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(Circulation Research. 1997;80:720-729.)
© 1997 American Heart Association, Inc.

Articles

C2 Region–Derived Peptides of ß-Protein Kinase C Regulate Cardiac Ca²⁺ Channels

Zhi-Hao Zhang, John A. Johnson, Long Chen, Nabil El-Sherif, Daria Mochly-Rosen, , Mohamed Boutjdir

From the Cardiology Division (Z.-H.Z., L.C., N.E.-S., M.B.), Department of Medicine, State University of New York, Health Science Center, and the Veterans Administration Medical Center, Brooklyn, New York, and the Department of Molecular Pharmacology (J.A.J., D.M.-R.), Stanford (Calif) University School of Medicine.

Correspondence to Dr Mohamed Boutjdir, Cardiology Division (IIIA), VA Medical Center, 800 Poly Place, Brooklyn, NY 11209. E-mail boutjdir.mohamed{at}brooklyn.va.gov

	Abstract

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Abstract We have previously shown that ₁-adrenergic activation inhibited ß-adrenergic–stimulated L-type Ca²⁺ current (I_Ca). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4ß-phorbol 12-myristate 13-acetate (PMA). To minimize Ca²⁺-induced Ca²⁺ inactivation, Ba²⁺ current (I_Ba) was recorded through Ca²⁺ channels in adult rat ventricular myocytes. We found that PMA (0.1 µmol/L) consistently inhibited basal I_Ba by 40.5±7.4% and isoproterenol (ISO, 0.1 µmol/L)–stimulated I_Ba by 48.9±7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue -phorbol 12,13-didecanoate (0.1 µmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (ßC2-2 and ßC2-4) specifically inhibit the translocation and function of C2-containing isozymes (-PKC, ß_I-PKC, and ß_II-PKC), but not the C2-less isozymes (-PKC and -PKC). We first used the pseudosubstrate peptide (0.1 µmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I_Ba was reduced to 16.8±7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I_Ba were then determined in the presence of C2-derived peptides or control peptides. When the pipette contained 0.1 µmol/L of ßC2-2 or ßC2-4, PMA-induced inhibition of basal I_Ba was 26.1±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated I_Ba was inhibited by 29.9±6.6% and 29.3±7.8% in the presence of ßC2-2 and ßC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled ßC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated I_Ba was almost completely abolished in cells dialyzed with both ßC2-2 and ßC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca²⁺ channel activity.

Key Words: Ca²⁺ current • receptor • phorbol ester • protein kinase C isozyme • cardiac myocyte

	Introduction

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Voltage-gated Ca²⁺ channels play an important role in normal and diseased myocardium.^{1 2} The sympathetic nervous system exerts an important modulatory effect on cardiac Ca²⁺ channels through both ₁- and ß-adrenergic receptors. ß-Adrenergic contribution to the sympathetic system has been studied extensively. In general, ß-adrenergic stimulation acts by increasing the levels of cAMP, which activates a cAMP-dependent protein kinase, which in turn phosphorylates the Ca²⁺ channel.² A parallel membrane-delimited pathway involving a direct stimulatory action of the G protein, G_s, on I_Ca has been also reported,³ although this remains controversial.⁴ The ₁-adrenergic regulation of cardiac function has been studied to a lesser extent. Even less attention has been directed to the interaction between ß- and ₁-adrenergic components of sympathetic regulation of cardiac Ca²⁺ channels. Our previous reports^{5 6} established the existence of a functional negative-feedback regulatory mechanism between ₁- and ß-adrenergic receptors vis-à-vis Ca²⁺ channels, and this may, at least in part, involve PKC.⁶

₁-Adrenergic activation leads to the generation of inositol trisphosphate and diacylglycerol.^{7 8} Diacylglycerol activates PKC isozymes, which phosphorylate several proteins, including the Ca²⁺ channel protein.⁹ Conflicting viewpoints regarding the effects of PKC on Ca²⁺ channels still remain: increase,¹⁰ decrease,^{11 12} increase followed by a decrease,^{11 13} and no effect¹⁴ were reported. To bypass the inositol trisphosphate pathway, we used the phorbol ester PMA to stimulate PKC isozymes. Two subfamilies of PKC isozymes are stimulated by PMA. These are C2-containing cPKC isozymes (-PKC, ß_I-PKC, ß_II-PKC, and -PKC) and the C2-less nPKC isozymes (-PKC, -PKC, -PKC, and -PKC).¹⁵ Although -PKC¹⁶ and -PKC¹⁷ are not present in the heart, the presence of other PKC isozymes in the heart remains debatable (reviewed in Reference 18¹⁸ ). Nevertheless, it is clear that multiple isozymes are present in cardiac myocytes and can concomitantly be activated by PMA. In the present study, we show that PMA inhibited L-type Ca²⁺ channel activity. To determine which PKC isozyme mediated this PMA-induced regulation of the channel, we have dialyzed myocytes with peptide inhibitors of PKC. These peptides specifically inhibit the translocation and function of C2-containing isozymes, but not the C2-less isozymes in intact cells.^{19 20}

The proposed mechanism of inhibition induced by these peptides is as follows: Hormone- or PMA-induced activation of PKC is associated with translocation of the enzyme from the cell soluble to the cell particulate fraction.^{21 22} In addition to binding to lipids, this translocation reflects association of activated PKC isozymes with proteins in the particulate fraction (see review in Mochly-Rosen²³). Mochly-Rosen's group suggested that there are isozyme-specific anchoring molecules termed RACKs that anchor each activated isozyme to its site of action. The binding sites for RACKs on the C2-containing cPKC isozyme have been mapped to the C2 domain^{20 24} and, specifically, to three small regions within this domain.¹⁹ Synthetic peptides corresponding to these regions inhibit cPKC binding to RACKs in vitro and inhibit PKC translocation in intact cells.¹⁹ Because translocation is required for PKC to carry out its function,²⁵ presumably because it brings the activated enzyme to the vicinity of its substrate, inhibition of translocation results in inhibition of the function of that enzyme.²³ The C2-derived peptides do not interfere with the translocation¹⁹ or function²⁶ of C2-less enzymes. For example, in the neonatal cardiac myocyte, PMA-induced translocation of ß_I-PKC into the nucleus and ß_II-PKC to the perinucleus and cell membrane was completely blocked by the introduction of the ßC2-4 peptide into these cells. In contrast, PMA-induced translocation of -PKC and -PKC was undisturbed.¹⁹ In the present study, these peptides have been applied intracellularly to adult cardiac myocytes to demonstrate that cPKC mediates PMA-induced inhibition of the L-type Ca²⁺ channel activity.

	Materials and Methods

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Isolation of Cardiac Myocytes
Cardiac myocytes were obtained from hearts of Wistar rats (200 to 250 g) by enzymatic dissociation according to the method of Wittenberg et al,²⁷ with some modifications.^{6 28} The heart was perfused with a HEPES-buffered solution containing (mmol/L) NaCl 117, KCl 5.7, NaHCO₃ 4.4, NH₂PO₄ 1.5, MgCl₂ 1.7, HEPES 20, glucose 11, creatine 10, and taurine 20, along with 21 mU/mL insulin, and the pH was adjusted to 7.4 with NaOH. The heart was then perfused with fresh buffer mixed with 1.5 mg/mL collagenase type A or B (Boehringer-Mannheim Corp) and 20 µmol/L Ca²⁺ for 40 to 50 minutes. The ventricles were then cut off and stirred to obtain cells. Cells were suspended in Petri dishes containing HEPES buffer with 1 mmol/L CaCl₂ and 0.5% bovine serum albumin (pH 7.4). All solutions used for perfusion were gassed with 100% O₂ and warmed to 37°C. After incubation for 30 minutes, a small aliquot of the medium containing single cells was transferred to a chamber mounted on the stage of an inverted microscope (Nikon Inc). Rod-shaped noncontracting cells with clear striations were used for the whole-cell voltage-clamp studies. Most experiments were carried out at temperatures of 22°C to 24°C and some at 35°C to 37°C using a temperature-controller device (by N. Datyner, Cell MicroControls). No differences in the effect of PMA on I_Ba/Ca were observed at either temperature range, except for the expected increase in the current kinetics at 35°C to 37°C.

Solutions and Drugs
The composition of external solutions was (mmol/L) NaCl 132, CsCl 5.4, BaCl₂ or CaCl₂ 1.8, MgCl₂ 1.8, NaH₂PO₄ 0.6, 4-aminopyridine 5, HEPES 10, dextrose 5, and sodium pyruvate 5, pH 7.4. Patch electrodes were filled with control internal solution containing (mmol/L) CsCl 139.8, K₂EGTA 0.1 or 10, MgCl₂ 4, CaCl₂ 0.062, Na₂-creatine phosphate 5, HEPES 10, Na₂ATP 3.1, and Na₂GTP 0.42, adjusted to pH 7.1 with KOH. C2 region–derived peptides of ß-PKC (ßC2-2 and/or ßC2-4, 0.1 µmol/L) were intracellularly applied, individually or in combination, with the pipette solution. As a negative control, we used the scrambled pseudosubstrate peptide (0.1 µmol/L) and, as a positive control, the pseudosubstrate peptide (0.1 µmol/L) that inhibits the activity of all the PKC isozymes. For these experiments, larger electrode tips (0.6 to 0.8 M) were used, and a time interval of 5 to 10 minutes was allowed before the application of PMA to ensure proper diffusion of the peptides into the cytoplasm. The effects of PMA on basal or ISO (0.1 µmol/L)–stimulated I_Ca/Ba were evaluated in the absence and presence of the peptide in the pipette solution. The concentrations of 0.1 µmol/L PMA^{10 11 12} and 0.1 µmol/L of each peptide were used throughout. Peptide ßC2-2 (M-D-P-N-G-L-S-D-P-Y-V-K-L, ß-PKC [186 to 198]), ßC2-4 (S-L-N-P-E-W-N-E-T, ß-PKC [218-266]), pseudosubstrate peptide (R-F-A-R-K-G-A-L-R-Q-K-N-V), control peptide, scrambled ßC2-4 (W-N-P-E-S-L-N-T-E), pentalysine (K-K-K-K-K), and scrambled pseudosubstrate peptide (R-A-L-Q-R-A-K-N-E-V-H-K-V-F-K-G-N-R) were synthesized at Protein and Nucleic Acid Facility, Stanford (Calif) University. All peptides used were over 90% pure. Peptides were dissolved in DMSO and stored at -20°C. The maximal concentration of DMSO in the recording solution was 0.1%. The same amount of DMSO was added to the control recording solution.²⁹ All chemicals were purchased from Sigma Chemical Co or otherwise indicated.

Electrophysiology
The whole-cell configuration of the patch-clamp techniques was used.³⁰ The seal and the breaking into the cell was first performed in the presence of Ca²⁺ ions, followed by switching to Ba²⁺ solution. We found that this method facilitated the formation of the seal and made the cells more tolerable to Ba²⁺. Five to 7 minutes was required for I_Ba to reach steady state. Therefore, the zero time shown in the figures represents at least 5 to 10 minutes after the breakthrough into the cell. Basal I_Ba is referred to as I_Ba recorded without prior ß-adrenergic stimulation. To record I_Ba, all K⁺ currents were blocked with intracellular and extracellular Cs⁺ and 4-aminopyridine.^{5 28 29} The fast Na⁺ current was blocked by a prepulse to -50 mV from a holding potential of -80 mV in the presence of tetrodotoxin (50 µmol/L) to ensure complete blockade. Cells were depolarized every 10 seconds from a holding potential of -80 mV to a prepulse level of -50 mV for 100 milliseconds and subsequently to a test pulse of 0 mV for 300 milliseconds. A programmable horizontal puller (model P-87, Sutter Instrument Co) was used to pull the electrodes. The volume of recording chamber was 0.5 mL, and the rate of superfusion was 3 mL/min. The junction potential was always compensated and was <5 mV. Membrane currents were recorded using a patch-clamp amplifier (model 3900A, Dagan Corp). Capacitive currents were elicited by a 10-mV depolarizing pulse from -80 mV and then compensated. Later on, the capacitive traces were fitted by a single exponential equation, and C_m was calculated according to the following equation: C_m=_c·I_o/E_m, where C_m is the membrane capacitance, _c is the time constant for cell membrane charge, I_o is the maximum capacitive current, and E_m is the clamp voltage. The average C_m was 140.6±6.7 pF (n=95).

Data Analysis
Membrane currents were digitally recorded and analyzed using pCLAMP software (version 6.0.1, Axon Instruments Inc). Origin (Microcal Origin v3.7, Microcal Software Inc) programs were used to generate figures. Data were presented as mean±SEM. Student's t test for paired data was used to compare control conditions with drug interventions. A value of P<.05 was considered statistically significant.

Western Blot Analyses
Rat hearts were removed, washed twice in chilled phosphate-buffered saline, and stored at -80°C. Hearts were then thawed, cut into small pieces, and homogenized in homogenization buffer (20 mmol/L Tris HCl, pH 7.4, 0.25 mol/L sucrose, 1 mmol/L each of EDTA and EGTA, and 20 µmol/L each of phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, and aprotinin). Approximately 300 µg of homogenate was then centrifuged for 30 minutes at 100 000g. The resulting supernatants were removed and saved. The particulate fractions were suspended in the same volume of homogenization buffer as the corresponding supernatant fractions with a tuberculin syringe and a 22-gauge needle. A small aliquot from each fraction was saved for Bio-Rad protein assay to confirm equal protein loading for each fraction. The remaining amounts of the supernatant and particulate fractions were combined with SDS Laemmli sample buffer, heated at 90°C for 5 minutes, and subjected to SDS-PAGE on 12% acrylamide gels. Western blot analyses with PKC isozymes were performed from Seikagaku Corp. Antisera for -PKC, -PKC, and -PKC isozymes were from Life Technologies Inc.

	Results

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Sensitivity of Ca²⁺ Channels to Dihydropyridines in Adult Rat Ventricular Myocytes
Although only L-type Ca²⁺ channels have been reported in adult rat ventricular myocytes,^{31 32} we tested the sensitivity of the current recorded, under our experimental conditions, to dihydropyridines and cobalt. Cells were routinely depolarized from a holding potential of -80 to -50 mV for 100 milliseconds, followed by a test pulse to 0 mV for 300 milliseconds, every 10 seconds. The currents recorded were increased by Bay K 8644 (1 µmol/L) and inhibited by nisoldipine (2 µmol/L) and cobalt (5 mmol/L). These characteristics are those of L-type I_Ca. Fig 1 illustrates these effects.

View larger version (14K): [in this window] [in a new window]   Figure 1. Effects of dihydropyridines and cobalt on IBa. Panel A shows superimposed current tracings of IBa recorded from a holding potential of -80 to -50 mV for 100 milliseconds, followed by a test pulse to 0 mV for 300 milliseconds every 10 seconds during control and during the steady state effect of Bay K 8644 (1 µmol/L). In panels B and C, the same protocol was used to test the effects of 2 µmol/L nisoldipine (B) and 5 mmol/L cobalt (C) on IBa recorded from two different cells. Note that only the test pulse is shown.

PMA Inhibits Basal I_Ca and I_Ba
As stated earlier, the reported effects of PMA on basal I_Ca are contradictory.^{10 11 12 13 14} Therefore, we tested the nature of the effects of PMA (0.1 µmol/L) on basal I_Ca in adult rat ventricular myocytes. PMA consistently inhibited I_Ca in all the cells studied by an average of 51.4±3.0% (n=10, P<.02). Fig 2A shows a representative example of the effects of PMA on I_Ca.

View larger version (21K): [in this window] [in a new window]   Figure 2. PMA inhibits basal Ica and IBa in adult rat ventricular myocytes. Panel A shows ICa recorded from a cell superfused with control external solution for 10 minutes in the presence of normal pipette solution. Superfusion of the cell with 0.1 µmol/L PMA resulted in a slow decrease in Ica from 1.55 to 0.78 nA during steady state. PMA inhibited Ica by 49.7%. Con indicates control conditions. In panel B, the experimental settings are the same as those in panel A except for the use of equimolar concentrations of Ba2+ (1.8 mmol/L) as a charge carrier in another cell. Substitution of Ca2+ with Ba2+ resulted in the expected slowing in the current inactivation rate. Similarly, a 10-minute superfusion of PMA (0.1 µmol/L) reduced IBa from 0.87 to 0.48 nA (44.8%). Two superimposed tracings of Ica (panel A) and IBa (panel B) represented by a and b during the time indicated on the graph are shown in the inset. Panel C shows the effect of PMA on ISO-stimulated IBa in a rat ventricular myocyte. IBa was recorded for 5 minutes in the presence of normal pipette solution. Application of 0.1 µmol/L ISO increased IBa from 0.8 to 2.74 nA in 10 minutes. Addition of PMA (0.1 µmol/L) to ISO-containing solution inhibited IBa from 2.74 to 1.43 nA (47.8%). The steady state effect was reached at 8 minutes of PMA application. The inset shows selected IBa tracings at the times indicated by a, b, and c on the graph.

Whereas Ca²⁺ ions are known to precipitate inactivation of I_Ca, Ba²⁺ ions do not substitute for Ca²⁺ in mediating this Ca²⁺-dependent inactivation³³ and subsequent reduction in peak I_Ca.³⁴ To minimize Ca²⁺-induced inactivation of I_Ca, equimolar substitution of Ca²⁺ by Ba²⁺ was performed. All the following experiments were then carried out with Ba²⁺ as the charge carrier. This allowed us to distinguish the inhibitory effects of PMA mediated through PKC from the inhibition resulting from Ca²⁺-induced inactivation. Superfusion of cells with PMA also inhibited basal I_Ba by 40.5±7.4% (n=5, P<.05). These effects were gradual and required longer times (45 to 50 minutes) for partial reversibility. Although the effects of PMA were difficult to wash out, it is noteworthy that a steady state effect of PMA was always obtained. Fig 2B illustrates such effects.

PMA Inhibits ISO-Stimulated I_Ba
We previously showed that ₁-adrenergic activation inhibited ß-adrenergic–stimulated I_Ca.^{5 6} These effects were attenuated by calphostin C, a PKC inhibitor.⁶ To determine whether direct activation of PKC plays a role in this inhibition, we tested the effects of PMA on ISO-stimulated I_Ba. Superfusion of cells with ISO (0.1 µmol/L) resulted in the expected increase in I_Ba. Subsequent addition of PMA (0.1 µmol/L) to the ISO-containing solution resulted in the inhibition of I_Ba by 48.9±7.8% (n=6, P<.02). Fig 2C illustrates such effects.

-PDD Does Not Affect Basal and ISO-Stimulated I_Ba
The specificity of the effects of PMA on I_Ba was confirmed by comparing its effects with those of another phorbol ester, -PDD, which does not activate PKC.^{10 35} Superfusion of cells with 0.1 µmol/L -PDD did not significantly change either basal I_Ba (5.1±2.6%, n=5, P=NS) or ISO-stimulated I_Ba (7.3±3.6%, n=6, P=NS), indicating that the observed PMA effects mentioned above were mediated through PKC activation (Fig 3). Note that the time course of basal and ISO-stimulated I_Ba is stable over a period of 15 to 20 minutes of recording.

View larger version (19K): [in this window] [in a new window]   Figure 3. -PDD does not inhibit basal and ISO-stimulated IBa. In panel A, IBa was recorded in the presence of normal pipette solution. After an 8-minute superfusion of the cell with -PDD, a phorbol ester that does not activate PKC, there was no significant change in IBa (from 1.79 and 1.71 nA, -4.5%). Con indicates control conditions. Panel B shows that -PDD (0.1 µmol/L) similarly did not significantly change ISO-stimulated IBa (from 1.32 and 1.25 nA, -5.3%). Selected IBa tracings at the times indicated by a and b (panel A) and a, b, and c (panel B) on the graphs are shown in the insets.

Effects of PMA on Basal and ISO-Stimulated I_Ba Are Prevented by Pseudosubstrate Peptide, a PKC Inhibitor
To further substantiate the role of PKC in the PMA inhibitory effect, we tested the ability of a specific PKC inhibitor,³⁶ pseudosubstrate peptide, to antagonize the effects of PMA. This peptide is derived from the N-terminus autoinhibitory region of ß-PKC and was found to inhibit the catalytic activity of the enzyme.³⁶ Cells were dialyzed with 0.1 µmol/L pseudosubstrate for at least 10 minutes to allow for the peptide to access the cytoplasm. Large electrodes (0.6 to 0.8 M) were also used for dialysis experiments (see "Materials and Methods"). PMA perfusion of cells initially dialyzed with the pseudosubstrate peptide resulted in only 12.6±9.4% (n=6, P=NS) inhibition of basal I_Ba. Similarly, addition of PMA to ISO-containing solution in the presence of the pseudosubstrate peptide reversed the effects of PMA (Fig 4A). In these cells, PMA did not significantly change ISO-stimulated I_Ba (16.8±7.4%, n=5, P=NS). The same above experimental protocol was used in the presence of a scrambled pseudosubstrate (0.1 µmol/L) that was used as a negative control. The effects of PMA on ISO-stimulated I_Ba were not prevented by the presence of scrambled pseudosubstrate (Fig 4B). PMA inhibited ISO-stimulated I_Ba by 45.8±6.3% (n=5, P<.05) in the presence of scrambled pseudosubstrate. Also, the effects of PMA on basal I_Ba were not prevented by the scrambled pseudosubstrate peptide (the Table).

View larger version (24K): [in this window] [in a new window]   Figure 4. Pseudosubstrate peptide, but not scrambled pseudosubstrate peptide, prevents PMA-induced inhibition of ISO-stimulated IBa. In panel A, the cell was dialyzed with the pseudosubstrate peptide (0.1 µmol/L) that inhibits the activity of all the PKC isozymes. Application of ISO (0.1 µmol/L) resulted in the expected increase in IBa from 0.72 to 1.94 nA. Addition of PMA (0.1 µmol/L) to ISO-containing solution for 8 minutes resulted in only a 10.8% decrease in IBa. Con indicates control conditions. Panel B shows IBa recorded from another cell that was dialyzed with the scrambled pseudosubstrate (0.1 µmol/L) used as a negative control. Under these conditions, PMA (0.1 µmol/L) dramatically inhibited IBa by 50.4% (from 1.80 to 0.89 nA). Selected IBa current tracings at the times indicated on the graphs by a, b, and c are shown in the insets.

View this table: [in this window] [in a new window]   Table 1. Effects of PMA and Peptides on Basal and Stimulated IBa

Effects of PMA on Basal and ISO-Stimulated I_Ba Are Modulated by Specific Inhibitors of cPKC
We have previously shown that there are multiple PKC isozymes in cardiac cells that can be concomitantly activated by PMA.^{18 37} It is likely that each is involved in the regulation of a specific function. To identify the isozyme(s) that may mediate PMA-induced regulation of the L-type Ca²⁺ channel, we have used novel inhibitors specific for cPKC.¹⁹ These are short peptides derived from the C2 region of ß-PKC that antagonize the translocation and function of -PKC, ß_I-PKC, and ß_II-PKC but not the translocation of -PKC (Reference 19¹⁹ and J.A. Johnson and D. Mochly-Rosen, unpublished data, 1996). Two different cPKC peptide inhibitors, ßC2-2 (0.1 µmol/L) and ßC2-4 (0.1 µmol/L), were tested for their ability to antagonize the PMA-induced inhibition of I_Ba. In the presence of ßC2-2 or ßC2-4, PMA inhibited basal I_Ba by 26.1±4.5% (n=4, P<.05) and 23.6±2.2% (n=4, P<.05), respectively (Fig 5). Similarly, in the presence of ßC2-2 or ßC2-4, PMA inhibited ISO-stimulated I_Ba by 29.9±6.6% (n=6, P<.02) and 29.3±7.8% (n=6, P<.05), respectively (Fig 6). Note that in the absence of the peptides, the inhibitory effect of PMA was 48.9±7.8% (n=6, P<.02). However, in the presence of both peptides, ßC2-2 and ßC2-4, the inhibitory effect of PMA on basal and ISO-stimulated I_Ba was almost completely abolished (Fig 7). In these cells, the inhibitory effect of PMA on basal and ISO-stimulated I_Ba was 8.4±5.5% (n=3, P=NS) and 11.3±3.5% (n=4, P=NS), respectively.

View larger version (22K): [in this window] [in a new window]   Figure 5. The inhibitory effect of PMA on basal IBa is attenuated by peptides that are specific inhibitors of cPKC. In panel A, IBa was recorded from a cell that was dialyzed with a peptide (0.1 µmol/L) derived from the C2 region of ß-PKC (ßC2-2) known to inhibit the translocation and function of cPKC.19 Superfusion with PMA (0.1 µmol/L) for 17 minutes resulted in 27.7% inhibition of IBa (from 0.94 to 0.68 nA). Con indicates control conditions. Panel B shows IBa recording from another cell that was dialyzed with a peptide (0.1 µmol/L) derived from another C2 region of ß-PKC (ßC2-4) shown to also inhibit the translocation and function of cPKC.19 Under these conditions, PMA (0.1 µmol/L) also inhibited IBa by 26.6% (from 0.64 to 0.47 nA). Selected IBa current tracings at the times indicated on the graphs by a and b are shown in the insets.

View larger version (22K): [in this window] [in a new window]   Figure 6. The inhibitory effect of PMA on ISO-stimulated IBa is attenuated by peptides that are specific inhibitors of cPKC. In panel A, IBa was recorded from a cell that was dialyzed with a peptide (0.1 µmol/L) derived from the C2 region of ß-PKC (ßC2-2) shown to inhibit the translocation and function of cPKC.19 Application of ISO (0.1 µmol/L) for 8 minutes resulted in the expected increase in IBa from 0.33 to 1.08 nA. Addition of PMA (0.1 µmol/L) to ISO-containing solution for 8 minutes resulted in 30.6% inhibition of IBa (from 1.08 to 0.75 nA). Con indicates control conditions. Panel B shows IBa recording from another cell that was dialyzed with a peptide (0.1 µmol/L) derived from another C2 region of ß-PKC (ßC2-4) shown to also inhibit the translocation and function of cPKC.19 Under these conditions, PMA (0.1 µmol/L) also inhibited ISO-stimulated IBa by 34.3% (from 2.12 to 1.4 nA). Selected IBa tracings at the times indicated on the graph by a, b, and c are shown in the insets.

View larger version (22K): [in this window] [in a new window]   Figure 7. Complete antagonism of PMA-induced inhibitory effect on basal and ISO-stimulated IBa by combined ßC2-2 (0.1 µmol/L) and ßC2-4 (0.1 µmol/L). Panel A shows that application of PMA (0.1 µmol/L) to a cell that was dialyzed with both ßC2-2 (0.1 µmol/L) and ßC2-4 (0.1 µmol/L) peptides reduced IBa by 10.2% from 0.61 to 0.54 nA. Con indicates control conditions. Panel B shows that PMA (0.1 µmol/L) reduced ISO-stimulated IBa by 11% from 2.1 to 1.86 nA in another cell that was also dialyzed with both ßC2-2 (0.1 µmol/L) and ßC2-4 (0.1 µmol/L). Selected IBa tracings at the times indicated by a and b (panel A) and a, b, and c (panel B) on the graphs are shown in the insets.

Effects of PMA on Basal and ISO-Stimulated I_Ba Are Not Affected in the Presence of Control Peptides
Negative control experiments were performed using scrambled ßC2-4 peptide or pentalysine (another control peptide used in the laboratory of D. Mochly-Rosen). PMA exerted its inhibitory effect on basal and ISO-stimulated I_Ba when cells were dialyzed with either 0.1 µmol/L scrambled ßC2-4 peptide (Fig 8) or 0.1 µmol/L pentalysine (Fig 9). PMA inhibited basal I_Ba by 42.0±3.5% (n=6, P<.02) and 35.8±5.0% (n=3, P<.05) in the presence of the scrambled ßC2-4 peptide and pentalysine, respectively. Similarly, PMA inhibited ISO-stimulated I_Ba by 37.8±2.5% (n=6, P<.02) and 42.0±6.2% (n=4, P<.02) in the presence of the scrambled ßC2-4 peptide and pentalysine, respectively. Therefore, the inhibitory effect of PMA on basal and ISO-stimulated I_Ba was not altered in the presence of any of the control peptides.

View larger version (21K): [in this window] [in a new window]   Figure 8. Lack of antagonism of PMA-induced inhibitory effect on basal and ISO-stimulated IBa by scrambled ßC2-4 control peptide. Panel A shows IBa recorded from a cell that was dialyzed with scrambled ßC2-4 control peptide (0.1 µmol/L). Application of PMA (0.1 µmol/L) inhibited IBa by 46.8% from 0.62 to 0.33 nA (see inset). Con indicates control conditions. Panel B shows recording of ISO-stimulated IBa from another cell dialyzed with scrambled ßC2-4 control peptide. PMA (0.1 µmol/L) superfusion inhibited ISO-stimulated IBa by 36.5% from 2 to 1.27 nA (see inset). Selected IBa tracings at the times indicated by a and b (panel A) and a, b, and c (panel B) on the graphs are shown in the insets.

View larger version (21K): [in this window] [in a new window]   Figure 9. Lack of antagonism of PMA-induced inhibitory effect on basal and ISO-stimulated IBa by the control peptide, pentalysine. Panel A shows that 8 minutes of superfusion of PMA (0.1 µmol/L) reduced IBa by 38.8% from 1.16 to 0.71 nA in a cell dialyzed with the control peptide pentalysine (0.1 µmol/L). Con indicates control conditions. Panel B illustrates the effect of PMA (0.1 µmol/L) on ISO-stimulated IBa from another cell dialyzed with pentalysine (0.1 µmol/L). PMA reduced IBa by 35.6% from 1.49 to 0.96 nA. Selected IBa tracings at the times indicated by a, b, and c on the graphs are shown in the insets.

The summary of the effects of PMA on basal and ISO-stimulated I_Ba in the absence and presence of the peptides is shown in the Table.

Effects of PMA on I_Ba Are Not Modified by OA
To investigate whether phosphatases interfere with PKC activation and subsequent Ca²⁺ channel phosphorylation, the effects of PMA on basal I_Ba were investigated in the presence of 1 µmol/L OA, an inhibitor of type 2A and type 1 phosphatases.² Cells were first superfused with OA, followed by OA+PMA in the absence and presence of both ßC2-2 and ßC2-4. In the absence of ßC2-2 and ßC2-4, exposure of cells with OA alone resulted in slight but not significant increase in basal I_Ba (7.6±3.5%, n=6, P=NS). Addition of PMA to OA-containing solution inhibited I_Ba by 45±4.4% (n=6, P<.02). When cells were dialyzed with both ßC2-2 and ßC2-4, OA similarly increased I_Ba, but not significantly (8.0±3.8%, n=6, P=NS). However, addition of PMA to OA resulted in 16.8±9.8% (n=6, P=NS) inhibition compared with the steady state effect of OA and 9.4±6.7% (P=NS) inhibition compared with control. These effects are not significantly different from the effects of these peptides on PMA-induced inhibition in the absence of OA (Table).

These data indicate that OA-induced inhibition of dephosphorylation by type 2A and type 1 phosphatases does not influence the effects of ßC2-2 and ßC2-4 on the PMA-induced inhibition of I_Ba, suggesting that the C2-derived peptides effectively inhibit the PKC isozyme(s) that mediates PMA regulation of the channel activity.

Detection of PKC Isozymes in the Adult Rat Heart
As mentioned above, the presence of ß-PKC in the adult rat heart has been controversial.^{16 37 38 39 40 41} The ability of ßC2-2 and ßC2-4 to attenuate PMA effects is consistent with the existence of a functional ß-PKC and/or -PKC in these cells. To confirm the presence of the ß-PKC isozyme in the adult rat ventricle, Western blot analysis was conducted on cytosolic and particulate cell fractions as described in "Materials and Methods" and elsewhere.¹⁸ As shown in Fig 10, ß-PKC was found in the cytosol of adult cardiac cells and migrated on SDS-PAGE as an 78-kD protein. This protein comigrated with ß-PKC purified from adult rat brain (leftmost lane). Furthermore, -PKC, -PKC, -PKC, and -PKC were also detected by Western blot. The -PKC and -PKC isozymes migrated at molecular masses of 78 kD, whereas the mass of -PKC was 90 kD. The immunoreactivity of -PKC consisted of two species of 78 and 69 kD.

View larger version (25K): [in this window] [in a new window]   Figure 10. Detection of PKC isozymes in the rat heart. Adult rat hearts were homogenized and fractionated into cytosolic (C) and particulate (P) fractions by centrifugation. Total cytosolic (90-µg) and particulate (160-µg) fractions were combined with SDS-PAGE sample buffer, heated at 90°C for 5 minutes, and subjected to SDS-PAGE on 12% acrylamide gels. Proteins were next electrotransferred onto nitrocellulose paper, which was probed with PKC isozyme–selective antisera as indicated on the left side of the figure. -PKC, ß-PKC, and -PKC isozymes migrated at molecular masses of 78 kD, whereas the mass of -PKC was 90 kD. PKC -immunoreactivity consisted of two species of 78 and 69 kD. Purified PKC from rat brain is shown in the leftmost lane as a reference. Data shown are from a single experiment typical of three experiments conducted with different heart preparations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study showed that direct activation of PKC by the phorbol ester PMA consistently inhibited basal and ß-adrenergic–stimulated I_Ba. This inhibitory effect was not mimicked by the biologically inactive phorbol ester analogue, -PDD, but was attenuated by C2 region–derived peptides that block the translocation and function of cPKCs, possibly ß-PKC and/or -PKC.

Nature of the Current Studied
Two types of voltage-activated I_Ca have been reported in mammalian cardiac cells.² Although several studies showed the existence of only L-type I_Ca in adult rat ventricular cells,^{31 32} we tested I_Ca sensitivity to dihydropyridines and cobalt under our experimental conditions. The results showed that the current recorded was enhanced by Bay K 8644 and blocked by nisoldipine and cobalt, consistent with L-type characteristics. At the single-channel level, similar findings using adult rat ventricular myocytes were found by our group⁶ and reported by Scamps et al.⁴²

Rundown of I_Ba
A time-dependent decline of whole-cell I_Ca has been reported in the literature for various cell types and is referred to as "rundown."⁴³ The inhibitory effect of PMA on basal I_Ba and on ISO-stimulated-I_Ba had average decay slopes of 82.1±4.6 pA/min (n=6) and 284.7±86 pA/min (n=7), respectively, compared with 7.1±3.5 pA/min (n=5) and 11.1±5.9 pA/min (n=6) during control experiments. Therefore, the inhibitory effect of PMA was far larger than could be accounted for by spontaneous rundown during control conditions. In addition, a steady state level of the effect of PMA was always reached.

Effects of PKC Activation on Ca²⁺ Channels
Phorbol esters have been reported to inhibit,^{11 12} have no effect,¹³ stimulate,¹⁰ or initially stimulate but later inhibit^{11 13} I_Ca in cardiac myocytes. PKC activation also produced inconsistent responses in noncardiac Ca²⁺ channels. Stimulation of Ca²⁺ channels by phorbol esters has been reported in Aplysia bag cell neurons,⁴⁴ neuroblastoma,⁴⁵ secretory RINm5f⁴⁶ cells, and frog sympathetic neurons.⁴⁷ Conversely, inhibition of Ca²⁺ channels by phorbol esters has also been reported in chick DRG cells⁴⁸ and PC-12 cells.⁴⁹ Under our experimental conditions, PMA consistently inhibited I_Ba in all cells studied. Therefore, it seems that the outcome of PKC activation is species and tissue dependent, probably because of the isozyme type present in each tissue.

In the present study, we demonstrated that one or more of the cPKCs present in these cells is responsible for the inhibitory effect of PMA on I_Ba. We currently cannot determine whether this is due to direct phosphorylation of the channel or phosphorylation of channel regulatory protein(s). Also, we cannot determine which of the cPKC isozymes mediates this effect because the C2-containing isozymes are equally inhibited by the C2-derived peptides.¹⁹ Nevertheless, we showed that a combination of the two C2 region–derived peptides inhibited 77% of the effect of PMA (PMA-induced inhibition of ISO-stimulated I_Ba was reduced from 48.9% to 11.3%). For comparison, the pseudosubstrate peptide inhibitor at an equal concentration inhibited only 65.6% of the effect of PMA (PMA-induced inhibition of ISO-stimulated I_Ba was reduced to 16.8%). These data suggest that cPKC and not nPKC mediated these effects of PMA. Blockade of type 2A and type 1 phophatases by OA did not significantly alter the PMA-induced response on I_Ba, probably because the 0.1 µmol/L PMA causes maximal phosphorylation of the Ca²⁺ channels or regulatory protein(s) by cPKC. In addition, OA did not reverse the effects of the C2-derived peptides on PMA, probably because they blocked the access of cPKC to the channel or associated regulatory protein(s); if there was no residual PKC-mediated phosphorylation activity, the presence or absence of phosphatase activity should not alter the Ca²⁺ channel activity.

Because cPKC isozymes depend on Ca²⁺ for their activity,⁵⁰ some further consideration should be made. The inhibitory effect was observed in the presence of extracellular Ba²⁺ used as a charge carrier through Ca²⁺ channels with 10 or 0.1 mmol/L EGTA in the pipette. The obvious question that arises is how the cPKCs were activated. Biochemical studies from J.A. Johnson and D. Mochly-Rosen at Stanford University (unpublished data, 1997) showed that BaCl₂ could substitute for CaCl₂ at equimolar concentrations when PKC-induced ³²P incorporation into the substrate histone IIIs was monitored. This suggests that Ba²⁺ ions can substitute for Ca²⁺ ions in the modulation of phosphotransferase activity of the classical PKC isozymes and therefore could account for the activation of PKC under our experimental conditions. In addition, phorbol esters are known to increase the affinity of cPKC for Ca²⁺⁵¹ (that was not completely buffered), resulting in its full activation probably even at very low concentrations of Ca²⁺.

₁-Adrenergic Activation Versus PKC Activation
The electrophysiological effects of ₁-adrenergic activation on basal I_Ca are also somewhat inconclusive. At least eight published reports including one from our group found no effect of ₁-adrenergic stimulation on I_Ca, using the patch-clamp techniques.^{5 52 53 54 55 56 57 58} An increase in I_Ca by ₁-adrenergic stimulation was reported in voltage-clamped bovine ventricular trabeculae⁵⁹ and recently in neonatal ventricular myocytes.⁶⁰ Whether receptor activation leads to an increase or no effect, direct PKC activation by PMA consistently inhibited Ca²⁺ channels. It is not surprising that ₁-adrenergic activation and exogenous PMA activation of PKC lead to different effects on Ca²⁺ channels. Several possibilities have been proposed and could account for these differences. Hartzell⁶¹ suggested that the different actions of phorbol esters compared with receptor stimulation may be due to the nonselective stimulation of all PKC isozymes present in the heart, whereas ₁-adrenergic stimulation activates only a subpopulation of the total PKC. Alternatively, the extent of activation by PMA may be greater and more sustained. This is consistent with the finding that myocardial cell ₁-adrenergic stimulation causes an increase in PKC activity, which is markedly less than that determined by a tumor-promoting phorbol ester.⁶² In addition, receptor stimulation leads to the generation of other second messengers. Thus, PKC isozyme activation in response to PMA may differ from those activated by ₁-adrenergic receptor stimulation.

The observed effects of PMA on Ca²⁺ channels were specific and related to the activation of PKC, since the inactive phorbol ester, PDD, had no effect on basal and ISO-stimulated I_Ba. In addition, the effects of PMA were antagonized by a known general PKC inhibitor, pseudosubstrate peptide, but not by the scrambled version of this peptide. PKC inhibition of ß-adrenergic–stimulated Ca²⁺ channels may prove to be relevant in physiological and pathological conditions where norepinephrine release generates mixed ₁- and ß-adrenergic responses. Under these circumstances, ₁-adrenergic stimulation could provide a negative-feedback loop and counteract the ß-adrenergic effect to enhance Ca²⁺ channels, as was recently reported for cardiac chloride channels.⁶³ The characterization of the role of PKC isozyme(s) in this regulation is of obvious therapeutic implications. In this regard, Ishii et al⁶⁴ have recently developed an oral ß-PKC–specific inhibitor that ameliorated vascular dysfunction in diabetic rats.

	Selected Abbreviations and Acronyms

 

cPKC, nPKC = conventional and novel PKC DMSO = dimethyl sulfoxide IBa = Ba2+ current ICa = Ca2+ current ISO = isoproterenol OA = okadaic acid -PDD = -phorbol 12,13-didecanoate PKC = protein kinase C PMA = 4ß-phorbol 12-myristate 13-acetate RACK = receptor for activated C kinase

	Acknowledgments

 
This study was supported by Veterans Administration Medical Research Funds and National Institutes of Health grant HL-55401 (Dr Boutjdir) and by National Heart, Lung, and Blood Institute grant 43380 (Dr Mochly-Rosen). We would like to thank Dr Brett Premack for reviewing the manuscript and the animal laboratory staff and N. Stergiopoulos for their assistance.

Received May 20, 1996; accepted February 3, 1997.

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