a computer (PC-98XA, NEC, Japan). The signals were simuhaneously fed to a data recorder (R-2 10, TEAC, Japan) as a back-up. The current density of the Ix tail was obtained by dividing the amplitude of the IK tail by the surface area of cell. The width and length of cells were measured using a scale on eyepieces of a microscope. The thickness of cells was assumed as 10 pm (Taniguchi et al., 1981). We calculated the surface area in assumption that the cells were a rectangular solid. Intracellular dialysis was performed by the same method as that of Matsuda and Noma (1984). Briefly, the test internal solution was sucked into the pipette from a plastic container through fine polyethylene tubing, with a negative pressure of 40 to 50 cm HzO. The flow rate was approximately 0.06 ml/min, counted as drops in the waste chamber. Three drops (about 0.3 ml) of the fluid were sufficient to exchange the intra-pipette fluid completely. The pipette potential was adjusted to zero with normal Tyrode solution both in the pip-
Enhancement ette and the recording chamber. When a gigaseal was established, the pipette solution was changed to the control internal solution and then the cell membrane was ruptured to make the whole-cell recording mode. Therefore, the junction potential was 0 mV. All data are presented as mean + standard errors (S.E.).
of Zg by PKC
7!!7
external solution. The membrane potential was held at - 30 mV, clamped to + 50 mV for 3 s, and then repolarized to - 30 mV. A small1 outward current at the holding potential of -30 mV was observed. The calculated COIIcentration of intracellular calcium ([Ca’+]i) was 0.1 nM (PCs 10). The Ca’+ current was blocked by 3 PM nifedipine. Application of TPA at 1 nM increased both the activation and deactivation of Ix at 2 min after the start of its Results administration [Fig. l(a)]. The amplitude of Eflectsof TPA andPKC on IK the tail current of Ix, defined as the difference IK can be isolated from other membrane currbetween the peak current of the tail and the ents by using a Na+- and K+-free external holding current, was 0.53nA and 0.69 nA, solution, a Na+-free internal solution and a before and after the application of 1 nM TPA, Ca2+ channel blocker such as nifedipine respectively. In six cells, 1 nM TPA increased (Tohse et al., 1987; Tohse, 1990). Figure 1 the amplitude of the tail current bly shows the concentration-related effect of TPA 31.9 + 3.5% over the control value. When 10 on the isolated IK in the Na+- and Kf-free nM TPA was applied to another single cell, thie increase in Iz became more marked [Fig. Is 1 (b)]. The amplitude of the tail current was 0.71 nA and 1.33 nA, before and after the application of 10 nM TPA, respectively. In five (a) cells, the increase in the amplitude of ta.il current was 60.0 + 16.6% over the control value. On the other hand, 0.1 rm TPA produced no change in Ix, the tail current being 95.5 + 3.1 y. (n = 4) of the control value. The effective concentration range of TPA was comparable to the dissociation constant of TPA binding to PKC (Dunphy et al., 1980; Driedger and Blumberg, 1980; Ashendel et al., 1983), implying that the effect of TPA was mediated by PKC. A suppressing effect of H-7, an inhibitor of PKC (Hidaka et al., 1984), on the increase in IK induced by TPA supports this assertion. Application of 10~~ H-7 to single cells decreased Zk and its tail current gradually, with the decrease reaching a steady state within 5 to 7 min after application.. When 1 nM TPA was added to the external solutian in the presence of 10 pM H-7, TPA did not exert its stimulatory effect on 1, as shown in Figure 2. Figure 2(a) shows families of 1,; traces elicited by depolarizing pulses for 3 s up to +70 mV. In the presence of H-7, TPA ____-_ - -------____------___----- _____---_________---_ -_. failed to increase Ix (compare the current FIGURE 1. Effects ofTPA at 1 no (a) and 10 IIM (b) on traces depicted in panels 2 and 3). In five cells, the isolated Z,. Current traces were elicited by depolarizthe tail current of Ix in response to a deing pulses for 3 s to + 50 mV from the holding potential of polarizing pulse to +50 mV did not increase,, -30 mV. The [Ca’+]i was kept at Z’Ca 10. Each current but even slightly decreased by 9.1 + 6.4%, inI trace was obtained in the absence (unmarked) and at 5 the presence of H-7. Figure 2(b) shows the min after the application of TPA (marked). The broken lines indicate zero current level. current-voltage relation oflx. Treatment with1
N. Tohse et 41.
728
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FIGURE 2. (a) Effect of TPA on Ik in the presence of 10 PM H-7. Current traces of Zk were recorded by depolarizing pulses to 10 different potentials by 10 mV steps between -20 and + 70 mV. Duration of the pulse was 3 s, and holding potential was -30 mV. The horizontal bar in left corner indicates zero current level. The [Caz+li was kept at PCs 10. 1, Control; 2, at 30 min after the application of 10 mr H-7; 3, at 5 min after the application of 1 tm TPA in the presence of 10 PM H-7. (b) Current-voltage relations ofthe I, tail current obtained in each condition in (a). The amplitude of the tail current was defined as the difference between a peak of the tail current and a steady-state holding current. Tail current shown from 1 (0), 2 (0) and 3 (0) of (a). H.P., holding potential.
H-7 alone, or the combined treatment of H-7 with TPA produced no change in the relation. The synthetic diacylglycerol OAG is known to activate PKC by extracellular application (Kaibuchi et al., 1983; Nishizuka, 1984). Application of OAG 125 PM produced an increase in 1x [Fig. 3(a)]. In three cells, OAG increased the amplitude of the tail current of Ix, in response to a depolarizing pulse to +50 mV, by .58.4 + 8.8% over the control value. The OAG produced little change in the current-voltage relation of Ix [Fig. 3 (b)]. The increase of Ix by OAG, without affecting the voltage-dependency of I,, is qualitatively the same as that produced by TPA (Tohse et al., 1987). These findings, using activators and an inhibitor of PKC, provide pharmacological
evidence to support the contention that PKC mediates the increase in I,. This approach, however, remains indirect. To overcome this drawback, PKC [type III(a)], purified from bovine brain, was directly applied to single cells using the intracellular dialysis method. Merits of the dialysis method for applying large molecules such as enzymes to the intracellular space have already been reported by Kameyama et al. (1985). The PKC used in the present study was a non-activated form. Therefore, in order to transform the non-activated PKC into the active form, 1 nM TPA was perfused externally before and during the intracellular application of protein kinase C. Figure 4 shows a typical example of the effect of protein kinase
Enhancement
729
of & by PKC
InA
fnA)
20 CmV)
FIGURE 3. (a) Effects of OAG different potentials by lo-mV steps - 30 mV. The horizontal bar in left at 5 min after the application ofOAG. presence (0 ) of OAG. The amplitude and a steady-state holding current.
(125 PM) on ZK. Current traces of 2, were recorded by depolarizing pulses to nine between -20 and +60 mV. Duration of pulse was 3 s, and holding potential was corner indicates zero current level. The [Ca*+]. was kept at PCs 10. 1, Control; 2, (b) Current-voltage relations of the Z, tail c&rent obtained in absence (0) and of the tail current was defined as the difference between a peak of the tail current H.P., holding potential.
C on 1~. The single cell was clamped at - 30 mV and ZK was elicited by depolarizing pulses for 3 s to +50 mV. The application of 1 nM TPA also increased IK by 24.6% as measured in the amplitude of tail current. When the IK increase by TPA reached a steady state, protein kinase C (10 pg/ml) was intracellularly applied to the single cell through the inner tube of the patch pipette. The amplitude of the tail current of 1~ was gradually increased until 13 min after the start of application, and reached an almost stable level, being increased 5.5-fold compared with the level of IK obtained in the presence of TPA only. In five cells, PKC increased the amplitude of the tail
current by 150.9 f lOl.lo/o over the value obtained in the presence of TPA. These findings provide direct evidence that activation of PKC increases IK. The relationship between the action of PKC and the Ca’+ -sensitivity of IK We have reported that IK is increased by [Ca*+]i elevation (Tohse et al., 1987; Tohse, 1990) and it is known that the activation olf PKC is dependent upon Ca2+ (Takai et al., 1979). These findings together with the present results, suggest the possibility that the Ca2+-sensitivity of IK may be influenced by
730
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FIGURE 4. Time course oflx increase produced by an internal application of 10 pg/ml type III (a) protein kinase C. The amplitude of 2~ tail current was measured with a holding potential of -30 mV and a test potential of - 50 mV. The [Ca’+]i was kept at PCs 9. At time 0, external perfusion of the cell with 1 no TPA was started. After 7 min, dialysis of the cell with protein kinase C was started. Examples of the current record are shown above the graph (A-D) for the time indicated in the graph. The broken lines indicate zero current level.
the activation of PKC. Therefore, we examined the effects of PKC on the Ca’+sensitivity of Zk. In the previous study, Ix increased at [Ca’+]i of PCs 8 and higher concentrations in the absence of TPA. In the present study, the concentration-response curve of [Ca’+]i for the increase in Zk was constructed in the presence of 1 nM TPA (Fig. 5). Since continued measurement of the current was not possible using a range of different calcium within a single cell, a few concentrations of [CaZ+li were examined in one single cell at a time. To avoid the cell-to-cell deviation of data, the current density of the Ix tail elicited by repolarization from +50 mV to - 30 mV was used as an index of Ix activation by PKC. The current density of, the Ix tail obtained in the presence of TPA at PCs 11 was 5.67 + 1.07 PA/cm’ (a = 5), which is almost the same as that in the absence of TPA. At PCs 10, 9, and 8, TPA produced 1.53-, 1.66-, and 1.34-fold increases, respectively. At PCs 7, the current density of the 1~ tail in the absence and presence of TPA reached a similar level of about 15 PA/cm’. Effects of [Ca2+]i higher than PCs 7 on Zk were not
investigated because at those [Ca2+]i the cells developed a contracture. These data indicate that TPA apparently shifts the concentration-response curve of the Zk tail in the direction of lower [Ca’+]i. The effect of protein kinase C on the Ca2’ current and the inward-rectifier X’ current Recently, it has been suggested that activation of PKC might increase Zc, in cardiac cells (Dosemeci et al., 1988; Lacerda et al., 1988). Therefore, we examined effects of PKC on currents other than Ix. Figure 6(a) shows Zca elicited by depolarizing pulses for 300 ms from -37 mV to + 3 mV in normal Tyrode solution at a [Ca’+]i of PCs 10. The amplitude of Zca was defined as the difference between the peak inward current and the current at the end of depolarizing pulse. The application of 1 nM TPA decreased the amplitude of Zc, from 0.89 to 0.80 nA. In five cells, however, this decrease was only 6.5 +_ 4.3%, and thus statistically insignificant. Figure 6(b) shows the inward-rectifier K+ current (ZKrec,) in normal Tyrode solution. An outward component of
Enhancement T
of IK by PKC
73 1
fecting other membrane currents. In the present study, we used three different procedures in order to activate protein kinase C; external application of TPA and OAG, and intracellular application of PKC [type III (a)] purified from bovine brain. Phorbol esters provide a useful pharmacological tool for activating PKC (for references, see the review by Ashendel, 1985). Among them, TPA is reported to be the most potent and a concentration of 10 rig/ml (16 nM) is sufficient to obtain full activation of PKC in vitro (Castagna et al., 1982). In the intact cell, however, TPA at concentrations higher than 50 rig/ml often perturbs the membrane structure and results in the degradation of membrane phospholipids (Yamanishi et al., 1983; Nishizuka, 1984). Hockberger et al. (1989) reported that 8 8 7 II IO 9 micromolar levels of phorbol esters decreased Intracellular calcium (pco) Ca’ + current in chick sensory neuron independently of their effect as activation of PKC. FIGURE 5. Concentration-response curve for the increasing effect of [Ca’+J on ZK in presence of TPA. Therefore, when TPA is used to activate PKC, Ordinate gives current density of the tail current of ZK its concentration should be chosen carefully in elicited by return to the holding potential of -30 mV order to avoid this nonspecific membrane acfrom a depolarizing pulse of +50 mV for 3 s. Squares tion. The TPA-induced increase in 1, deindicate the mean values and numerals in parentheses give the number ofcells used in the presence of 1 nM TPA. scribed in the present study was obtained with Circles are the current density of the Z, tail obtained in the external application of TPA at concentthe same condition except in absence of TPA, taken from rations of 1 to 10 nM. This concentration range a previous study (Tohse, 1990). Vertical bars give S.E. is a very reasonable one for TPA to act as a Curves were fitted by eye. selective activator of PKG. Another phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10 I Krect was elicited by hyperpolarizing pulses for nM) has been reported to enhance IK in ven300 ms to - 47 mV from a holding potential of tricular cells (Walsh and Kass, 1988). Fur-37 mV, and its inward component was thermore, H-7, possessing an inhibitory effect elicited by hyperpolarizing pulses to -87 mV. on PKC (Hidaka et al., 1984), clearly blocked It is evident that neither component of IKrect the TPA-induced increase in 1~. Physiological was affected by 1 nM TPA. In six cells, TPA activators of PKC are 1,2-diacylglycerols produced a 5.3 + 5.2% decrease in the outwhich are generated by phospholipase C from ward component and a 3.5 + 8.0% decrease polyphosphoinositides in the plasma memin the inward component, respectively. In brane. The membrane-permeable OAG normal Tyrode solution, however, TPA con(Kaibuchi et al., 1983), at a concentration of sistently enhanced the time-dependent out125 PM, markedly increased tK in guinea-pig ward current elicited by depolarizing pulses to ventricular myocytes. These lines of evidence +43 mV (figure not shown). These findings strongly support the proposal that activation indicate that activation of PKC does not affect of PYC modulates 1~ channels. Further conI& and IKrect. firmation of this is provided by the effect on Ib; of PKC applied internally by cell dialysis. Type III (LX) PKC, which is the major subspecies expressed in cardiac tissue (Kosaka et Discussion al., 1988), markedly increased & in the pre,The major finding in the present study is that sence of TPA. An activity of type II (/?) PKC the activation of PKC increases IK in isolated was also observed in cardiac tissue (Kosaka et guinea-pig ventricular myocytes without af- al., 1988). We have yet to examine the effect of (61
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FIGURE 6. (a) Effect of TPA on Za in normal Tyrode solution. Current traces were obtained by depolarizing pulse for 300 ms to + 3 mV from a holding potential of - 37 mV. The [Ca’+]i was PCs 10. Left, control; right, at 5 min after the application of 1 IIM TPA. (b) Effect of TPA on I,,,,, in normal Tyrode solution. Current traces were obtained by hyperpolarizing pulses for 300 ms to -47 and -87 mV from a holding potential of -37 mV. The [Ca2+]i was PCs 10. Left, control; right, at 5 min after the application of 1 nM TPA. The broken lines in both (a) and (b) indicate zero current level. -
this subspecies on ZK, but this is an important experiment to determine if there is a functional difference between the two enzymes. To our knowledge, this is the first demonstration of ion channel modulation by application of a resolved PKC subspecies to intact mammalian cardiac cells. Based on these findings we conclude that the activation of PKC increases Zx in ventricular cells of guinea-pig. Recently, Apkon and Nerbonne (1988) showed that TPA decreased the delayed outward current in rat ventricular cells. However, they did not isolate the current from the transient outward current. The discrepancy between these findings may be caused by the difference of experimental conditions or species differences. Hirasawa and Nishizuka (1985) have proreceptor hypothesis”, in posed the “mobile which PKC may phosphorylate functional proteins bound to the plasma membrane. Thus, it is quite possible that PKC phosphorylates
IK channels
in the
membrane
and
modu-
lates its function. A number of reports have shown that phorbol esters and purified PKC modulate ion channels in various types of cells
(Baraban et al., 1985; DeRiemer et al., 1985; Farley and Auerbach, 1986; Madison et al., 1986; Llano and Marty, 1987; Strong et al., 1987; Brown and Higashida, 1988). In cardiac cells, TPA has been suggested to modulate ionic currents including Ix (Tohse et al., 1987; Apkin and Nerbonne, 1988; Dosemeci et al., 1988; Lacerda et al., 1988; Satoh and Hashimoto, 1988). Dosemeci et al. (1988) and Lacerda et al. ( 1988) reported that TPA increased Zca or produced a dual effect of increasing and decreasing on Zca in cultured ventricular cells of neonatal rats. Satoh and Hashimoto (1988) described that TPA increased Zc, and decreased ZK in sino-atria1 nodal cells of rabbits. These results, however, were obtained by application of TPA at relatively high concentrations ( > 100 nM), at which TPA is reported to perturb the membrane structure (see above). In the present study, we used TPA at low concentrations (< 100 nM) and found that TPA increases Zx but does not affect Zca or ZKrect in ventricular cells. From the present study, we conclude that the activation of PKC selectively increases ZK in guinea-pig ventri-
Enhancement cular cells. Recently, Hartmann et al. (1988) reported that TPA had no significant effect on Ca2+ current in feline ventricular myocytes. Walsh and Kass (1988) reported results consistently with the present study using another phorbol ester, PDBu. We have previously reported that Zk in guinea-pig ventricular cells is sensitive to Ca2+ (Tohse et al., 1987; Tohse, 1990). Intracellular CaZf may increase Ix, not only by activating PKC in the presence of TPA or second messengers, but also by a direct action on the channels. The present study revealed that TPA apparently shifts the concentration-response curve of [Ca’+]i for Ix, in the direction of lower [ Ca2 ‘Ii, although effects of high [Ca’+]t on Ix remain obscured. This result raises the possibility that the activation of PKC might enhance the [Ca2+]isensitivity of Zx. Thus, an intriguing future study would be to investigate the possible modulation of the Ca’+-sensitivity of Zx by PKC-mediated channel phosphorylation. The role of PKC in cardiac function remains unknown, although PKC activity has been observed in cardiac tissues (Wise et al., 1982; Yuan et al., 1987; Kosaka et al., 1988).
of ZK by PKC
733
Phorbol esters have been reported to produlce negative inotropic effects on cardiac muscle cells (Leatherman et al., 1987; Yuan et al., 1987; Dosemeci et al., 1988). According to our results, PKC increases ZK without afecting Ic, thereby shortens the or 4heet and probably action potential duration (APD). This shortening in APD would restrict Ca’+-influx during the plateau phase (Morad and Trautwein, 1968). Therefore, the negative inotropic effect produced by activation of PKC may be related to the APD-shortening. Further study is required to resolve the physiological funlction of cardiac PKC. Acknowledgements We thank Professors Y. Nishizuka and l-l. Irisawa for their encouragement, Professor N. Sperelakis for critical reading of the manuscript, Mrs M. Ohara, 0. Nagata and K. Komeichi for their technical assistance and Miss M. Kobayashi for providing excellelnt secretarial assistance. N.T. is a fellow of the Japan Society for the Promotion of Science for Japanese Junior Scientist, M.S.S. is a recipient of a Research Fellowship from the JSPS.
References APKON M, NERBONNE JM (1988) al-adrenergic agonists selectively suppress voltage-dependent K+ currents in rat ventricular myocytes. F’roc Natl Acad Sci USA 85: 8756-8760. AWENDEL CL (1985) The phorbol ester receptor: a phospholipid-regulated protein kinase. Biochem Biophys Acta 82!2: 219-242. ASHELDEL CL, STALLER JM, BOUTWELL RK (1983) Identification of a calcium- and phospholipid-dependent phorbol
ester binding activity in the soluble fraction of mouse tissues.Biochem Biophys Res Commun 111: 340-345. BARABAN JM, SNYDER SH, ALGAR BE (1985) Protein kinase C replates ionic conductance in hippocampal pyramidal neurons: electrophysiological effectsof phorbol esters. Proc Nat1 Acad Sci USA 82: 25382542. BROWN DA, HIGA~HIDA H (1988) Inositol 1,4,5-t&phosphate and diacylglycerol mimic bradykinin effectson mouse neuroblastoma x rat glioma hybrid cells. 3 Physiol397: 185-207. CASTAGNA M, TAKAI Y, KAIBUCHI K, SANO K, KIKKAWA U, NISHIZUKA Y (1982) Direct activation ofcalcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.J Biol Chem 257: 7847-7851. DERIEMER SA, STRONG JA, ALBERT KA (1985) Enhancement of calcium current in Aplysa neurones by phorbol ester and protein kinase C. Nature (Lond) 313: 313-316. DOSEMECI A, DHALLAN RS, COHEN NM, LEDERER WJ, ROGERS TB (1988) Phorbol ester increases calcium current arld simulates the effectsof angiotensin II on cultured neonatal rat heart myocytes. Circ Res 62: 347-357. DRIEDGER PE, BLUMBERG PM (1980) Specific binding of phorbol ester tumor promoters. Proc Natl Acad Sci USA 77: 567-571. DUNPHY WG, DELCLOS KM, BLUMBERG PM (1980) Characterization of specific binding of PH] phorbol 12,13dibutyrate and PH] phorbol 12-myristate 13-acetate to mouse brain. Cancer Res 40: 3635-3641. DUNPHY WG, KOCHENBURGER RJ, CASTAGNA M, BLUMBERG PM (1981) Kinetics and subcellular localization ofspecific PH] phorbol 12,13-dibutyrate binding by mouse brain. Cancer Res 41: 2640-2647. FAB~ATO A, FABLATO F (1979) Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 75: 463-505. FARLEY J, AUERBACH S (1986) Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning. Nature (Lond) 319: 22@223. HAMILL OP, MARTY A, NEHER E, SAKMANN B, SIGWORTH FJ (1981) Improved patch-clamp techniques for highresolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391: 85-100.
734
Enhancement
of ZR by PKC
HA, MAZZOCCA NJ, KLE~MAN RB, HOUSER SR (1988) Effects of phenylephrine on calcium current and contractility of feline ventricular myocytes. Am Physio1255: H1173-H1180. HIRASAWA K, NISHIZUKA Y (1985) Phosphatidylinositol turnover in receptor mechanism and signal transduction. Ann Rev Pharmacol Toxic01 25: 147-170. HIDAKA H, INAGAKI M, KAWAMOTO S, SAKAIR Y (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036-5041. HOCKBERGER P, TOSELLI M, SWANDULLA D, Lux HP (1989) A diacylglycerol analogue reduces neuronal calcium carrents independently of protein kinase activation. Nature (Lond) 338: 34@342. ISENBERC G, KLOCKNER IJ (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”. Plhigers Arch 395: 618. KA~BUCHI K, TAKAI Y, SAWAMURA M, HOSHIJIMA M, FUJIWARA T, NISHIZUKA Y (1983) Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. J Biol Chem 258: 67016704. KAMEYAMA M, HOFFMANN F, TRAUTWEIN W (1985) On the mechanism of/l-adrenergic regulation of the Ca channel in the guinea-pig heart. Pfliigers Arch 405: 285-293. KIKKAWA U, ONO Y, OGITA K, FUJII T, ASAOKA Y, SEIUGLJCHI K, KOSAKA Y, IGARASHI K, NISHIZUKA Y (1987) Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain. FEBS Lett 217: 227-231. KOSAKA Y, OGITA K, ASE K, NOMURA H, KIKKAWA U, NISHIZUKA Y (1988) The heterogeneity of protein kinase C in various rat tissues. Biochem Biophys Res Commun 151: 973-981. LACERDA AE, RAMPE D, BROWN AM (1988) Effects of protein kinase C activators on cardiac Ca’+ channels. Nature (Lond) 355: 249-25 1. LEATHERMAN GF, KIM D, SMITH TW (1987) Effects of phorbol esters on contractile state and calcium flux in cultured chick heart cells. Am J Physiol253: H205-H209. LJ..ANO I, MARTY A (1987) Protein kinase C activators inhibit the inositol’triphosphate-mediated muscarinic current responses in rat lacrimal cells. J Physiol394: 239-248. MADISON DV, MALENKA RC, NICOLL RA (1986) Phorbol esters block a voltage-sensitive chloride current in hippocampal pyramidal cells. Nature (Lond) 321: 695697. MATSUDA H, NOMA A (1984) Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea-pig. J PhysioI357: 553-573. MORAD M, TRAUTWEIN W (1968) The effect of the duration of the action potential on contraction in the mammalian heart muscle. Pfliigers Arch 299: 6682. NISHIZUKA Y (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (Lond) SO& 6933698. SATOH H, HASHIMOTO K (1988) On electrophysiological responses to phorbol esters which stimulate protein kinase C in rabbit sino-atria1 node cells. Naunyn Schmiedeberg’s Arch Pharmacol337: 308-315. SHEARMAN MS, NAOR Z, SEKIGUCHI K, KISHIMOTO A, NISHIZUKA Y (1989) Selective activation of the y-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites. FEBS Lett 243: 177-182. STRONG JA, Fox AP, TSIEN RW, KACZMAREK LK (1987) Stimulation of protein kinase C recruits covert calcium channels in AprySia bag cell neurons. Nature (Lond) 325: 7 14-7 17. TAKAI Y, KISHIMOTO A, ISAWA Y, KAWAHARA Y, MORI T, NISHIZUKA Y (1979) Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J Biol Chem 2% 3692-3695. TANIGUCHI J, KOKOBUN S, NOMA A, IRISAWA H (1981) Spontaneously active cells isolated sino-atria1 and atrioventricular nodes of the rabbit heart. Jpn J Physio131: 547-558. TOHSE N (1990) Calcium sensitive delayed rectifier potassium current in guinea-pig ventricular cells. Am J Physiol, 257: H1200-H1206. TOHSE N, KAMEYAMA M, IR~SAWA H (1987) Intracellular Gas+ and protein kinase C modulate K2+ current in guineapig heart cells. Am J Physiol253: H1321-H1324. TSIEN RY, RINK T (1980) Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium. Biochim Biophys Acta 599: 623-638. WALSH KB, KASS RS (1988) Regulation of a heart potassium channel by protein kinase A and C. Science 242: 67-69. protein kinase from heart. J Biol Chem WISE BC, RAYNOR RL, Kvo JF (1982) Phospholipid-sensitive Ca ‘+-dependent 257: 84818488. YAMANISHI J, TAKAI Y, KAIEXJC~I K, SANO K, CASTAGNA M, NISHIZUKA Y (1983) Synergistic functions of phorbol ester and calcium in serotonin release from human platelets. B&hem Biophys Res Commun 112: 778-786. YUAN S, SUNAHARA EA, SEN AK (1987) T umor-promoting phorbol esters inhibit cardiac functions and induce redistribugion of protein kinase C in perfused beating rat heart. Circ Res 61: 372-378. HARTMANN
