Yonago Acta medica 1999;42:31–39

Activated Protein Kinase C Attenuates Ca2+ Overloading and Reoxygenation Hypercontracture in Isolated Rat Cardiomyocytes following
Chemical Hypoxia
Shigeto Miyasaka, Takashi Ichiba, Hiroaki Kuroda and Shigetsugu Ohgi
Second Department of Surgery, Faculty of Medicine, Tottori University, Yonago 683-0826,
Japan
The aims of this study were i) to test the effects of protein kinase C (PKC) activation on
the intracellular Ca2+ concentration of rat cardiomyocytes during chemical hypoxiareoxygenation, ii) to determine the contribution of the sarcoplasmic reticulum (SR)and the Na+-Ca 2+ exchange on the regulation of intracellular Ca2+ following PKC
activation and iii) to test the role of PKC-dependent intracellular pH changes inintracellular Ca2+ regulation. We used the isolated adult rat cardiomyocyte perfusion
model. Cardiomyocytes were loaded with the Ca2+-fluorescent probe Fluo-3 and the
pH-fluorescent probe SNARF1. Cells were subjected to 50 min of glucose-free and
NaCN chemical hypoxia followed by 30 min of simulated reoxygenation. The activationof PKC significantly inhibited the hypoxia-induced increase of Fluo-3 fluorescentintensity (control; 692 ± 100% and activated PKC; 322 ± 43%: P < 0.05). This inhibitory
effect was not affected by the inhibition of the SR Ca2+ uptake induced by thapsigargin,
but was cancelled by the inhibition of the Na+-Ca2+ exchange with dichlorobenzamil(thapsigargin 337 ± 47%; dichlorobenzamil 609 ± 100%). PKC activation also attenuatedthe decrease in intracellular pH during chemical hypoxia, even in the presence of the
Na+-H+ exchange inhibitor amiloride (control; 6.54 ± 0.02, PKC; 6.72 ± 0.03, PKC +amiloride; 6.73 ± 0.03). We concluded that the PKC attenuation of Ca2+ overloading to
rat cardiomyocytes during chemical hypoxia-reoxygenation does not depend on theCa2+ uptake by the SR, but does require a Na+-Ca2+ exchange. Since PKC attenuatedthe increasing intracellular H+ during chemical hypoxia, a low H+ concentration may
be important for the maintenance of Ca2+ extrusion via the Na+-Ca2+ exchange.
Key words: Ca 2+ overload; intracellular pH; isolated rat cardiomyocytes; Na+-Ca 2+ exchanger;
protein kinase C

Ischemic preconditioning reduces myocardial
injury induced by sustained ischemia-reperfusion, with preservation of myocardial function
and infarct size reduction. Previous findings
suggest that the activation of protein kinase C(PKC) plays a role in the cardioprotective effectof ischemic preconditioning (Liu et al., 1994; Li
and Kloner, 1995; Kitakaze et al., 1996). PKC
activation by Ca2+ or pharmacologic agents
mimics ischemic preconditioning (Miyawaki

and Ashraf, 1977; Armstrong and Ganote,
1994; Tsuchida et al., 1994; Miyawaki et al.,1996). It has been proposed that PKC attenuates an increase in intracellular Ca2+ concentration during ischemia/reperfusion via i) intracellular alkalization (MacLeod and Harding,1991), ii) activation of the sarcoplasmic reticulum (SR) calcium pump (Movesesian et al.,1984), iii) modification of the Na+-Ca2+ exchange (Steenbergen et al., 1993) and iv) inhib-

Abbreviations: amil, amiloride; [Ca2+]i, intracellular Ca2+ concentration; %[Ca2+]i,percentage of the preischemic fluorescene intensity; DCB, dichlorobenzamil; Fluo-3, Fluo-3 acetoxymethylester; %L, percentageof the prehypoxic length; PKC, protein kinase C; pHin, intracellular pH; PMA, phorbol 12-myristate 13acetate; SNARF1, SNARF1 acetoxymethylester; SR, sarcoplasmic reticulum;thaps, thapsigargin

31

S. Miyasaka et al.

ition of the L-type Ca2+ channel (Leatherman etal., 1987; Zheng et al., 1992). However, theprecise mechanism underlying the cardioprotection afforded by PKC activation is unknown.
The present study i) examined how PKC
affects the intracellular Ca 2+ concentration([Ca2+]i) and cell length of isolated rat cardiomyocytes during chemical hypoxia-reoxygenation, ii) tested whether the SR and Na+-Ca2+
exchanger on the sarcolemma contribute to theattenuation of Ca2+ overloading during ischemia under the activation of PKC and iii) examined how PKC affects intracellular pH (pHin)and the regulation of the intracellular Ca 2+
during chemical hypoxia-reoxygenation.

Loading of Fluo-3 and SNARF1
The culture medium was changed from KB
solution to Medium 199 (GIBCO, Grand Island,NY) containing 1% bovine serum albumin (wt/vol), the Ca2+-selective fluorescence probe 10µmol/L Fluo-3 acetoxymethylester (Fluo-3)(Dojindo, Kumamoto, Japan). Pluronic F127
[0.075% (wt/vol)] (Molecular Probes, Inc.,
Evgene, OR) was used as a detergent for cellpermeation of the dye (Satoh et al., 1994; Lopezet al., 1996). Cells were loaded for 30 min at
37˚C, followed by incubation for 30 min withTyrode solution to allow hydrolysis of the Fluo3. The fluorescent pH indicator SNARF1 acetoxymethylester (SNARF1) (Molecular Probes)
at 10 µmol/L was loaded in the same way as
Fluo-3 (Seksek et al., 1991; Zaguilan et al.,1991).

Materials and Methods

Isolation of rat cardiomyocytes
Ventricular myocytes were isolated from adult
rat hearts by enzymatic dissociation (Satoh etal., 1994; Siegmund et al., 1994). In brief, 8week-old male Wistar rats weighing 200 to 250
g were anesthetized by inhalation of diethyl
ether. After an injection of 50 U of heparin into
the inferior vena cava, the hearts were quickly
excised and attached to a Langendorff apparatus. Each heart was perfused through the coronary artery with i) Ca2+-free Tyrode solution(136.5 mmol/L NaCl, 5.4 mmol/L KCl, 0.53
mmol/L MgCl2, 5.5 mmol/L HEPES, 5.5 mmol/
L glucose, adjusted to pH 7.4 with NaOH) at
37˚C for 3 min, ii) Tyrode solution containing
collagenase type I (0.04 g/100 mL, Sigma
Chemical Co, St. Louis, MO) for 20 min and iii)
modified Kraftbruhe (KB) solution (70 mmol/L
KOH, 40 mmol/L KCL, 20 mmol/L KH2PO4, 3
mmol/L MgCl2, 50 mmol/L glutamic acid, 10
mmol/L glucose, 10 mmol/L HEPES, 0.5mmol/L EGTA, titrated to pH 7.4 with KOH).
The ventricles were excised and minced inmodified KB solution. The cells were filteredthrough a stainless steel mesh (150 µm) andincubated for 2 h.
The Guidelines for Animal Experimentation
at the Faculty of Medicine, Tottori University,
were followed.

Apparatus and measurement of [Ca2+]i ,cell length and pHin
Cardiomyocytes attached to a glass-bottomed
dish (content volume 1 mL) on the stage of an
inverted conforcal laser microscope (InSIGHT,
Meridian Instruments Far East, Tokyo, Japan)
were perfused (0.6 mL/min) with Tyrode
solution. The temperature of the dish wasmaintained at 37˚C by a temperature controller(Medical Systems Corp, NY).
The ion-selective fluorescent probes were
excited by an argon laser, at wave lengths of
488 nm for Fluo-3 and 514 nm for SNARF1.
The light emitted from the cells was monitored
by a CCD camera (Meridian Instruments) using
interference filters (530 nm for Fluo-3 and 580
nm and 644 nm for SNARF1). Images wererecorded by a video cassette recorder (SONY,Tokyo) at 30 frames per second.
The fluorescence intensity of Fluo-3 wasanalyzed by the InSIGHT PLUS-IQ analysis
system (Meridian Instruments). Because Fluo3 has no spectral shift on binding Ca2+, the absolute value of [Ca2+]i can not be measured (Minta
et al., 1989; Hayashi et al., 1994). Changes in[Ca2+]i are expressed as the percentage of thepreischemic fluorescence intensity (%[Ca2+]i).
32

Activated PKC attenuates Ca2+ overloading

Normoxic perfusion

Protocol 1
Control
PMA

Chemical hypoxia

Reoxygenation

PMA
0.1 µmol/L

PMA + thaps

thaps 100 nmol/L

PMA + DCB

DCB 2 µmol/L

Protocol 2
ControlPMA
amil 2 mmol/L

PMA + amil
–35

–30

0

50

80

(min)

Time
Fig. 1. Schematic representation of the 2 cardiomyocyte treatment protocols. Cells were perfused with 100%
oxygenated Tyrode solution during normoxic perfusion and reoxygenation periods. Chemical hypoxia wasachieved by the perfusion of Tyrode solution with 2 mmol/L NaCN without glucose at pH 6.7. In theactivated PKC group, cells were perfused with Tyrode solution with 0.1 µmol/L PMA for 5 min from –35 to–30 min. amil, amiloride; DCB, dichlorobenzamil; PKC, protein kinase C; PMA, phorbol 12-myristate 13acetate; thaps, thapsigargin.

The fluorescence intensity of SNARF1 wasanalyzed by the InSIGHT PLUS-IQ analysis
system with a ratio of 580/644 nm. The in situ
calibration of SNARF1 was conducted by thenigericin-K + method (Seksek et al., 1991;
Zaguilan et al., 1991). The fluorescence intensity ratio of 580/644 nm was converted to pHinusing a published calibration curve.
Cell length was measured from the Fluo-3
fluorescence image by the InSIGHT PLUS-IQ
analysis system, and changes in cell length are
expressed as the percentage of the prehypoxic
cell length (%L).
The %[Ca2+]i, pHin and %L were calculated
from the mean of 30 frames (1 s) at each experimental point.

were then subjected to chemical hypoxia for 50
min and reoxygenation for 30 min. The chemical hypoxic solution was based on Tyrode solution but contained 2 mmol/L NaCN, no
glucose and a pH of 6.7.
PMA: Cells were exposed to 0.1 µmol/L
phorbol 12-myristate 13-acetate (PMA) (Wako,
Osaka, Japan) for 5 min before normoxic perfusion. The cells were subsequently perfusedwith oxygenated Tyrode solution and subjected
to chemical hypoxia-reoxygenation in the same
way as the controls.
PMA + thaps: The procedure was similar
to the PKC group except that 100 nmol/Lthapsigargin (thaps) (Funakoshi, Tokyo) was
included in the perfusion solution administeredduring chemical hypoxia (Wu and Feher, 1995;
Meldrum et al., 1996).
PMA + DCB: The procedure was similar to
the PKC group except that 20 µmol/L 2-4dichlorobenzamil (DCB) (Molecular Probes)
was included in the perfusion solution administered during chemical hypoxia (Schiffman etal., 1990; Kawada et al., 1992).
Fluo-3 signals were obtained 1 min before

Experimental protocol 1
Effects of PKC activation on Ca2+ over-loading
and cell length during chemical hypoxiareoxygenation (Fig. 1).
Control: Cardiomyocytes loaded with Fluo3 were perfused with Tyrode solution equilibrated with 100% O2 for 30 min. The cells
33

S. Miyasaka et al.

(%)
900

Chemical hypoxia

Reoxygenation

800
700

%[Ca2+]i

Fig. 2. The time course ofchanges in %[Ca 2+ ] i duringchemical hypoxia-reoxygenation in untreated (open circles, n= 11) and activated PKC-treated(closed circles, n = 11) rat cardiomyocytes. Data are mean ±SEM of control and PMAtreated cells. PKC, protein
kinase C; PMA, phorbol 12myristate 13-acetate.

Control

600

PMA
500
400
300
200
100
0
pre

5

10

15

20

25

30

35

40

45

50

5

10

15

20

25

30

(min)

Time

oride (amil) (an inhibitor of the Na+-H+ exchanger) was administered during chemical hypoxia.
The data were collected at the same point as
those for protocol 1.

chemical hypoxia as prehypoxic data and forevery 5-min period during chemical hypoxiareoxygenation.

Experimental protocol 2
Statistical analysis

Effects of PKC activation on intracellular acidosis during chemical hypoxia-reoxygenation(Fig. 1).
Control: Cells loaded with SNARF1 underwent the same procedure as the control group in
protocol 1.
PMA: Cells loaded with SNARF1 underwent
the same procedure as the PMA group in protocol 1.
PMA + amil: This procedure was similar tothat used for PMA, except that 2 mmol/L amil-

All data are expressed as mean ± SEM. The significance of differences among groups was determined using an analysis of variance with
Fisher’s PLSD post hoc test. Values of P < 0.05
were considered significant. All statistical analysis was performed using the software program
StatView version J-4.5 on a Macintosh computer.

(%)

Chemical hypoxia

Reoxygenation

100
90

%L

80

Fig. 3. The time course of %changes in initial cell length
during chemical hypoxia-reoxygenation in untreated (opencircles, n = 11) and activatedPKC (closed circles, n = 11) rat
cardiomyocytes. PKC, protein
kinase C; PMA, phorbol 12myristate 13-acetate.

70
60

Control
50

PMA

40
30
20
pre

5

10

15

20

25

30

35

40

45

50

5

10

15

20

25

30

(min)

Time

34

Activated PKC attenuates Ca2+ overloading

1000

*

100

*

90

800

80

700

70

600

60

%L

%[Ca2+]i

900

(%)

500

(%)

*
*

**

50

400

40

300

30

200

20

100

10
0

0

Control

PMA

PMA +
thaps

PMA +
DCB

Control

Fig. 4. Intracellular Ca2+ concentration changes inisolated rat cardiomyocytes treated with PMA, thaps
or DCB. The PMA group and the PMA + thaps
group showed lesser increases of %[Ca2+]i than thecontrols, but the PMA + DCB group showed no significant difference from the controls at the end ofchemical hypoxia. *P < 0.05. DCB, dichlorobenzamil; PMA, phorbol 12-myristate 13-acetate;
thaps, thapsigargin.

PMA

PMA +
thaps

PMA +
DCB

Fig. 5. Cell length changes in isolated rat
cardiomyocytes treated with PMA, thaps or DCB. In
the PMA group and the PMA + thaps group, cell
lengths were maintained compared with the controls,
but in the PMA + DCB group, the cell lengths were
not significantly different from those of the controls
at the end of reoxygenation. *P < 0.05. DCB, dichlorobenzamil; PMA, phorbol 12-myristate 13-acetate;
thaps, thapsigargin.

of [Ca2+]i in the PMA group was considerably
attenuated from 35 to 50 min of chemical hypoxia
compared with controls. Finally, the %[Ca2+]iof the control and PMA groups reached 692 ±
100% (n = 11) and 322 ± 43% (n = 11) respectively in chemical hypoxia (P < 0.05) (Fig. 4).
After reoxygenation, %[Ca2+ ] i decreased inboth groups and reestablished preischemic
levels within 15 min.
Ischemic cell shortening (rigor shortening)
was observed 15 min after the initiation ofchemical hypoxia in both groups (Fig. 3). The
cell shapes changed rapidly from rod to squarewith a length reduction to about 62% of theinitial length. At the end of chemical hypoxia,
the %L values of the control and PMA groups
were reduced to 62.9 ± 1.7% and 62.5 ± 1.5%,
respectively. In the control cells, hypercontraction occurred at the initiation of reoxygenation. In contrast, hypercontraction was not observed in the PMA group, and some cells slightly recovered their length. At 30 min after reoxygenation, the %L values of the control and
PMA groups were 40.8 ± 2.6% and 61.8 ± 2.1%
(P < 0.05), respectively (Fig. 5).

Results

Isolated cardiomyocytes
We selected the following cells, each loadedwith fluorescent dye to use as material in theexperiment: i) cells that were rod-shaped, ii)
those with clear cross-striations and iii)
quiescent cells. The mean initial cell length
was 106 ± 3 µm (n = 42), and there were no
significant differences in cell length among
these 4 groups. The cells which had entirely
balled up into blebbed forms with an absent
sarcomere pattern were defined as hypercontractured cells.

Effects of PKC activation on [Ca2+]i andcell length
In the control and PMA groups, [Ca 2+] i increased significantly 25 min after the inductionof chemical hypoxia (Fig. 2). The %[Ca2+]irose continuously to the end of chemical
hypoxia in both groups. However, the increase35

S. Miyasaka et al.

7.6

Chemical hypoxia

Reoxygenation

7.4

7.2

Fig. 6. The time course ofchanges in pHin during chemical hypoxia-reoxygenation in
untreated rat cardiomyocytes(open circles, n = 10 ), thosewith activated PKC (closed circles, n = 9 ), and those with activated PKC and inhibited Na+H– exchanger (squares, n = 9 ).
amil, amiloride; PKC, protein
kinase C; PMA, phorbol 12myristate 13-acetate.

pHin

7

6.8

6.6

Control
PMA

6.4

PMA + amil
6.2
pre

2

5

10

15

20

25

30

35

40

45

50

5

10

15

20

25

30

(min)

Time

7.25 ± 0.03 (n = 9), respectively. In all groups,
a decrease of pHin was observed during chemical hypoxia (Fig. 6).
In the PMA and PMA + amil groups, intracellular acidification was significantly reduced
compared with the control group at 25 min before reoxygenation. The final pHin values during chemical hypoxia of the control, PMA and
PMA + amil groups were 6.54 ± 0.02, 6.72 ±0.03 and 6.73 ± 0.03, respectively (Fig. 6).
In the PMA + amil group, the effect of PKC
activation was observed despite inhibition of
the Na+-H+ exchanger.

Contribution of SR and the Na+-Ca2+ exchanger
In the PMA + thaps group, the increase of%[Ca2+]i during chemical hypoxia was attenuated. The PMA + thaps value of %[Ca2+]i at 50
min of hypoxia was 337 ± 47% (n = 10), with nosignificant difference between the PMA and
PMA + thaps group (Fig. 4).
The alteration of %L in the PMA + thaps
group was similar to that of the PMA group;
hypercontraction was not observed after reoxygenation, and the final %L was 69.4 ± 1.78%,
not significantly different from the PMA groupvalue (Fig. 5). These results indicate that theeffects of PKC activation were not abolisheddespite the inhibition of SR Ca2+ uptake.
In the PMA + DCB group, the %[Ca2+]i at
50 min of hypoxia and final %L were 609 ±
100% and 39.4 ± 4.6% (n = 10), respectively.
These values were not significantly different
from those of the controls (Figs. 4 and 5).
These results suggest that the effects of PKC
activation were abolished by inhibition of the
Na+-Ca2+ exchanger.

Discussion
This study demonstrated that PKC activation by
PMA affects the [Ca2+]i, L and pHin of isolated
rat cardiomyocytes during chemical hypoxia. Intracellular Ca2+ is a very important factor in cell
damage during ischemia-reperfusion (Smithand Allen, 1988). A Ca2+ overloading of cardiomyocytes accelerates ATP depletion andactivates Ca2+-dependent enzymes, leading toirreversible changes including hypercontraction
at reperfusion (Nicotera et al., 1989; Ladilov etal., 1997). The reduction of a Ca2+ overload may
thus reduce reperfusion injury, preserve cardiac
function and limit infarction size.
The results of the present study indicate thatthe activation of PKC reduced the increase of[Ca2+]i during chemical hypoxia. This suggests

Effects of PKC activation on pHin duringchemical hypoxia
The pHin values before chemical hypoxia in thecontrol, PMA and PMA + amil groups were
7.30 ± 0.03 (n = 10), 7.33 ± 0.02 (n = 9) and36

Activated PKC attenuates Ca2+ overloadingplay an important role in the effect of PKC.
Since the SR Ca2+ pump requires ATP to function, its activation by PKC might increase ATP
consumption, and the pump would thus not
function during severe ischemic ATP depletion.
However, when the Na +-Ca2+ exchanger
was inhibited, the effects of PKC activation
were abolished. This indicates that the attenuation of a Ca2+ overload by PKC depends on the
Na+-Ca2+ exchanger. The Na+-Ca2+ exchanger
is a passive Ca2+ transport system that is dependent on the electrical membrane potential andthe intra- and/or extra-cellular concentrations of
Ca2+ and Na+. The Na+-Ca2+ exchanger is thus
a potentially important mechanism in theregulation of Ca2+ under the condition of severe
ATP depletion.
The activation of PKC also reduced intracellular acidification during chemical hypoxia.
Meldrum and colleages (1997) and Rehring andcolleages (1996) reported that the activation of
PKC by PMA or α1 adrenoreceptor stimulation
limits ischemic acidosis in rat hearts. This effect was independent of the Na+-H+ exchanger,
because it occurred despite Na+-H+ exchanger
inhibition. Macleod and Harding (1991) proposed that PKC does not activate the Na+-H+
exchanger directly but rather the Na+-H+ exchanger function was modified by the change in
pHin. The mechanisms underlying the reduction of intracellular acidification following
PKC activation are unknown, although Murryand coworkers (1990) showed that preconditioning reduced the production of lactate, resulting
in a drop in cytosolic pH during ischemia.
Kohmoto and colleagues (1990) andCoravoeuf and coworkers (1981) suggested that
intracellular acidosis impairs Ca2+ extrusion via
the Na+-Ca2+ exchanger. Intracellular H+ may
directly inhibit the Na + - Ca 2+ exchanger(Philipson et al., 1982), and may also indirectly
inhibit it by increasing the Na+ influx via the
Na+-H + exchanger (Baker and McNaughton,1976). If activated PKC attenuates intracellularacidosis by stimulation of the Na+-H+ exchanger, the intracellular Na+ would increase and inhibit the Na+-Ca2+ exchanger. Thus, PKC may
not directly stimulate the Na+-Ca2+ exchange
but rather may inhibit it via a reduction of H+

that activating PKC might protect the myocardium from ischemia and reperfusion injury, as
previously reported (Miyawaki and Ashraf,
1977; Armstrong and Ganote, 1994; Tsuchida
et al., 1994; Miyawaki et al., 1996).
Cell-length changes of isolated cardiomyocytes may be useful as an index for systolicand diastolic function of the myocardium. We
observed that the rat cardiomyocytes shortened
to 65% of their initial length during chemical
hypoxia. This hypoxic shortening is reversible,
and myocytes have the ability to undergo twitch
contractions if the cells have suffered no irreversible ischemic damage. When the ischemic
injury was more severe, reoxygenation caused asudden further contraction of the cells at
reperfusion, to 36% of their initial length.
Hyper-contracted cells never recover theirlength and have no contraction ability (Stern etal., 1985). Pathologically, hypercontraction
may prevent recovery of cardiac function. The
lengths of the present PKC-activated cells weremaintained after reoxygenation compared withthe controls, which suggests that activated PKC
may protect myocardial function. Meldrum andcolleagues (1996) demonstrated the effects ofactivated PKC on cardiac function and theleakage of creatinine kinase during ischemiareperfusion, using the Langendorff model. Our
results support their study; we examined thesame phenomena at the cellular level.
Several mechanisms for the attenuation of
Ca2+ overload during ischemia by activated
PKC have been proposed. Movsesian and coworkers (1984) reported that PKC stimulates
Ca2+ uptake from cytosol by the SR. Steenbergenand coworkers (1993) suggested that PKC
activation modifies the Na+-Ca2+ exchanger.
Lacarda and colleagues (1988) proposed that
activated PKC inhibits the L-type Ca2+ channel.
However, the precise mechanism of how PKC
attenuates a Ca2+ overload remains uncertain.
In the present study, we tested the contributionof Ca2+ uptake by SR and Ca2+ extrusion by the
Na+-Ca2+ exchanger under the activation of PKC.
The inhibition of Ca2+ uptake from the cytosol into the SR by thapsigargin did not block theeffects of activated PKC during chemical hypoxia. This result suggests that the SR did not
37

S. Miyasaka et al.

sodium-calcium exchange. J Physiol 1981;311:
605–622.
4 Hayashi H, Satoh H, Noda N, Terada H, Hirano
M, Yamashita Y, et al. Simultaneous measurement of intracellular Na+ and Ca2+ during K+-free
perfusion in isolated myocytes. Am J Physiol
1994;266:C416–C422.
5 Kawada T, Yoshida Y, Sakurai H, Imai S.
Myocardial Na + during ischemia and accumulation of Ca 2+ after reperfusion: a study withmonesin and dichlorobenzamil. Jpn J Pharmacol
1992;59:191–200.
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K, Funaya H, Shinozaki Y, et al. Role of activation of protein kinase C in the infarct sizelimiting effect of ischemic preconditioning
through activation of ecto-5'-nucleotidase. Circulation 1996;93:781–791.
7 Kohmoto O, Spitzer KW, Movsesian MA, Barry
WH. Effects of intracellular acidosis on [Ca2+]itransients, transsarcolemmal Ca 2+ fluxes, andcontraction in ventricular myocytes. Circ Res
1990;66:622–632.
8 Lacarda AE, Rampe D, Brown AM. Effects ofprotein kinase C activators on cardiac Ca 2+channels. Nature 1988;335:249–251.
9 Ladilov YV, Siegmund B, Balser C, Piper HM.
Simulated ischemia increases the susceptibility of
rat cardiomyocytes to hypercontracture. Circ Res
1997;80:69–75.
10 Leatherman GF, Kim D, Smith TW. Effect ofphorbol esters on contractile state and calcium
flux in cultured chick heart cells. Am J Physiol
1987; 253:H205–H209.
11 Li Y, Kloner RA. Does protein kinase C play arole in ischemic preconditioning in rat hearts?
Am J Physiol 1995;268:H426–H431.
12 Liu Y, Ytrehus K, Downey JM. Evidence thattranslocation of protein kinase C is a key event
during ischemic preconditioning of rabbit myocardium. J Mol Cell Cardiol 1994;26:661–668.
13 Lopez JR, Jahangir R, Jahangir A, Shenk WK,
Terzic A. Potassium channel openers prevent
potassium-induced calcium loading of cardiac
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ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular
myocytes. J Physiol 1991;444; 481–498.
15 Meldrum DR, Cleveland JC Jr, Meng X,
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17 Meldrum DR, Cleveland JC, Sheridan BC,

production. Our present results suggest that a
reduced pHin leads to a reduced Na+ influx via
the Na+-H+ exchanger and limits increases in[Ca2+]i by maintaining the function of the Na+Ca2+ exchanger. This hypothesis is the same asthat of Steenbergen (1993), who reported
similar ionic changes using a nuclear magnetic
resonance (NMR) analysis of ischemicallypreconditioned guinea pig hearts.
Since the signal transduction pathways
following PKC activation are complex, there
are other possible mechanisms for cardioprotection from Ca 2+ overload and reoxygenation hypercontracture. Ladilov and coworkers (1997) suggested that the prevention of
the ischemic dephosphorylation of contractile
proteins reduces the susceptibility of cells to
reoxygenation hypercontracture. The reductionof myofilament sensitivity to calcium may be
mediated by PKC-dependent pathways andcontribute to the reduction of the susceptibilityof isolated cardiomyocytes to reoxygenation
hypercontracture.
In summary, the activation of PKC attenuates the Ca2+ overload of rat cardiomyocytes
and facilitates the maintenance of cell length
during chemical hypoxia-reoxygenation. These
effects do not depend on the Na+-H+ exchanger
or SR, but require the Na+-Ca2+ exchanger function and a reduction of intracellular acidosis.

Acknowledgments: We would like to thank Prof. J.
Hasegawa, Dept. of Clinical Pharmacology and Prof.
Y. Hiji, the First Dept. of Physiology, Faculty of
Medicine, Tottori University for their appropriate
advice and valuable suggestions to this study.

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(Received December 15, Accepted December 22, 1998)

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