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Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata 1900, Argentina
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Angiotensin II (ANG II) evokes positive inotropic
responses in various species. However, the effects of this peptide on
L-type Ca2+ currents (ICa) are still
controversial. We report in this study that the effects of ANG II on
ICa differ depending on the mode of patch-clamp
technique used, standard whole cell (WC) or perforated patch (PP). No
significant effects of ANG II (0.5 µM) were observed when WC in cells
dialyzed with high EGTA was used. However, when the intracellular
milieu was preserved using PP, ANG II induced a significant 77 ± 6% increase in ICa (
2.2 ± 0.3 in
control and 3.9 ± 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low
Ca2+ buffer capacity (EGTA 0.1 mM), ANG II was able to
induce an increase in ICa (3.5 ± 0.3 in
control vs. 4.8 ± 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor
chelerythrine (50 µM) or calphostin C (1 µM). The above results
allow us to conclude that strong intracellular Ca2+
buffering prevents the physiological actions of ANG II on cardiac ICa, which are also dependent on activation of PKC.
cardiac myocytes; perforated patch
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ANGIOTENSIN II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on the cardiac L-type Ca2+ current (ICa) are still controversial. Early studies using multicellular preparations described an increase in ICa after ANG II treatment (7, 17). However, more recent observations using isolated myocytes reported contradictory results: increase (3, 4, 15), no effect (1, 12), and even decrease (8) in ICa induced by ANG II were reported. Kaibara et al. (15) reported that in rabbit ventricular myocytes, ANG II induced an increase in ICa only after stimulation of Na+/H+ exchanger (NHE) and subsequent intracellular alkalization. On the other hand, Ikenouchi and co-workers (12), using the same cell type and species, detected a significant 0.2-pH unit increase in intracellular pH (pHi) after ANG II application without changes in ICa or intracellular Ca2+ transients.
ANG II type-1 receptors (AT1), together with
1-adrenoceptors and endothelin (ET)-1 receptors, belong
to a family of G protein (Gq)-coupled receptors
linked to phospholipase C (PLC) activation and consequent production of
inositol trisphosphate and diacylglicerol, which, in turn, activates
Ca2+-dependent (classic) and Ca2+-independent
(novel) isoforms of protein kinase C (PKC). For the cellular
responses that involve these pathways, the preservation of the
intracellular milieu (as intact as possible) might be required for
observation of the physiological effects of these hormones on
ICa.
Similar to the phenomenon recently described for
1-agonists (23, 40) and ET
(18), we report in this study that the effects of ANG II
on ICa may differ depending on the mode of
patch-clamp technique used, standard whole cell (WC) or perforated
patch (PP). Altering the Ca2+ buffer capacity of the
pipette solution in the WC recordings allowed us to conclude that
differential control of intracellular Ca2+
(Ca
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Myocyte isolation. Cat myocytes were isolated according to the technique previously described (2, 26) with some modifications. Briefly, the hearts were attached via the aorta to a cannula, excised, and mounted in a Langendorff apparatus. They were then retrograde perfused at 37°C at a constant perfusion pressure of 70-80 mmHg with Krebs-Henseleit solution (K-H) of the following composition (in mM): 146.2 NaCl, 4.7 KCl, 1.35 CaCl2, 10 HEPES, 0.35 NaH2PO4, 1 MgSO4, and 10 glucose (pH adjusted to 7.4 with NaOH). The solution was continuously bubbled with 100% O2. After a stabilization period of 4 min, the perfusion was switched to a nominally Ca2+-free K-H for 6 min. Hearts were then recirculated with collagenase (118 units/ml), 0.1 mg/ml pronase, and 1% BSA in K-H containing 50 µM CaCl2. Perfusion continued until hearts became flaccid (15-25 min). Hearts were then removed from the perfusion apparatus by cutting at the atrioventricular junction. The desegregated myocytes were separated from the undigested tissue and rinsed several times with a K-H solution containing 1% BSA and 500 µM CaCl2. After each wash, myocytes were left for sedimentation for 10 min. Myocytes were kept in K-H solution at room temperature (20-22°C) until use. Only rod-shaped myocytes with clear and distinct striations and an obvious marked shortening and relaxation on stimulation were used. Experiments were performed at room temperature.
ICa recordings.
Isolated cat ventricular myocytes were placed in a perfusion chamber
and superfused with bath solution at a flow rate of 1.5 ml/min. The PP
and standard WC configurations of the patch-clamp technique (2,
9, 19) were used for voltage-clamp recordings with a patch-clamp
amplifier (Axopatch 200A; Axon Instruments, Foster City, CA). Patch
pipettes were pulled with a PP-83 puller (Narishige; Tokyo, Japan) and
fire polished with an MF-83 Microforge (Narishige) to a final
resistance of 1-3 M
when filled with pipette solution. The tip
of the pipette was positioned above the cell, and its potential and
capacitance were nullified. WC currents (filtered at 1 kHz) were
digitally recorded directly to hard disk via an analog-to-digital
converter (Digidata 1200, Axon Instruments) interfaced with an IBM
clone computer running pCLAMP and Axotape software (Axon Instruments).
Data analysis was performed with pClamp (Clampfit).
80 mV was used in all protocols to prevent slow
inactivation and to minimize current rundown (25). No
L-type ICa rundown was observed in the PP
recordings, whereas only 10-15% of peak current diminution was
present after 30-40 min of cell dialysis in the WC recordings
[16 ± 6 and 12 ± 5% in cells dialyzed with 0.1 mM
(n = 5) and 5 mM (n = 4) EGTA,
respectively]. A 500-ms prepulse to 40 mV, used to inactivate
Na+ channels and potential T-type Ca2+
channels, preceded the depolarizing test pulses to different potentials. Under the present recording conditions, no WC currents were
detected in the absence of extracellular Ca2+
concentration ([Ca2+]o) (not shown).
In six cells superfused with Na+-free external solution,
the amplitude of the control peak density current registered at 0 mV
(2.48 ± 0.73 pA/pF; PP recordings) was similar to the one
recorded in the presence of Na+ (see Figs. 2 and 3). The
currents evoked by the test pulses after the 500-ms prepulses of 40
mV exhibited activation and inactivation kinetics consistent with those
of L-type ICa. In addition, acute application of
either 0.5 or 1 µM nifedipine to the bath solution reduced the WC
current evoked at 0 mV by 42.5 ± 7.4% (n = 4)
and 87.4 ± 7.8% (n = 4), respectively.
Accordingly, these observations strongly suggest that the currents
measured have a minimal or no contamination with currents other than
L-type ICa.
Nystatin produced good intracellular access after 15-20 min of
seal formation. The ICa amplitude was measured
as peak inward current with reference to the current measured at the
end of the test pulse. For each cell, capacitative current was recorded
to determine the membrane capacitance, and the currents were normalized for cell capacitance. The average cell capacitance was 132.5 ± 5.7 pF (n = 19).
The superfusion medium used to measure ICa had
the following composition (in mM): 5 CsCl, 133 NaCl, 1 MgCl2, 1.2 MgSO4, 10 HEPES, 10 tethraethylammonium chloride (TEA), 1.35 CaCl2, and 10 glucose; pH was adjusted to 7.4 with NaOH. The internal (pipette) solution used for the PP recordings contained (in mM): 140 CsCl, 1 MgCl2, 10 NaCl, 1 EGTA, 10 HEPES, and 0.4 mg/ml nystatin;
pH was adjusted to 7.2 with NaOH. The pipette solution used for the standard WC recordings contained (in mM): 140 CsCl, 1 MgCl2, 5 Na2ATP, 5 EGTA (CaMaterials. Collagenase type B was purchased from Worthington Biochemical (Lakewood, NJ); pronase was from Boerhinger Mannheim (Mannheim, Germany); BSA was essentially fatty acid free; ANG II, calphostin C, and chelerythrine were from Sigma (St. Louis, MO); and HOE-642 was a gift from Hoechst (Frankfurt, Germany). All other chemicals were of the purest reagent grade available.
Statistics. All data are presented as means ± SE. Comparisons within groups were assessed by a paired Student's t-test. ANOVA was used when required, as indicated. A value of P < 0.05 was taken to indicate statistical significance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Figure 1A shows lack of
effect of ANG II in representative traces of WC L-type
ICa evoked by pulses to 0 mV (250 ms) delivered at 0.2 Hz from a holding potential of 80 mV and followed by a 500-ms
prepulse to 40 mV. Out of a total of 24 cells, ANG II (0.5 µM)
produced no effect in ICa in 12 cells, a slight
increase in 4 cells, and a decrease in 8 cells. On average, no
statistically significant effects of ANG II were observed when WC was
used in these experiments (Fig. 1B).
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In contrast to the results obtained using WC, when the intracellular
milieu was preserved using PP (Figs. 2
and 3), ANG II (0.5 µM)
induced a significant and consistent 73 ± 5% (n = 13) increase in ICa. A similar increase in
ICa was induced by 100 nM ANG II
(n = 3, data not shown). Analysis of the amplitude of the end-pulse current showed no significant differences between control
(7 ± 10 pA) and ANG II [3 ± 9 pA, n = 13, not significant (NS)]. Figure 2A shows the time course of
the effect of ANG II on the peak ICa evoked at 0 mV. The representative traces corresponding to the points indicated in
Fig. 2A are shown at bottom. Application of ANG
II to the bath induced an increase in ICa that
started after 2 min, reached a maximum value after 8-10 min, and
slightly decreased to a steady-state value after 12-15 min. Figure
2B shows average data of this time course collected from
five myocytes.
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The effects of the AT1 receptor antagonist losartan (Los; 2 µM) in the presence of ANG II are shown in Fig. 3. The time course of
the effects of ANG II and Los on ICa is shown in
Fig. 3A. During the maximum ICa
enhancement induced by ANG II, the addition of Los to the bath solution
in the continuous presence of the peptide induced a slow decrease in
ICa that reached a steady-state value (20%
above control) after 10 min of exposure of the myocyte to the
AT1 receptor antagonist. Pretreatment of the myocytes with Los (2 µM) prevented the increase in ICa
induced by ANG II (n = 3, data not shown). In contrast,
pretreatment of the cells with the AT2 receptor antagonist
PD-123,319 (2 µM) did not modify the response to ANG II
(n = 3, data not shown). Figure 3B depicts the average current density-voltage relations at control, after 7-8 min of ANG II treatment, and after 10 min of the addition of
Los in the continuous presence of ANG II. A significant increase in
current density was observed in the range of voltage between 20 and
+40 mV after application of ANG II to the bath solution, which was
almost completely reversed by the addition of Los.
As can be observed in Fig. 3B, the control average peak
current density recorded at 0 mV was smaller than the one recorded under WC. A more important contribution of the
Ca3.7 ± 0.6 pA/pF in control vs. 5.9 ± 0.9 pA/pF after
ANG II; n = 5, P < 0.05).
Figure 3B also demonstrates that ANG II produces a
Los-sensitive clear change in the shape of the current-voltage relation of ICa, consistent with a negative shift (10
mV) in the voltage at which ICa is maximal
(VI peak). To investigate the
reason for this negative shift, the kinetics and voltage dependence of activation and inactivation were studied.
The time course of activation of ICa was
examined by calculating the time to peak current evoked at 0 mV. A
significant acceleration in the time to peak current was observed after
ANG II treatment (16.6 ± 1.9 ms in control vs. 13.2 ± 1.6 ms after ANG II; n = 13, P < 0.05).
The time course of inactivation of ICa was also examined. From peak current to end-pulse current, traces were fitted by
a double exponential function of the form
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1 and 2 are their respective time
constants. A0 is a constant that represents the
current at time = 
. Fast (1) and slow
(2) inactivation time (t) constants were not
different in either the absence or presence of ANG II. At 0 mV, time constants were 17.5 ± 3.4 ms (1) and 78.9 ± 9.3 ms (2) in control and 20.4 ± 3.4 ms
(1) and 93.5 ± 13.8 ms (2) after ANG II
addition to the extracellular solution (n = 13, NS).
Figure 4 shows the voltage dependence of
steady-state activation and inactivation of ICa
in the absence and presence of ANG II. ANG II induced a statistically
significant negative shift in the activation curve. The voltage at
which 50% of activation is present (V0.5) was
8.5 ± 1.2 mV in control and 11.8 ± 1 mV in the presence of ANG II
(n = 6, P < 0.05). No differences were observed
in the activation slope factors (k) before (3.7 ± 0.4 mV)
and after (3.5 ± 0.4 mV) addition of ANG II to the bath solution. A
nonstatistically significant slight positive shift in the inactivation curves was observed after ANG II (V0.5 and
k values were 13.9 ± 1.5 and 4.1 ± 0.6 mV in control and
13.1 ± 0.6 and 3.4 ± 0.2 mV after ANG II, respectively). The
evaluation of the steady-state activation and inactivation parameters
suggests that ANG II not only increases
ICa but also alters the gating
properties of the L-type Ca2+ channels,
resulting in a widening of the steady-state window current.
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Kaibara et al. (15) reported that cardiac
ICa enhancement induced by ANG II is due to the
intracellular alkalization produced by the stimulation of the NHE.
Under the present recording conditions, in which no bicarbonate was
included in the extracellular solution, the most important modulator of
pHi is the NHE. One possible explanation for the lack of
effect of ANG II in the WC experiments could be that intracellular
dialysis with pipette solution containing the pH buffer HEPES (10 mM)
might be preventing the increase in pHi induced by the
peptide. However, in PP experiments, pretreatment of the cells with the
NHE inhibitor HOE-642 (1 µM) did not prevent the increase in
ICa induced by ANG II (at 0 mV, 71 ± 7%;
n = 5). Figure 5 shows an
example of these experiments. After application of HOE-642, a slight
decrease in ICa, likely due to intracellular acidification or direct inhibition of the channels by the drug, was
observed. Only two out of five cells exhibited this behavior, and no
effect of HOE-642 on basal ICa was observed in
the other three myocytes. Under the continuous presence of the NHE
inhibitor, ANG II produced a Los-sensitive enhancement of
ICa of a similar magnitude to the one observed
in the absence of the blocker. Although simultaneous measurements of
pHi were not performed in the present PP recordings, this
concentration of HOE-642 has been widely accepted as an NHE inhibitor
in the heart (6, 29, 31, 37).
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Because Ca
In experiment 1, measurements of ICa
were performed in myocytes dialyzed with the more rapid and efficient
Ca2+ chelator BAPTA (10 mM), and the results are shown in
Fig. 6. Figure 6A shows
representative traces of ICa recorded under
these conditions, before and after exposure of the myocyte to ANG II. Average data are shown in Fig. 6B. ANG II failed to induce
ICa enhancement in all the cells studied under
these conditions. Indeed, two out of six myocytes dialyzed with BAPTA
showed a sustained decrease in ICa after ANG II.
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In experiment 2, the cells were dialyzed with low
Ca2+ buffer capacity (EGTA 0.1 mM). The myocytes were also
dialyzed with a higher concentration of HEPES (30 mM) with the
objective of efficiently clamping pHi at a
constant value. It is important to note that this is the same level of
HEPES that prevented the increase in ICa induced
by ANG II in the study of Kaibara and co-workers (15). Under these conditions, ANG II was able to induce an increase in
ICa, as shown in the representative traces of
Fig. 7A. Figure 7B
depicts the average current density-voltage relation for
ICa before and after addition of ANG II to the
bath solution. This peptide induced a significant increase in the
current in the voltage range between 25 and +40 mV.
ICa recorded at 0 mV was 38 ± 4% higher in the
presence of ANG II than in its absence. This value was lower than the
one obtained using PP and is in the order of previously reported data
in which WC was used (3, 4 15). These results allow us to
conclude that the increase in
ICa induced by ANG II is a
mechanism dependent on Ca
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Although in these present experiments, ANG II also produced a change in
the shape of the current density-voltage relation, a significant shift
in VIpeak was not evident. As
was the case for the PP recordings, in these WC recordings, the
kinetics of activation measured as the time to peak current was also
significantly increased by ANG II (at 0 mV, 11.3 ± 1.8 ms in control
vs. 10.1 ± 1.8 ms after ANG II; n = 13, P < 0.05). The inactivation kinetics remained unaltered after ANG II. At 0 mV, inactivation time constants were 12.4 ± 2.2 ms (1)
and 71.8 ± 7.4 ms (2) in control and 12 ± 1.8 ms
(1) and 64.9 ± 5.3 ms (2) after ANG
II(n = 13, NS).
Because Ca1, 16 ± 1.5 ms; 2, 89 ± 9 ms;
n = 24) were higher but not statistically different
(repeated measures ANOVA for unpaired values) than the ones obtained
with 0.1 mM EGTA (see above). Because EGTA is a poor subsarcolemmal
Ca2+ buffer (38), this discrepancy was likely
due to the fact that, as the L-type ICa was
augmented, the increased Ca2+ entry would lead to an
accumulation of Ca2+ (ions) in the vicinity of the inner
mouth of the channels. This would accelerate inactivation of the
channel and outweigh the effect of providing a low cytosolic
Ca2+. Moreover, when the cells were dialyzed with the fast
Ca2+ buffer, (BAPTA, which is reported to be efficient in
chelating subsarcolemmal Ca2+; Ref. 38), the
rate of inactivation was significantly slowed (1, 35.4 ± 1.9 ms; 2, 166 ± 19 ms; n = 6, P < 0.05, repeated measures ANOVA for unpaired values).
Activation of cardiac AT1 receptors by ANG II leads to the
stimulation of several PKC isoforms, including
Ca2+-dependent (classic) (32) and
Ca2+-independent (novel) (16, 32) types.
Although it was previously suggested (3, 4) that
ANG II induced enhancement of ICa because of
stimulation of PKC, no convincing evidence was provided linking the
hormone, the receptor, and the kinase. Figure
8A shows representative traces
of ICa before and after bath application of ANG
II recorded under WC in myocytes dialyzed with the PKC inhibitor
calphostin C (1 µM) or chelerythrine (50 µM). The increase in
ICa was prevented when the cells were dialyzed
with these PKC inhibitors. The overall results of these experiments are
shown in Fig. 8B. PP was also used in three myocytes
pretreated for 60 min with calphostin C (1 µM). Under these
conditions and in the continuous presence of calphostin C in the bath,
ANG II failed to induce an increase in ICa (at 0 mV, 2.3 ± 0.6 pA/pF in control and 2.6 ± 0.7 pA/pF after ANG II;
NS). Despite the fact that calphostin C has been reported to be a
direct ICa blocker (10), in the
present experiments, this compound did not affect basal ICa. Indeed, other studies (8, 36)
obtained similar results. The reason for this controversy is not
apparent to us. Nevertheless, we cannot completely deny the possibility
that the lack of specificity of the organic kinase inhibitory agents
used in the present study could enable them to act through pathways
other than PKC. Specific peptide inhibitors of PKC would be
useful tools to strengthen the present data.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study demonstrates that ANG II activation of
AT1 receptors increases cardiac ICa
by stimulation of PKC and via a Ca2+-dependent and
pHi-independent mechanism. An important contribution of our
study is that to observe these effects, it is necessary to allow for
certain levels of Ca
Controversial results were previously obtained by several researchers when the effects of ANG II on ICa were studied in isolated myocytes. This peptide was reported to cause an increase (3, 4, 15), no effect (1, 12), and even a decrease (8) in basal ICa. One possible explanation for these discrepant results could be differences in the effect of the peptide in different types of cardiac cells or the species involved. An alternative explanation could be related to the fact that most of these studies attempted to measure the effect of ANG II on ICa using the WC mode of the patch-clamp technique. As in these previous studies, in the present work, ANG II induced variable results when the cells were dialyzed with a conventional pipette solution containing a millimolar concentration of the widely used Ca2+ chelator, EGTA. Under these conditions, only a few cells exhibited an increase in ICa on ANG II exposure. Because EGTA is a slow Ca2+ buffer, we can speculate that in these cells, incoming and/or subsarcolemmal Ca2+ was not being properly chelated (38). Moreover, when the more rapid Ca2+ buffer, BAPTA (38), was dialyzed into the cells, none of the myocytes showed a positive response after ANG II.
The data presented herein raise the obvious question of whether studies
that failed to detect enhancement of ICa by ANG
II in ventricular myocytes were performed using pipette solutions with
high Ca2+ buffering capacity. Accordingly, no change in
ICa after ANG II was observed by Ai et al.
(1) or Ikenouchi et al. (12), and both used
high EGTA in the pipette solution. However, in the latter study,
Ca2+ was also added to the pipette solution, and the free
Ca2+ was estimated to be ~100 nM. On the other hand,
Allen et al. (3) reported ICa
augmentation after ANG II, using very low concentrations of EGTA in the
pipette. In contrast, De Mello (4) and Kaibara et al.
(15) observed that ANG II induced
ICa augmentation, using high EGTA in the
pipette. The reasons for this discrepancy are not apparent to us.
Perhaps the changes in Ca
Kaibara et al. (15) described an attractive mechanism to explain the ANG II-induced cardiac ICa enhancement. They proposed that ICa increases after ANG II as a result of activation of NHE and the subsequent intracellular alkalization. The ANG II-induced ICa enhancement observed in the present study does not support Kaibara et al. conclusions (15), because this effect was still present in the presence of NHE blockade in the PP experiments and in the presence of high pH buffer capacity in the pipette solution in the WC experiments.
The NHE blocker, HOE-642, was used in our study, whereas Kaibara et al. (15) used amiloride derivatives in their work. Whether these different experimental conditions could explain the discrepant results observed is not apparent to us. We should not disregard that amiloride derivatives are less specific blockers of NHE than HOE-642, and, among other currents, inhibition of ICa by these drugs was previously reported (27). This independence of pHi changes of the ICa enhancement induced by ANG II can be also supported by the experiments of Ikenouchi et al. (12): despite a significant increase of 0.2 pH units in pHi after ANG II, no changes in ICa were detected. Moreover, Le Grand et al. (21) reported an ANG II-induced enhancement of ICa during superfusion of the cells with an Na+-free solution, a situation in which the NHE cannot be functional.
Similar to what was reported for 
-adrenoceptor agonists (14,
34), ANG II induced a negative shift in
VI peak consistent with a negative
shift in the voltage dependence for steady-state activation. Although
Allen et al. (3) have previously reported a negative shift
in the V0.5 of activation after ANG II, no
change in the shape of the current-voltage relation was observed. The voltage dependence for activation appears to be governed by the properties of the charge movement of the voltage-sensing moiety of the
channel. Josephson and Sperelakis (14) related the
negative shift in the ICa
VI peak and voltage dependence of
activation observed after isoproterenol with parallel changes in the
gating charge movements produced by phosphorylation of the channels. Whether ANG II produces similar changes in the gating charge movement of ICa remains to be investigated.
As was previously reported by Allen et al. (3), in the present study, we did not detect changes in the time constants of ICa inactivation after ANG II. This is in contrast to ICa augmentation after PKA (39) or Ca2+-calmodulin (CaM) kinase II activation (5). The latter signaling molecules shift the discrete modes of gating of single-channel currents from a short-opening mode (mode 1) to a long-opening mode (mode 2). The presence of mode 2 slows the inactivation kinetics of the WC currents. Thus, on the basis of the present data, ANG II and PKC phosphorylation may not involve a modal gating shift. Furthermore, this hypothesis is supported by single-channel recordings from cell-attached patches performed by Kaibara et al. (15), in which ANG II increased the open probability of L-type Ca2+ channels without affecting the pattern of channel opening.
Direct activation of PKC by phorbol esters leading to increased ICa was previously reported (20, 22, 30). However, PKC involvement in the ANG II-induced ICa enhancement has not been studied carefully. Allen et al. (3) have shown that ANG II increased phosphoinositide hydrolysis, and they related these effects with a possible role of PKC in the ICa enhancement induced by the peptide. De Mello (4) has recently demonstrated that PKC inhibition prevented the increase in ICa produced by ANG II administered intracellularly, a pathway insensitive to Los and probably involving receptors different than AT1. Thus our data assessing PKC inhibition represent a new piece of information for the cardiac ICa enhancement through the pathway involving extracellular ANG II, AT1 receptors, and PKC activation.
In the present study, we have shown that either Ca
In summary, results of this study suggest the following. 1)
Changes in pHi do not seem to contribute to the ANG
II-induced cardiac ICa enhancement.
2) A physiological level of Ca
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ACKNOWLEDGEMENTS |
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The technical assistance of Mónica Rando and Cristina Taraborrelli is gratefully acknowledged. E. A. Aiello and H. E. Cingolani are Established Investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de Argentina.
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FOOTNOTES |
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This study was supported by a grant (PEI 0321/98) from the CONICET de Argentina.
Address for reprint requests and other correspondence: E. A. Aiello or H. E. Cingolani, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, 60 y 120, La Plata 1900, Argentina (E-mail: cicme{at}atlas.med.unlp.edu.ar).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 July 2000; accepted in final form 17 November 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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