Ca’+ mobilization by extracellular ATP in rat cardiac myocytes: regulation by protein kinase C and A JING-SHENG

ZHENG,

ALLEN

CHRISTIE,

MATTHEW

N. LEVY, AND ANTONIO

SCARPA

Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 Zheng, Jing-Sheng, Allen Christie, Matthew N. Levy, and Antonio Scarpa. Ca2+ mobilization by extracellular ATP in rat cardiac myocytes: regulation by protein kinase C and A. Am. J. Physiol. 263 (Cell Physiol. 32): C933-C940, 1992.Activation of protein kinase C (PKC) modulates the mobilization of intracellular Ca2+ induced by extracellular ATP in rat ventricular myocytes. Pretreatment of myocytes with PKC activators attenuated both the ATP-induced Ca2+ transient and the noradrenergic potentiation of the Ca2+ response. Various PKC activators decreased both the basal CAMP level and the CAMP levels that had been elevated by norepinephrine, forskolin, or 3-isobutyl-l-methylxanthine. The inhibitory effects of PKC activators were reversed by the PKC inhibitor staurosporine. The ATP-induced Ca2+ response is an integrated response resulting from ATP eliciting an inward cation current (IA&, cellular depolarization, Ca2+ influx through Ca2+ channels, and Ca2+ release from the sarcoplasmic reticulum. We used the whole cell voltage-clamp technique to investigate which steps of this integrated response are affected by PKC. PKC activators did not significantly affect the IATP. In contrast, PKC activators decreased the basal Ca2+ current (I& or Ba2+ current and the ,&adrenergic-stimulated loa. These results suggest that PKC-induced suppression of the ATP-induced Ca2+ response and the ,&adrenergic-potentiated Ca2+ response is achieved at least partially by decreasing the intracellular CAMP level and loa. adenylate cyclase; adenosine triphosphate receptor; calcium channel RECENT STUDIES HAVE SHOWN that the plasma membrane of ventricular myocytes has ATP receptors that regulate transmembrane potential and cytosolic Ca2+ concentration (3,4, 27, 28). The addition of micromolar concentrations of ATP (or 2-Met-ATP) to acutely isolated cardiac myocytes results in a transient increase in cytosolic Ca2+, which has been hypothesized to occur following these successive steps: binding of ATP to a purinergic receptor, activation of an inward nonselective cation current (I ATP), membrane depolarization, activation of voltage-sensitive Ca 2+ channels, increased influx of Ca2+ from the extracellular space, and finally Ca2+induced Ca2+ release from the sarcoplasmic reticulum (SR) (3, 4, 27). In additional studies, we found that the transient increase of intracellular Ca2+ concentration induced by extracellular ATP is potentiated in the presence of norepinephrine (NE) and that potentiation can be fully accounted for by ,&adrenergic stimulation and subsequent increase in cytosolic adenosine 3’,5’-cyclic monophosphate (CAMP) (28). Activation of protein kinase C (PKC) may modulate various receptor-coupled effector systems, including the adenylate cyclase system. Depending on the cell type, PKC may either potentiate (12) or inhibit (24) an agonist-induced increase in CAMP concentration. Activation of PKC by phorbol esters or diacylglycerol evokes negative inotropic responses in various cardiac prepara0363-6143/92

$2.00 Copyright

tions (2, 15, 26). Such negative inotropic effects have been explained in multiple ways including the following: desensitization of the P-adrenergic receptors (19), decreased cytosolic Ca2+ concentration (2)) inhibition of Ca2+ accumulation in the SR (20), and modulation of the transmembrane calcium current (loa) (16,18,23). However, the action of PKC on the Ioa in cardiac myocytes is variable and controversial: decrease (23), increase (7), initial increase followed by decrease (16), or no change of calcium current (1, 6) has been reported. In this study, we have investigated the involvement of PKC in the Ca2+ mobilization induced by ATP and the potentiation of the Ca2+ response in the presence of NE. Our data indicate that the intracellular Ca2+ mobilization induced by ATP is depressed by activation of PKC. The CAMP transduction system and the L-type Ca2+ channels are inhibited by activation of PKC thereby leading to suppression of the ATP-induced Ca2+ mobilization. METHODS Preparation. Ventricular myocytes were prepared from Sprague-Dawley adult rats (250-350 g) by the method of Christie et al. (3) as previously described. The dispersed ventricular myocytes were suspended in Joklik solution that contained 1% bovine serum albumin (by weight) and 1.25 mM Ca2+. The viability of the cells in Joklik solution was typically 70-80% as visually determined by the percentage of rod-shaped cells. Preincubation of cells with modifying agents [such as NE, phorbol 12,13-dibutyrate (PDBU), or staurosporine] did not change the viability of the cells. Measurement of intracellular Ca2+ concentration. Intracellular Ca2+ concentration ( [Ca2+Ii) was measured by loading the ventricular myocytes with fura-2/acetoxymethyl ester and suspending them in Geigy solution containing the following (in mM): 120 NaCl, 10 NaHCO,, 10 Na-N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic acid (HEPES), 3.0 KCl, 1.2 KH,PO*, 1.0 MgSO,, 1.25 CaCl,, and 10 glucose, as previously described (4, 27). The viability of the cells in Geigy’s solution was the same as that in the Joklik solution. The pH of the solution was maintained at 7.4 by titrating with HCl. Cell suspension (1.5 ml; 5 x lo4 cells/ml) was placed in a thermostated cuvette (37°C) under continuous stirring. The fluorescence was excited by ultraviolet light at 340 nm, and light emission was monitored at 510 nm in a fluorimeter designed and built by Analytical Bioinstrumentation Cleveland, Case Western Reserve University. [ Ca2+]i was calculated according to the equation of Grynkiewicz et al. (9). More detailed calibration protocols and considerations for dye leakage distribution in compartments were described by De Young and Scarpa (4). Because of quantitation uncertainties in myocytes, the basal level of cytosolic Ca2+ had some day to day variability. On the other hand, all the experiments were carried out, within the same preparation, in pairs (control and modifying agents), and under those conditions resting cytosolic Ca2+ concentrations were qualitatively similar.

0 1992 the American

Physiological

Society

c933

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

c934

PROTEIN

KINASE

C AND

CA”

Measurement of CAMP. The intracellular CAMP concentration was assessedby radioimmunoassay procedures previously described by Zheng et al. (27). Whole cell voltage-clamp experiments. An AXOPATCH-1B system (Axon Instruments, Burlingame, CA) was used to record transmembrane currents. Pipette resistances were 2.5-5 MQ. An AT-type computer and pCLAMP software (Axon Instruments) were used for data acquisition and analysis. Generally, the analog currents were sampled at 16 kHz after they were filtered at 2 kHz. In the voltage-clamp experiments, scaled hyperpolarizing steps of one-fourth amplitude were used to subtract linear leak current from the records. The standard extracellular solution contained the following: 140 mM N-methyl-Dglucamine, 1.5 mM CaCl,, 2 mM MgC12, 10 mM HEPES, and 10 mM glucose (pH 7.40). The standard intracellular solution consisted of the following: 140 mM CsCl, 1 mM MgC12, 10 mM HEPES, 10 mM ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid, 2 mM CaCl, (free calcium concentration: 108 nM, as calculated by a computer program, IONSLASP, provided by R. B. Taylor, Univ. of Texas at San Antonio), 2 mM MgATP, 20 mM phosphocreatine, and creatine phosphokinase (100 U/ml), pH 7.20, unless otherwise indicated. To avoid leakage of ATP from the patch pipette and the subsequent stimulation of ATP receptors, the tip of the pipette was filled with the described intracellular solution without ATP, and the electrode was backfilled with the described ATP-containing intracellular solution. Solutions were applied to the bath at a flow rate of 5 ml/min through a gravity-fed perfusion system. All experiments were performed at 25°C. Perforated patch experiments. A modification of the method described by Horn and Marty (13) was used. Nystatin (Sigma) was freshly dissolved in dimethyl sulfoxide solution (5 mg/ml) and subsequently added to the intracellular solution to a final concentration of 50-100 pg/ml. Both stock nystatin solution and nystatin-containing intracellular solution were sonicated to facilitate solvation. Stock nystatin was kept at -20°C for up to 1 wk. The final nystatin-containing intracellular solution was kept at 4°C for up to 3 h. The electrode tip was usually filled with nystatin-free intracellular solution, and the electrode was backfilled with a nystatin-containing intracellular solution. After forming a gigaohm seal, the series resistance and capacitance of the cell were monitored every 3-5 min. A greater capacitance and a lower series resistance than the initial values indicated that nystatin had diffused to the membrane patch. Before current recording, the access resistance was typically .3.5-8 MR, which slightly exceeds the range in whole cell recording (typically 2.5-5 Ma). Perforated patch recording provides electrical access to the cell and maintains the intracellular Ca2+ buffering system intact but does not cause the loss of intracellular substances involved in channel activity. This method was used to study the effects of PKC on the basal activity of Ca2+ channel. Statistical analysis. Data are expressed as means k SE. The mean values of control and experimental groups were compared by Student’s t test. A value of P c 0.05 was considered to be significant. RESULTS

Effects of PKC on Ca2+ homeostasis. Addition of ATP (10 PM) to a suspension of adult rat ventricular myocytes loaded with fura- transiently increased the [Ca2+]; by 20% (Fig. 1A). The observed change will be referred to as “the ATP-induced Ca2+ response.” This is an integrated response due to the ATP-induced depolarization of quiescent cells, followed by L-type Ca2+ channel opening and Ca2+ release from the SR. Because of the addition of ATP to a large population of cell/volume, the peak Ca2+ tran-

HOMEOSTASIS

IN MYOCYTES

B -

ATP

ss

146

-134

.OAG/ATP

ATP

+% 0

PDBWATP

- 108

n. I

1 min

I

Fig. 1. Effects of phorbol 12,13-dibutyrate (PDBU) on the ATP-induced Ca2+ response in suspensions of ventricular myocytes loaded with fura-2. A: addition of ATP (10 PM) increased intracellular Ca2+ concentration ([Ca’+]J from a basal level of 105-126 nM (20% increase). After pretreatment with 25 PM 1-oleoyl-2-acetyl-sn-glycerol (OAG) for 10 min, same amount of ATP increased [Ca2+]i from 105 to 119 nM (13% increase). OAG inhibited ATP-induced Ca2+ response by 35%. B: in a different cardiac preparation, ATP increased [Ca”+]i from 108 to 146 nM (35% increase). After pretreatment with 200 nM PDBU for 10 min, ATP increased [Ca2+]i from 108 to 134 nM (24% increase). PDBU inhibited ATP-induced Ca2+ response by 31%. Similar results were obtained in each experimental condition with at least 8 experiments.

sient occurred within lo-20 s, consistent with what was previously reported (3, 4, 27, 28). To investigate the effects of PKC on the ATP-induced Ca2+ response, we used several different PKC activators, such as 1 -oleoyl-2-acetyl-sn-glycerol (OAG) and PDBU. When a different aliquot from the same cell preparation was pretreated with OAG (25 PM) for 10 min, ATP increased [Ca2+]; only 13% (Fig. 1A). OAG inhibited the ATP-induced Ca2+ response by 35%. In a different cell preparation, ATP increased [Ca2+]i from 108 to 146 nM (35% increase). After pretreatment with PDBU for 10 min, ATP increased [Ca2+]; from 108 to 134 nM (24% increase; Fig. 1B). This represents a 3 1% inhibition of the ATP-induced Ca2+ response by PDBU. This decrease occurred within 1 min after pretreatment with PDBU and it reached a maximum after 10 min of pretreatment (data not shown). We also investigated the effect of PKC activators on ,&agonist or 8(4-chlorophenylthio)-CAMP (CPT-CAMP) potentiated response. In a different cell preparation, ATP increased [ Ca2+]; by 17% (Fig. 2A). When cells were pretreated with NE (300 nM) for 2 min, the resting Ca2+ concentration did not significantly change. In some experiments, a small transient decrease (Cl0 nM) was observed. The subsequent addition of ATP (10 PM) increased [ Ca2+]i by 75% (Fig. 2A). Thus, as previously reported (27), NE potentiated the ATP-induced Ca2+ response. Activation of PKC, either before or after (Fig. 2A) ,&agonist stimulation, decreased the noradrenergic potentiation of the Ca2+ response. When a different aliquot from the same cell preparation was pretreated with 300 nM NE for 2 min followed by an additional lo-min preincubation with NE and OAG (25 PM), ATP (10 PM) increased [Ca2+]i by 50% (Fig. 2A). This represents a 34% decrease of the NE-potentiated Ca2+ response. When myocytes were preincubated with OAG for 10 min followed by an additional 2-min preincubation with NE and OAG, ATP increased [Ca2+]i only 34%

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

PROTEIN

KINASE

C AND

cA2+

HOMEOSTASIS

6

A

-177

z +-.93 2

- 159 - 142

z

NE/ATP NE/OAG/ATP

- 124

OAG/NE/ATP

cpt-cAMP/ATP

+r;%I 2

cpt-cAMP/OAG/ATP

ATP -

-106

106

flI ,I min,

Fig. 2. Effects of OAG on norepinephrine (NE)- or 8-(4-chlorophenylthio)-CAMP (CPT-CAMP)-potentiated Ca2+ mobilization induced by ATP. A: ATP increased [Ca2+]i from 106 to 124 nM (17% increase). After pretreatment with NE (300 nM) for 2 min, addition of ATP (10 PM) increased [Ca*+]i from basal level of 106-186 nM (75% increase). However, if myocyte suspensions were stimulated by NE (300 nM) 2 min and followed by incubation of OAG (25 PM) and NE for an additional 10 min, ATP only increased [Ca2+]i from 106 to 159 nM (50% increase). If myocyte suspensions were pretreated with OAG (25 PM) for 10 min followed by incubation of OAG and NE (300 nM) for additional 2 min, ATP increased [Ca2+]i from 106 to 142 nM (34% increase). Although there were small changes in percentage of beating cells in presence of NE, at any time, percentage of beating cells was ~3% of total population. B: in same cardiac preparation after pretreatment with CPT-CAMP (500 PM) for 2 min, addition of ATP (10 PM) increased [Ca2+]i from basal level of 106-177 nM (67% increase). However, if myocyte suspensions were stimulated by CPT-CAMP (500 PM) 2 min and followed by incubation of OAG (25 PM) and CPT-CAMP for an additional 10 min, ATP only increased [Ca2+]i from 106 to 139 nM (31% increase). OAG inhibited CPT-CAMP potentiation by 54%.

(Fig. 2A). This represents a 55% inhibition of the NEpotentiated Ca2+ response. Similar results were obtained in each experimental condition with at least six experiments. Another active phorbol ester [ 12-O-tetradecanoylphorbol-13-acetate (TPA)] also decreased both the ATP-induced Ca2+ response and the potentiation of the Ca2+ response by NE (data not shown). Similar results were observed when CPT-CAMP was added to potentiate the ATP-induced Ca2+ increase. In the same cell preparation as in Fig. 2A, after 2 min of pretreatment with CPT-CAMP (500 PM), ATP increased [Ca2+]i by 67% (Fig. 2B). However, if the myocytes had been stimulated first by CPT-CAMP for 2 min

IN MYOCYTES

c935

and then incubated with CPT-CAMP and OAG for an additional 10 min, ATP increased [Ca2+]i only 31%. This represents a 54% inhibition of the potentiation by CPT-CAMP of the ATP-induced Ca2+ response (Fig. 2B). Qualitatively, similar results were observed in three experiments. Table 1 summarizes the effects of PKC activation on the ATP-induced Ca2+ response prior to application of CPT-CAMP. Neither PDBU nor CPT-CAMP significantly modified the basal intracellular Ca2+ concentration. However, PDBU significantly decreased the ATPinduced Ca2+ response (P < 0.05). After pretreatment with CPT-CAMP for 2 min, ATP increased [Ca2+]i 105 t 20% (Table 1). When cells were pretreated with PDBU for 10 min followed by incubation with PDBU and CPTCAMP for an additional 2 min, ATP increased [Ca2+]i by only 45 t 6%. Thus PKC inhibited the potentiation effect of CPT-CAMP by 57%. Although the magnitudes of NE or CPT-CAMP-potentiated ATP-induced Ca2+ response varied in batches of myocytes prepared on different days, the PKC inhibition of the potentiated Ca2+ response remained relatively constant. Pretreatment with PDBU or OAG also attenuated the Ca2+ responses that had been potentiated by forskolin or 3-isobutyl-lmethylxanthine (IBMX; data not shown). Therefore, activation of PKC suppresses the ATP-induced Ca2+ response and attenuates the potentiation of Ca2+ response by NE or by other agents that increase the intracellular CAMP concentrations. Effects of PKC on the intracellular CAMP level. To investigate the effect of PKC activation on the adenylate cyclase system, several agents that increase the intracellular CAMP level through different mechanisms were used. The aim of this experiment was to assess whether PKC activators act mainly to modulate the basal CAMP level, the CAMP levels that had been elevated by various agents, or both. Figure 3A shows that the basal CAMP concentration of isolated myocytes was 2.50 t 0.17 pmol/mg protein. Pretreatment with PDBU or OAG for 10 min decreased the basal CAMP levels 24% and 18%, respectively. NE increased the CAMP level 146% after 2 min of incubation and maintained CAMP at a high level for 10 min after addition. In the presence of PDBU or OAG, NE increased the CAMP level only 70% and 67%, respectively (Fig. 3A). Forskolin, an activator of adenylate cyclase, increased CAMP level 190% after 2 min of

Table 1. Effects of 200 nM PDBU and 500 PM CPT-CAMP on Ca2+ mobilization

response induced by ATP (Control

1)

10 PM ATP

Pretreatment with PDBU for 10 min Before Addition of ATP

Pretreatment with CPT-CAMP for 2 min Before Addition of ATP (Control 2)

Pretreatment with PDBU for 10 min Followed by PDBU and CPT-CAMP for an Additional 2 min Before Addition of ATP

8 7 6 6 Basal Ca, nM 171t7 149*13* 162t16* 160&26* Ca-ATP, nM 220tll 175+13t 324*30-f 231+38$ %Increase 27t2 18+2t 105+20§ 45&6t$ Values are means t SE. Basal Ca is resting intracellular Ca2+ concentration ( [Ca2+]i) before addition of ATP. Ca-ATP is peak value of [Caz+]i after addition of ATP. %Increase is calculated by following equation: (Ca-ATP - basal Ca) x loo/basal Ca. P values were calculated by Student’s t test for paired or unpaired samples. * P > 0.05 when compared with control 1. t P < 0.05 and § P < 0.005 when compared with control 1. # P < 0.05 when compared with control 2.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

C936

6

PROTEIN

KINASE

C AND

CA’+

HOMEOSTASIS

IN

MYOCYTES

control El +PDBU m

+OAG

200 nM

+PDBU

25 uM

J control

NE

control

forskolin (1 fl)

(50 nM)

L

contrd

m

+PDBU 200 nM

z

I=m

contrd +PDBU 200 nM

I

+PDBU 200 nM

E4 i 9(

$

l-

Fig. 3. Effects of PDBU and OAG on basal intracellular CAMP level and CAMP level stimulated by NE, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). A: basal CAMP level was 2.50 t 0.17 pmol/mg protein (n = 10). NE (50 nM) increased CAMP level to 6.15 t 0.42 pmol/mg protein. Pretreatment with PDBU (200 nM; n = 8) and OAG (25 PM; = 4) not only decreased basal CAMP Lvel to 1.89 t 0.17 and 2.05 & 0.18 pmol/mg protein, respectively, but also decreased CAMP level stimulated by NE (50 nM) to 4.24 & 0.21 and 4.18 t 0.27 pmol/mg protein, respectively. Similar inhibitory effects of PDBU were also seen when cells were stimulated by 1 PM forskolin (B; n = 7) or 100 pM IBMX (C; n = 8). D: inhibitory effect of PDBU was reversed by staurosporine. Bars, means t SE. *P < 0.05 and **p < 0.005 when we compared intracellular CAMP levels under control conditions and that with PDBU pretreatment.

I control

IBMX (100 UM)

NE

control

incubation (Fig. 3B). However, forskolin increased CAMP only 72% in the presence of PDBU (Fig. 3B). Pretreatment with IBMX (an inhibitor of phosphodiesterase) for 10 min increased the CAMP level 160% (Fig. 3C). However, after pretreatment with PDBU for 10 min, IBMX increased the basal CAMP level only 81%. These data suggest that PKC decreased both the basal CAMP level and the CAMP level that had been stimulated by NE, forskolin, and IBMX. Other active phorbol esters, such as TPA, had similar inhibitory effects on CAMP production, whereas an inactive phorbol ester, 4cu-phorbol12,13-didecanoate (4aPDD), did not (data not shown). We also determined whether the PKC inhibitor, staurosporine, would attenuate the effects of PKC activators. Figure 30 shows that staurosporine prevented PDBU from decreasing the CAMP content. NE (1 PM) and IBMX (100 PM) increased CAMP level from a basal value of 2.54 t 0.15 (n = 11) to 5.07 t 0.34 (n = 11) and 7.59 t 2.21 (n = 6) pmol/mg protein, respectively. Pretreatment with 200 nM PDBU for 10 min decreased the basal CAMP level to 1.96 t 0.11 (n = 11) and decreased the CAMP level stimulated by NE or IBMX to 3.64 t 0.34 (n = 11) and 4.21 t 0.47 (n = 6) pmol/mg protein, respectively. After pretreatment with 100 nM staurosporine for 10 min, followed by incubation with staurosporine and PDBU for an additional 10 min, IBMX increased the CAMP level from a basal value of 2.87 t 0.70 (n = 11) to 7.01 t 0.40 pmol/mg protein (n = 7). In the presence of staurosporine and PDBU, the CAMP level stimulated by NE was 7.05 t 0.69 pmol/mg protein (n = 1 l), which is significantly higher than the CAMP level stimulated by NE alone (P < 0.005; Fig. 3D).

IBMX

Electrophysiological effects of PKC. One of the early events leading to the ATP-induced Ca2+ mobilization response is the activation of 1ATP by ATP (3,27). Therefore, the effects of PDBU on this current were investigated to determine if PDBU modulated IATP and subsequently attenuated the ATP-induced Ca2+ response. In whole cell voltage-clamp experiments, the addition of ATP (100 PM) to a cell clamped at -70 mV evoked an lATP (Fig. 4). The inset shows that under control conditions, IATP was 434 t 38 pA (n = 13). After cells were preincubated with PDBU for lo-20 min, IATP was 455 t 57 pA (n = 8), which is not significantly different from the control value. This experiment indicates that PKC ATP 0

-

-lOO-2oo-. (200 did)

2 e-300-

H

24-400-

-500 1

2 Set

-6004

Fig. 4. Effect of PDBU on ATP-induced nonselective cation inward current (I*&. Addition of ATP (100 PM) to a cell clamped at -70 mV produces IATP. Inset: statistical data on 1*TP under control condition (434 t 38 PA) and after pretreatment with 200 nM PDBU for lo-20 min. Bars, means & SE.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

PROTEIN

KINASE

C AND

CA*+

does not modulate the ATP-induced Ca2+ response by inhibiting IATP. A major source of Ca2+ responsible for the ATPinduced Ca2+ mobilization is the extracellular space. The extracellular Ca2+ enters the cell through Ca2+ channels upon cellular depolarization due to activation of &rP. Therefore, the following experiments were designed to assess whether PKC modulates Ca2+ channels. The perforated patch recording technique was used to study the effects of PKC on the basal Ca2+ channel activity. Ba2+ (2 mM) was used as the charge carrier (Fig. 5). In the perforated patch recording, the transmembrane potential was held at -70 mV, and the test pulse was stepped to -10 mV every 10 s to monitor the peak Ba2+ current (&). After basal IBa remained constant, addition of PDBU (200 nM) decreased the peak IBa. After washout of PDBU, the lBa did not return to the control level, but it remained constant for more than 15 min. We found that 2.5 min after PDBU addition, basal lna was decreased by 27 t 2% (n = 4). Similar results were obtained using Ca2+ as the charge carrier (data not shown). When we substituted 4cu-PDD (2 PM, an inactive form of phorbol ester) for PDBU, no decrease of lna or loa was seen (data not shown). These data indicate that PKC modulates basal loa. Several investigators (14, 22) have shown that P-agonists stimulate calcium channels by a CAMP-dependent mechanism, and we have determined that @-agonist potentiation of the ATP-induced Ca2+ response is due to an increase of CAMP level (27). Because PDBU decreased the basal and the ,&adrenergic-stimulated CAMP levels, we investigated the effect of PDBU on the stimulatory effect of NE on loa. We used the whole cell voltage-clamp technique with Ba 2+ (2 mM) as the charge carrier. Cells were held at -70 mV, and the potential was changed in lo-mV depolarizing steps to generate current-voltage relationships for &. Figure 6A shows NE enhanced IBa when cells were depolarized over the range of -30 mV to +60 mV. NE enhanced the peak lna from 1,313 t 164 to 2,101 t 301 pA at -10 mV. In cells that were pretreated with 200 nM PDBU for 10 min, NE increased lBa from 1,063 t 65 to 1,482 t 98 pA at -10 mV (Fig. 6B). Figure 6C shows that pretreatment of myocytes with PDBU

4d

-0.5. crc+.Ful/c--* washout

-1.5

I

5 min

Fig. 5. Effect of PDBU on Ba*+ current (IBa) during perforated patch recording. Membrane potential was held at -70 mV and stepped to -10 mV every 10 s. Addition of PDBU (200 nM) decreased peak 1na. After washout, 1na remained stable for more than 15 min.

HOMEOSTASIS

c937

IN MYOCYTES

CONTROL

-60-40 0 204060

+ PDBU

200 nM

-20

NE

OAG/NE

Fig. 6. Effect of PDBU on I Ba stimulated by NE. A: in whole cell voltage-clamp studies, Ba *+ (2 mM) was used as charge carrier. Cells were clamped at -70 mV, and depolarizing pulses were applied in IO-mV increments to generate current-voltage relationships. NE (1 PM) increased peak In, from 1,313 & 164 to 2,101 & 301 pA at -10 mV (n = 4, mean & SE). B: cells that were pretreated with 200 nM PDBU for 10 min, NE increased peak Ina from 1,063 & 65 to 1,482 t 98 pA at -10 mV (mean & SE). c: statistical data of &a stimulated by NE without (n = 4) or with PDBU (n = 3). NE increased peak Ina by 60 t 5%. However, after pretreatment with PDBU (200 nM), NE increased peak Ina by only 38 t 2%. Bars, means t SE.

significantly decreased the stimulatory effect of NE on the calcium channel (P < 0.05). Figure 7 shows the time courses of NE stimulation and PDBU inhibition of loa. The currents shown are from two different cells voltage clamped in the whole cell configuration with Ca2+ as a charge carrier. The membrane potential was held at -70 mV and stepped to 0 mV to elicit &. The intracellular solution contained 108 nM free Ca2+ and an ATP regenerating system with 2 mM ATP. Under these conditions, addition of isoproterenol (ISO; 1 PM) maximally stimulated calcium channels and increased loa by 62% (Fig. 7A). In the presence of ISO, addition of PDBU (200 nM) decreased the stimulatory effect of IS0 on loa by 45%. We also found that TPA (200 nM to 1 PM) or OAG (25 PM) decreased the loa stimulated by IS0 (300 nM to 1 PM) or NE (300 nM to 1 PM; data not shown). The inhibitory effects of PKC activators on ,&agonist-stimulated loa can be seen when intracellular solution contained a physiological concentration of Ca2+, with or without ATP. However, when the

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

C938

PROTEIN

KINASE

C AND

cA2+

HOMEOSTASIS

Fig. 7. Effects of PDBU on Ca2+ current (Ica) stimulated by isoproterenol (ISO). A : in whole cell configuration with Ca2+ as charge carrier, cell was clamped at -70 mV and depolarized to 0 mV every 10 s. We monitored time course of peak Ica. Intracellular solution contained free Ca2+ = 108 nM, and an ATP regeneration system with 2 mM ATP (see detail in METHODS). Addition of IS0 (1 PM) increased loa by 62%. Subsequent addition of PDBU (200 nM) decreased Ica in presence of ISO. Individual current traces recorded at indicated time are shown in inset. B: in absence of intracellular Ca2+ and ATP, PDBU did not decrease Ica that had been previously stimulated by ISO. Individual current traces recorded at indicated time are shown in inset.

+ isoproterenol + PDBU

4 min

IN MYOCYTES

**

intracellular solution was devoid of Ca2+, PDBU did not decrease the Zoa stimulated by IS0 (Fig. 7B). These data are consistent with the idea that the effect of PKC is Ca2+ dependent. Because these data demonstrate that PKC decreases both basal Zoa and P-agonist-stimulated Zca, we conclude that the calcium channel is a main site of action for PKC inhibition of the ATP-induced Ca2+ mobilization. DISCUSSION

PKC regulates agonist-induced responses by affecting one or more steps in agonist-response cascades. PKC modulates cellular responses at the level of receptors (19), G proteins (lo), phospholipase C effector systems (21), adenylate cyclase (24), and directly on ion channels (17). Although cross talk between PKC and the adenylate cyclase system has been characterized in various cell types, it has not been quantitatively addressed in cardiac myocytes. In the present study, we investigated the role of PKC in the regulation of the ATP-induced Ca2+ mobilization and the potentiation of the Ca2+ mobilization by NE. PKC not only suppresses the ATP-induced mobilization of intracellular Ca 2+ but it also attenuates the potentiation of Ca2+ mobilization by NE and other

agents that increase the intracellular Interactions

CAMP level.

between PKC and adenylate cyclase system.

Various agents increase the intracellular CAMP levels in cardiac myocytes through different mechanisms: NE stimulates P-receptors, forskolin activates adenylate cyclase directly, and IBMX inhibits phosphodiesterase (PDE) thereby retarding the degradation of CAMP. To investigate the interaction of PKC with the adenylate cyclase effector system, agents that increase the intracellular CAMP level were used. Our data indicate that activation of PKC by OAG and phorbol esters modulates the basal and P-stimulated adenylate cyclase effector system in cardiac myocytes. The effects of OAG and phorbol esters were not due to nonspecific interactions with the adenylate cyclase effector system because multiple PKC activators had the same effect, inactive phorbol ester was ineffective, and the effects of PKC activators were inhibited by staurosporine. The CAMP level stimulated by NE in the presence of staurosporine and PDBU was significantly higher than the CAMP level stimulated by NE alone. This might be due to removal of tonic PKC inhibition of adenylate cyclase, removal of tonic PKC stimulation of PDE, or inhibition of protein kinase A (PKA) by staurosporine.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

PROTEIN

KINASE

C AND

CA2

NE, by stimulating ,&receptors, increased the intracellular CAMP level (Fig. 3), increased the transmembrane loa, and potentiated the ATP-induced Ca2+ response (Fig. 1). In principle, the effect of PDBU and OAG on NE effect may be partially due to desensitization of P-adrenergic receptors in the cardiac membrane (19). However, this is unlikely because PKC also suppressed the CAMP levels stimulated by forskolin and IBMX that bypass the ,&receptors. Our data suggest that PKC may inhibit adenylate cyclase activity, increase PDE activity, or both. It has shown CPT-CAMP potentiates the ATP-induced Ca2+ response, presumably by direct activation of PKA followed by activation of Ca 2+ channels (27). Therefore, a post-CAMP modulation mechanism is also involved in the effects of PKC in the cardiac myocytes, possibly at the level of calcium channel or PKA. Electrophysiological effects of PKC. The ATP-induced Ca2+ mobilization cascade involves the activation of a depolarizing nonselective cation current and subsequent activation of L-type calcium channels. Because pretreatment with PKC activators did not significantly modulate 1ATP (Fig. 4), our data do not support the idea that modulation of the ATP-induced Ca2+ mobilization response by PKC occurred at the level of the ATP receptor (i.e., by inhibiting I ATP) or by desensitization of the ATP receptor itself. Previously (27)) we have shown noradrenergic potentiation of the ATP-induced Ca2+ response involves CAMP as a second messenger. However, CAMP does not modulate IATP. Thus we have not been able to identify a second messenger system that modulates the initial event in the ATP-induced Ca2+ mobilization (i.e., IATP). However, we found that PDBU decreased both the basal intracellular CAMP level and basal IBa and loa. Because intracellular CAMP might be required for the regulation of basal Ca2+ channel activity, the inhibitory effect of PKC on basal loa is at least partially accounted for by a decrease of the basal CAMP level. We also found that after washout of PDBU, lna remained inhibited for more than 15 min. Lack of reversibility can be accounted for by the lipophilic property of PDBU. The irreversible effects of PDBU were also seen when Ca2+ was used as the charge carrier. Our results are consistent with the finding that in chick heart cells the negative inotropic effect induced by TPA was not reversed after TPA was washed out (17). The PKC activators, PDBU or TPA, not only decreased the basal loa but also decreased the loa that was maximally stimulated by the ,&adrenergic agonists, NE or IS0 (Fig. 7). The effects of PKC are Ca2+ dependent because the inhibitory effect of PKC was seen only when intracellular Ca2+ was present. It is likely that, under these conditions, cellular ATP remained at concentrations above those required for phosphorylation by kinases. The inhibitory effect of PKC on loa was consistent with other studies that phorbol esters decrease cardiac contractility in various preparations (2, 7, 17, 26). Many studies have shown that activation of PKC modulates ion channel activity in various preparations. In cardiac myocytes, the modulation of PKC on Ca2+ channel is variable. In neonatal cardiac myocytes, PKC increases (7) or increases and then decreases Ioa (16). In contrast, PKC was reported to have no effects on loa

HOMEOSTASIS

IN MYOCYTES

c939

in guinea pig ventricular myocytes (6). The diverse effects of PKC can be explained by use of different cell types or different experimental conditions. Different cell types may contain different PKC isozymes. It is generally accepted that PKC is a Ca2+ and phospholipid-dependent enzyme. Different experimental conditions (such as different Ca2+ buffering condition) may also influence the activity of PKC. Rogers et al. (20) have shown that in cardiac myocytes, CAMP stimulates the rate of oxalate-supported 45Ca uptake into the sarcoplasmic reticulum by twofold, whereas TPA and PDBU inhibit this process by 45%. These data suggest that a decrease of the SR function can, at least partially, account for the negative inotropic effect of PKC (20). Under our experimental conditions, we cannot eliminate the possibility that PKC may inhibit the ATPinduced Ca2+ response by modulating the Ca2+-induced Ca2+ release mechanism In conclusion, we have identified some of the mechanisms responsible for the inhibitory effects of PKC on the ATP-induced Ca2+ response in myocytes. Binding of ATP to a purinergic receptor elicits IATp, which depolarizes the cell membrane and activates the L-type Ca2+ channel. The influx of calcium through the calcium channels increases [Ca2+]i and releases Ca2+ from the SR. NE potentiates the ATP-induced Ca2+ response mainly by increasing the Ica. Activation of PKC inhibits this cascade mainly by decreasing calcium current and CAMP level. We cannot rule out the possibility that PKC also desensitizes P-receptors, inhibits PKA activity, or interferes with Ca2+ release from the SR. Based on the results of this study and of previous studies (27,28), we conclude that at least two second messenger systems regulate the ATP-induced mobilization of intracellular Ca 2+. Activation of the adenylate cyclase effector system potentiates the ATP-induced Ca2+ response, whereas activation of PKC suppresses both the ATP-induced Ca2+ response itself and the potentiation of the Ca2+ response by NE and other agents that increase the intracellular CAMP levels. Hence, the interaction between PKC and the adenylate cyclase system may regulate the intracellular CAMP level and the [Ca2+]i, which, in turn, would regulate cardiac contractility. The authors thank Drs. C. A. Obejero-Paz, G. Dubyak, S. Jones, and R. Harvey for valuable suggestions. This study was supported by National Heart, Lung, and Blood Institute Grants HL-10951, HL-18708, and HL-07653. Address reprint requests to A. Scarpa. Received 26 February 1992; accepted in final form 25 June 1992. REFERENCES 1. Apkon, M., and J. M. Nerbonne. cui-Adrenergic agonists selectively suppress voltage-dependent K+ currents in rat ventricular myocytes. Proc. Nut!. Acad. Sci. USA 85: 8756-8760, 1988. 2. Capogrossi, M. C., T. Kaka, C. R. Filburn, D. J. Pelto, R. G. Hansford, H. A. Spurgeon, and E. G. Lakatta. Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes. Circ. Res. 66: 1143-1155, 1990. 3 . Christie, A., V. K. Sharma, and S.-S. Sheu. Mechanism of extracellular ATP induced increase of cytosolic Ca2+ concentration in isolated rat ventricular myocytes. J. Physiol. Lond. 445: 360-388, 1992.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.

c940

PROTEIN

KINASE

C AND

cA2+

M. B., and A. Scarpa. ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+. Am. J.

4. De Young,

Physiol. 257 (Cell Physiol. 26): C750-C758, 1989. 5. De Young, M. B., and A. Scarpa. Extracellular

ATP activates coordinated Na+, Pi, and Ca2+ transport in cardiac myocytes. Am.

J. Physiol. 6. Dirksen,

260 (Cell Physiol. R., D. Williford,

29): C1182-C1190, and S.-S. Sheu.

1991.

Regulation of the guinea pig ventricular action potential by al-adrenergic receptor stimulation: possible role of protein kinase C (PKC) (Abstract). FASEB J. 3: A847, 1989.

A., R. S. Dhallan, 7. Dosemeri, and T. B. Rogers. Phorbol

N. M.

Cohen,

W. J. Lederer,

HOMEOSTASIS

Leatherman, G. F., D. Kim, and T. W. Smith. Effect of phorbol esters on contractile state and calcium flux in cultured chick heart cells. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H205-H209, 1987. 18. Levitan, I. B. Phosphorylation of ion channels. J. Membr. Biol.

17.

87: 177-190, 1985. 19. Limas, C. J., and

20.

21.

1991. 9. Grynkiewicz,

22.

A new generwith greatly improved fluorescence prop-

Poenie,

and

R. Y. Tsien.

ation of Ca2+ indicators erties. J. Biol. Chem. 260: 3440-3450,

1985. J. A. Garcia-Sainz.

Activation of protein kinase C inhibits hormonal stimulation of the GTPase activity of Gi in human platelets. FEBS Lett. 279: 316-318, 1991.

10. Gutierrez-Venegas,

11. Hockberger,

G.,

P.,

M.

and

Toselli,

D. Swandulla,

and

H.

D. Lux.

A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation. Nature Lond. 338: 340342, 1989. 12. Hollingsworth,

E. B.,

D.

Ukena,

and

J. W.

Daly.

The proenhances

tein kinase C activator phorbol-12-myristate-13-acetate cyclic AMP accumulation in pheochromocytoma cells. FEBS

Lett.

196: 131-134, 1986. 13. Horn, R., and A. Marty.

Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol.

92: 145-159, 14. Kameyama,

1988. M.,

F. Hofmann, and W. Trautwein. On the mechanism of P-adrenergic regulation of the Ca channel in guineapig heart. Pfluegers Arch. 405: 285-293, 1985. 15. Karmazyn, M., J. E. Watson, and M. P. Moffat. Mechanisms for cardiac depression induced by phorbol myristate acetate in working rat hearts. Br. J. Pharmacol. 100: 826-830, 1990. 16. Lacerda, A. E., D. Rampe, and A. M. Brown. Effects of protein kinase C activators on cardiac Ca2+ channel. Nature Lond. 335: 249-251,

1988.

Phorbol ester- and diacylglycerolof cardiac ,&adrenergic receptors. Circ.

C. Limas.

mediated desensitization

ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes. Circ. Res. 62: 347-357, 1988. 8. Dubyak, G. R. Signal transduction by P2-purinergic receptor for extracellular ATP. Am. J. Respir. Cell Mol. Biol. 4: 295-300, G., M.

IN MYOCYTES

Res. 57: 443-449, Rogers, T. R.,

1985. S. T.

Gaa,

C.

Massey,

and

A.

Dosemeci.

Protein kinase C inhibits Ca2+ accumulation mic reticulum. J. Biol. Chem. 265: 4302-4308,

in cardiac sarcoplas-

Ryu, S. Carpenter,

A.

H.,

U.-H. Kim, K.-P. Huang,

tion of phospholipase 17941-17945, Sperelakis,

M. and

I.

Wah,

1990. B. Brown,

G.

S. G. Rhee. Feedback regulaC-p by protein kinase C. J. Biol. Chem. 265:

1990. N., and

G. M. Wahler. Regulation of Ca2+ influx in myocardial cells by /3-adrenergic receptors, cyclic nucleotides and phosphorylation. Mol. Cell. Biochem. 82: 19-28, 1988. 23. Tseng, G.-N., and P. A. Boyden. Different effects of intracellular Ca and protein kinase C on cardiac T and L Ca currents. Am. J. 1991. 24. Wiener,

Physiol.

261

(Heart

Circ.

Physiol.

30):

H364-H379,

E., and A. Scarpa. Activation of protein kinase C modulates the adenylate cyclase effector system of B-lymphocytes. J. Biol. Yatani,

Chem. 264: 4324-4328, A., and A. M. Brown.

1989.

Rapid beta-adrenergic modulation of cardiac calcium channel currents by a fast G-protein pathway. Science Wash. DC 245: 71-74, 1989. 26. Yuan, S., F. A. Sunahara, and A. K. Sen. Tumor-promoting phorbol esters inhibit cardiac functions and induce redistribution of protein kinase C in perfused beating rat heart. Circ. Res. 61: 25.

27.

372-278, 1987. Zheng, J.-S., A. Christie, A. Scarpa. Synergism

transduction 28.

Physiol. 31): Cl28-C135, Zheng, J.-S., M. B. De A. Scarpa. Extracellular

myocytes are potentiated NY

Acad.

M. B. De Young,

M. N. Levy,

and

between cyclic AMP and ATP in signal and cardiac myocytes. Am. J. Physiol. 262 (Cell

Sci.

603: 448-451,

1992. Young,

E. Wiener,

M. N. Levy,

and

ATP-induced Ca2+ transients in cardiac by an increase in cellular CAMP. Ann. 1990.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.