(Received for publication, March 22, 1995; and in revised form, June 20, 1995)
From the
Angiotensin II (AngII) is a hormone that alters contractility as
well as myocyte growth in heart. Since many hormones that regulate
cardiac contractility have also been found to modulate intracellular pH
(pH) the goal of this study was to determine if
AngII altered pH
in cultured neonatal rat
ventricular myocytes. Changes in pH
were monitored
in single cells using the fluorescent pH indicator
carboxy-seminaphthorhodafluor-1. Application of 100 nM AngII
resulted in a rapid, receptor-mediated alkalinization of 0.08 ±
0.02 pH unit. The Na
/H
exchanger was
not involved since the response was
HCO
-dependent and amiloride-insensitive.
Ammonia rebound experiments showed AngII increased the initial rate of
recovery from an imposed acid load by 3.15-fold and showed that the
hormone led to the selective activation of the
Na
/HCO
symport. In
contrast, phorbol ester activation of protein kinase C led to the
selective activation of Na
/H
antiport
in these cells. Pharmacological studies showed that the alkalinization
was independent of the AngII receptor subtype 1 (AT
)
phosphoinositide signaling path. In contrast, AngII activation of the
symport was blocked by nanomolar AT
receptor antagonist PD
123319. Superfusion of the myocytes with exogenous arachidonic acid (5
µM) mimicked the AngII-mediated alkalinization, further
suggesting that the AT
signaling pathway underlies the
response. In summary, while most of the known actions of AngII in heart
are mediated through AT
receptors, activation of the
Na
/HCO
symport occurs
through a distinct alternative path that is likely related to fatty
acid production.
Regulation of intracellular pH, pH, is
important in all cells, as accumulation of metabolic acid and other
perturbations of pH
can have profound effects on
cellular homeostasis. In heart, acidosis depresses contractility in
cardiac myocytes by affecting virtually every step in the
excitation-contraction coupling (1) . Hence, in order to
preserve the proper functioning of the heart, cardiac myocytes possess
pH regulating transporters that maintain pH
within
very narrow limits(2) . There are three alkalinizing
transporters that have been characterized in cardiac myocytes including
the Na
/H
exchanger, the
Na
/HCO
symporter, and
the Na
-dependent
HCO
/Cl
exchanger (3, 4, 5, 6) . The
Na
-independent
HCO
/Cl
exchanger acts
as an opposing acidifying mechanism in such
cells(2, 7) . Although there is some species variation
in the expression of exchangers in heart, it is clear that resting
pH
results from a balance between the intrinsic
activities of the exchangers present.
It has been recently
recognized that there is a significant functional role for hormonal
modulation of these pH-regulating transporters as well. Since
cardioactive hormones that regulate contractility, such as
-,
-adrenergic agonists and endothelin, also
modulate pH
, it appears that the control of
pH
underlies part of the inotropic intracellular
signaling pathways in
heart(8, 9, 10, 11, 12) .
Although a frequently seen underlying mechanism involves activation of
the Na
/H
exchanger through
PKC(
)(12, 13) , any pH regulating
transporter is a potential target for hormone action. Indeed several
recent reports reveal that this is a likely phenomenon in heart,
although at present little information is known of the signal
transduction mechanisms that bring about these
responses(10, 14) .
AngII is a peptide hormone
known to regulate contractility as well as to control long term growth
in cardiac myocytes. In particular AngII-evoked stimulation of the
phosphoinositide/PKC pathway underlies, in part, the Ca mobilization and the hormone-evoked cardiac hypertrophy that have
been observed(15, 16, 17) . However, recent
studies have revealed that there is a parallel, independent
AngII-evoked activation of PLA
(18) . The
significance of this alternative path remains to be defined.
It is
within this context that we sought to determine whether AngII regulates
pH in neonatal rat ventricular myocytes. Our
results show unexpectedly that AngII induces an intracellular
alkalinization by the selective activation of the
Na
/HCO
symport. Further,
while the phosphoinositide pathway is not involved, the pH
response is mediated by an AT
receptor pathway
that is likely to involve ARA, possibly through PKC.
The superfusion cell chamber was a small-volume (<500 µl), flow- and temperature-controlled bath developed by Cannell and Lederer(21) . The bath volume was maintained by the use of a small flotation device attached to a piezoresistive element (SensoNor AE-801) and a feedbackflow-control circuit which controlled the outflow pump(21) . Superfusion was maintained at 400 µl/min with a constant temperature of 30 °C. Electrically actuated valves (Lee Valve Co. LFA 1201618H) were used that allowed for rapid changes of bath solutions in less than 10 s.
Fluorescence was measured from single c-SNARF-1-loaded cells using an inverted microscope (Nikon Diaphot) equipped for epifluorescence (Photon Technology International). The excitation wavelength (488 nm) was selected by the monochromator and the emission wavelengths (580 ± 10 and 640 ± 10 nm) were selected by interference filters. A 510-nm dichroic longpass mirror separated the excitation and emission beams in the microscope, and a 610-nm dichroic longpass mirror separated the two emission wavelengths directed to the photomultiplier tubes. Autofluorescence from the cells was found to be negligible.
pH was calculated from the ratio of the
emission intensity at 580 nm (I
) to that at 640 nm
(I
). I
/I
was calibrated in
each cell studied by the use of a high K
-nigericin
method to equilibrate pH
with pH
solutions of known pH from 6.6 to 7.8 (22) .
Calibration solutions contained 150 mM KCl, 5 mM NaCl, 25 mM HEPES, 10 mM glucose, 1 mM EGTA, and 1 µM nigericin. I
/I
was converted to pH
using the experimentally
derived calibration curve. After conversion, the calibrated
pH
signal was filtered by calculating a running
average over 40 point intervals. The data acquisition rate was 16
points/s.
The proton efflux rate carried by various
transporters was calculated from the equation: J =
(dpH
/dt), where
is
the buffering capacity (calculated above) and dpH
/dt is the initial rate of
pH
recovery from an acid load. For the analysis it
was necessary to fit experimental data into a continuous curve. The
following method was used. The data were divided into four segments.
The first three segments were 6-s intervals (100 points); the data
points for each of these segments were fit to a line using the
least-squares linear regression of the contributing points. The
remainder of the recovery was fit with a 2-exponential function. The dpH
/dt values were calculated by
differentiating the function at 15-s intervals, and its corresponding
pH
was taken as the mean pH
of the interval. The proton fluxes (J
) in Table 1were calculated by
multipling the dpH
/dt value at
pH
7.10-7.15 with the appropriate
.
Background loading due to metabolic acid production when all acid
extruding transporters are blocked was calculated to be 0.17 ±
0.05 mM/min (Table 1) and was not included in the
calculation of fluxes.
Figure 1:
Effects of AngII and phorbol ester on
pHi in cultured cardiac cells. Cultured rat myocytes, preloaded with
c-SNARF-1 and mounted in a superfusion bath on the stage of a
microscope, were superfused with HEPES buffer with (panels A and B) or without (panels C and D) 25
mM HCO added. Cells were also
superfused with 25 mM HCO
buffer
bubbled with 5% CO
containing 145 mM Na
(panel E) or 0 Na
(panel F) as
described under ``Experimental Procedures.'' Bars indicate buffer used in experiments in the panels below.
Fluorescence of single cells was monitored and pH
was determined as described under ``Experimental
Procedures.'' In panel A cells were exposed to 100 nM AngII at time indicated (arrow below trace). In panel
B similar cells were exposed to 100 nM TPA at the time
indicated. Traces in panels C and D show the results
of experiments identical to those in A and B,
respectively, except that the superfusion buffer did not contain
bicarbonate. In panels E and F, cells were stimulated
with 100 nM AngII at the indicated time in the presence (panel E) and absence (panel F) of extracellular
Na
.
The HCO dependence suggests that the Na
/H
exchanger is not involved. This hypothesis was explored in
parallel experiments with the phorbol ester, TPA, a PKC activator that
stimulates particular isoforms of the Na
/H
exchanger in many cells including cardiac
myocytes(6, 13, 26) . As shown in Fig. 1B, application of 100 nM TPA resulted in
a rapid increase in pH
(
pH
= 0.09 ± 0.01, n = 6) that is
nearly identical to that seen with AngII. However, in contrast to the
response for AngII, the TPA-evoked alkalinization was the same in the
absence or presence of HCO
(Fig. 1D,
pH
=
0.06 ± 0.01, n = 8). Thus, the underlying
mechanisms of alkalinization evoked by AngII and TPA are distinct.
Experiments with a Na/H
exchange
inhibitor, amiloride, provided further evidence for the lack of
involvement of this antiporter. A double-pulse protocol was developed
so that the effects of the inhibitor could be followed on a single
cell. In these sets of experiments, cells were challenged with 10
nM AngII instead of 100 nM. Although the pH
response was submaximal under these conditions, the protocol
permitted reversal of the AngII effect. Fig. 2A shows
the responses of a cell to two consecutive applications of 10 nM AngII. After the initial challenge with 10 nM AngII
(
pH
= 0.034 ± 0.002, n = 5), the cell was washed by constant superfusion with
hormone-free buffer for 15 min to reverse the initial pH
increase. Following this, a second challenge of 10 nM AngII resulted in a nearly identical pH
response (
pH
= 0.040
± 0.003, n = 5). Thus, there is little
``run down'' of the cell or desensitization of the myocytes
to AngII under the double-pulse protocol. In parallel experiments
amiloride (1 mM) was added after the first AngII stimulation.
As shown in Fig. 2B, the AngII response in
amiloride-treated cells was similar to the control value
(
pH
= 0.033 ± 0.003, n = 3 compared to the control 0.04 pH units). Control
experiments were performed with TPA (Fig. 2C) which
revealed that 1 mM amiloride completely blocked the TPA-evoked
alkalinization (
pH
= 0.003 ±
0.007, n = 3) as expected if the
Na
/H
exchanger was inhibited. The
amiloride-insensitive nature of the AngII effect is further evidence
that activation of the Na
/H
exchanger
is not an underlying mechanism.
Figure 2:
Effect of amiloride on AngII- and
TPA-evoked alkalinizations. Myocytes were superfused with
HEPES/bicarbonate buffer as described under ``Experimental
Procedures'' and Fig. 1. Panel A shows the effects
of AngII (10 nM) on pH in a double-pulse
protocol. AngII was first applied over several minutes as indicated by
the time bar. At the end of this exposure, the cell was
superfused for 15 min with buffer to reverse AngII alkalinization (data
not recorded; note break in trace). Reapplication of 10 nM AngII resulted in a nearly identical pH
response by the same cell. Shown is a representative trace
of five experiments where the first stimulation with 10 nM AngII yields an average
pH
=
0.034 ± 0.002, and the second challenge yields a
pH
= 0.040 ± 0.003 pH unit (n = 5). Panel B shows an identical experiment
except that 1 mM amiloride was added prior to the second AngII
application as shown by the time bar. Panel C shows
pH traces from experiments on two different cells; on the left, 100
nM TPA was added to the myocyte resulting in an alkalinization
of 0.09 ± 0.01 pH unit (n = 6). On the right,
100 nM TPA was added during superfusion with 1 mM amiloride resulting in a complete inhibition of the
alkalinization. The double-pulse protocol could not be used with TPA
because the response to this phorbol was not readily
reversible.
Figure 3:
AngII stimulates rate of pH recovery from an acid load. Panel A shows the
pH
versus time trace for a cell exposed
to a 2-min pulse of 10 mM NH
Cl (indicated by bar above trace) in HEPES buffer containing 25 mM HCO
. After a 10-min wash, the cell
was exposed to an identical second pulse of NH
Cl except
that at the point of maximum acid load (marked with an arrow)
100 nM AngII was added to the cells. The AngII-stimulated
rebound is labeled above the trace. The overshoot of the recovery phase
is accentuated by the dotted line marking basal
pH
. In panel B, the J
versus pH
relation was plotted
for the control (
) and 100 nM AngII-stimulated (
)
traces shown in panel A. Fluxes and pH
are calculated as described under ``Experimental
Procedures.'' Panel C shows results of parallel
experiments as in panel A that were performed in the absence
of HCO
. Panel D displays the J
versus pH
relation from the results in panel
C.
The
mechanism of the AngII effect was studied in more detail by analyzing
the net proton efflux, J, as a function
of pH
. As shown in Fig. 3B, AngII
had several effects on the pH
-flux relation. There
was a marked stimulation in the symport activity at pH
in the range of 7.10 to 7.15. Second, there was a shift in
the curve to the right in the region of resting
pH
, indicative of the activation of the symporter
by AngII at resting pH
. This is also illustrated
by the overshoot seen in the recovery phase of the NH
Cl
pulse response following AngII application (Fig. 3A)
and is consistent with the AngII-induced alkalinization observed in
resting cells (Fig. 1A).
Figure 4:
Effects of receptor-specific blockade on
the AngII-evoked pH response. Panel A depicts the pH
responses in the double-pulse
protocol with AngII in the presence of
HEPES/HCO
buffer as described in Fig. 2. Bars on top of panel indicate interval in which
the cell is superfused with AngII; bottom traces are aligned
so that application of AngII coincide with these bars. Panel B depicts results from a similar experiment in which the second
AngII stimulation is preceded by the application of 100 nM PD
123319 (an AT
receptor subtype antagonist), as indicated by
the bar below trace. Panel C shows an experiment in
which 10 nM DuP 753 (an AT
receptor type
antagonist) is present during the second AngII challenge. For the
experimental results displayed in panel D, myocytes were
incubated for 1 h at 37 °C in HEPES/HCO
buffer with 1 mM DTT. Cells were then loaded with
c-SNARF-1 and placed in a superfusion chamber where they were
challenged with 10 nM AngII in the continued presence of 1
mM DTT. Values for pH
on the y axis were calculated as described under ``Experimental
Procedures.'' Summary data are displayed in Table 2.
Additional experiments were
pursued to more clearly define the receptor specificity of the AngII
effect. DTT is known to selectively inactivate AT receptors
in isolated membranes(30) . In addition, recent studies by
Lokuta and Rogers (18) have shown that it will block the
AT
/phosphoinositide response in intact cardiac cells while
sparing the AT
signaling path. As seen in Fig. 4D, DTT-treated cells still displayed an
AngII-mediated alkalinization. Over many experiments there was no
difference in the AngII response in the presence or absence of DTT (Table 2). It is important to note that control experiments
demonstrated that DTT did not alter spontaneous beating behavior,
resting pH
, or the proton fluxes observed in
NH
Cl pulse experiments compared to nontreated cells (data
not shown). As an additional control it was found that DTT completely
blocked the contractile responses of the myocytes to 100 nM Ang II (Fig. 5), further functional evidence that the
AT
pathway was inhibited under these incubation conditions.
Taken together, these data indicate that the
AT
/phospholipase C signaling path is not involved and
further underscore the importance of the AT
-like signaling
mechanism in the alkalinization response.
Figure 5: Effects of DTT on spontaneous beating behavior of cultured myocytes. Cells were mounted in a superfusion chamber as described in Fig. 1and under ``Experimental Procedures.'' Contractile beating behavior of a single cell was monitored using a video dimensional analysis system as described previously(43) . Panels A and B show the contractile behavior of a cell before and 1.5 min after application of 100 nM AngII, respectively. Panels C and D depict the results from parallel experiments in which the cultures were preincubated for 1 h with DTT prior to the superfusion experiment. Shown is the beating behavior before (panel C) and 1.5 min after (panel D) application of AngII. DTT levels were maintained throughout this protocol.
Since stimulation of
AT receptors increases ARA production (18) , it is
possible that AngII increases pH
through
elevations in this fatty acid. To examine this issue, the effects of
exogenous ARA on pH
were studied. As shown in Fig. 6A, application of 5 µM ARA led to a
rapid increase in pH
with a magnitude nearly
identical to that seen with 10 nM AngII. The threshold dose of
ARA was in the range of 2 µM. The results from a large
number of experiments revealed an increase of
pH
= 0.044 ± 0.01 (n = 6) with 5
µM ARA. Similar to the AngII effect, Fig. 6B shows that the ARA-induced alkalinization was strictly
HCO
-dependent (
pH
= -0.010 ± 0.003, n =
3). The specificity of the ARA response was further characterized in Fig. 6C by the use of the non-metabolizable analogue
ETYA (5 µM) (31, 32) which evoked a
nearly identical pH
response (
pH
= 0.030 ± 0.003, n = 3). These
data suggest that ARA mobilization may underlie, in part, the effects
of AngII on pH
in the cardiac myocytes.
Figure 6:
Effect of arachidonic acid on
pH. Myocytes were superfused with 5 µM ARA in HEPES buffer in the presence (panel A) or absence (panel B) of 25 mM HCO
. Bars above traces indicate interval during which myocyte is
exposed to ARA. Panel C shows that application of 5 µM ETYA in the presence of 25 mM HCO
resulted in an alkalinization of nearly identical magnitude as
elicited by ARA. Summary data are displayed in Table 3.
pH
values are reported as the means ±
S.E. for three to six experiments.
It has
been shown that the cardiac AT/ARA signal transduction path
depends on extracellular Ca
in a characteristic
manner; i.e. the response is blocked by extracellular EGTA but
is insensitive to the Ca
channel inhibitor
nifedipine(18) . Thus if the AngII/pH
response is related to ARA production, then a similar
dependence on extracellular Ca
should prevail. Paired
pulse AngII stimulation experiments were performed in which 2 mM EGTA was added to the superfusion buffer (free extracellular
[Ca
]
o
= 100
nM) after the first AngII stimulation. Under these conditions
the second AngII response was completely inhibited (Table 2). In
contrast, addition of 1 µM nifedipine, which inhibits
L-type Ca
channels, had no effect on the
AngII-mediated alkalinization (Table 2). Thus the profile of
Ca
dependence for the AngII/pH
response is consistent with a role of ARA in the pathway.
Although the alkalinizing characteristics of TPA are distinct from those of ARA and AngII, PKC may still be involved in the response of these latter signaling molecules. To test this hypothesis, cells were preincubated with a PKC inhibitor, staurosporine (10 nM, 45 min at 37 °C), prior to loading with c-SNARF-1/AM. Spontaneous contractions of the cultured cardiac cells were maintained during this incubation. Control experiments indicated that this treatment inhibited PKC, as the effects of TPA were reduced to 10% of the normal response (Table 3). Under similar conditions, staurosporine inhibited the AngII- and ARA-mediated alkalinizations by 91 and 98% of control, respectively (Table 3). These results suggest that PKC, or perhaps another kinase, mediates the effects of TPA and AngII. Furthermore, they also suggest that the action of ARA is not direct but is also mediated by a kinase.
It is clear that intracellular pH is a crucial factor in
cardiac myocyte function, as it is in many
cells(1, 33, 34) . Thus it is not surprising
to observe that there is a wide array of acid transporters which are
responsible for the control and maintenance of pH(2) . Recent studies in heart show that hormone-evoked
changes in acid transporter activities are also important mechanisms in
the regulation of contractility(8, 9, 11) .
It is within this context that studies were initiated to determine if
myocyte pH
is regulated by AngII, a cardioactive
hormone that alters contractility as well as cardiac cell
growth(15, 17, 25, 35) . An initial
finding was that AngII rapidly alkalinizes myocytes through a
receptor-mediated process. More focused experiments revealed the
unexpected finding that, although the Na
/H
exchanger is a widely seen target for
-adrenergic, endothelin signaling, and activated PKC
in heart(8, 9, 11, 13) ,
AngII-evoked alkalinization is mediated by a selective activation of
the Na
/HCO
symport.
Finally, it is likely that a previously described AT
-like
signal transduction pathway involving fatty acid production is
responsible for the hormone-evoked alkalinization.
Several observations demonstrate that the AngII-evoked alkalinization is caused by a direct activation of an acid transporter rather than indirectly through changes in contractility(25, 36, 37) . When the beating frequency was fixed with field stimulation or when contractility was completely blocked with nifedipine, AngII-evoked alkalinization was still observed. In addition, when cells were pretreated with DTT, under conditions which block the AngII-mediated increase in frequency, an identical alkalinization was still observed. Finally, flux studies revealed that an activation of an acid transporter was a likely mechanism underlying the alkalinization. Taken together, these data support the view that the action of AngII on heart cells includes a second messenger-mediated activation of one or more acid transporters.
It has been shown that in mammalian heart pH regulation is
accomplished principally through two alkalinizing transporters, the
Na/H
exchanger and the
Na
/HCO
symport (3, 29) . Several results in the present study support
the conclusion that AngII selectively activates the
Na
/HCO
symport. First
the AngII alkalinization is strictly Na
- and
HCO
-dependent, as well as
amiloride-insensitive. Although the lack of specificity of amiloride
adds an element of caution, control experiments revealed that the
Na
/H
exchanger was in fact inhibited
under the conditions used. In addition, through the use of ammonia
rebound protocols it was possible to measure the acid flux generated by
each transporter and to demonstrate the selective activation of a
HCO
-dependent proton flux by AngII. While
it is possible that another previously unidentifed cardiac
Na
- and HCO
-dependent
transporter is involved, the evidence reported here taken together with
the work of other groups support the conclusion that the
Na
/HCO
symport underlies
the response.
Parallel experiments with phorbol ester, TPA, further
defined the selectivity of AngII action. This agent can activate
distinct Na/H
exchanger isoforms via
PKC in several cell types, including rat heart myocytes(13) .
The phorbol ester effects reported here were consistent with the view
that PKC-mediated Na
/H
exchange
activation had occurred. Thus, although the increases in resting
pH
evoked by AngII and TPA are of nearly identical
magnitude and time course, the underlying molecular mechanisms are
clearly distinct.
Since there is little information on intracellular
signaling pathways that regulate the
Na/HCO
symport, a focus
of this study was to identify such underlying mechanisms. A series of
experiments in the present study revealed that the AngII stimulation of
the Na
/HCO
symport is
also independent of the AT
-phospholipase C path. The
AT
receptor ligand, PD 123319 (100 nM), completely
inhibited the alkalinization at doses that have no effect on AngII
evoked phosphoinositide turnover (18) . DTT pretreatment, which
has been shown to selectively inhibit the AT
-phospholipase
C pathway, had no effect on the AngII-evoked alkalinization. There was
one conflicting pharmacological result; the AT
receptor
antagonist DuP 753 also inhibited alkalinization. However, these data
should be interpreted with caution since detailed studies in a previous
report reveal that low doses of this antagonist can partially inhibit
the AT
-like pathway in intact heart cell
cultures(18) . Thus the accumulated evidence support the view
that the signaling pathway that leads to increases in pH
is independent of the AT
/phospholipase C system.
The observations that ARA, and a nonmetabolizable analogue, ETYA,
evoke a HCO-dependent alkalinization are
consistent with previous reports that describe a role for ARA as an
important signaling molecule in heart. For example, studies have shown
that this fatty acid can alter contractility, ion channel activity, and
Ca
homeostasis in heart cells (31, 38, 39, 40) . It is not yet
known what role
pH
may play in these various
responses. Interestingly, some of these actions appear to be mediated
by PKC(39, 40) . Consistent with this view,
experiments with staurosporine reported here suggest that activation of
PKC appears to be necessary for ARA-, as well as for AngII-induced
alkalinization. Although control experiments revealed that PKC was
inhibited under the incubation conditions used, these data should be
interpreted with caution since staurosporine can also inhibit other
kinases(41) . However, taken together these data extend the
results of others that implicate kinases in the action of ARA in heart.
Alkalinization is a signaling motif that has been seen in cardiac
cells with -adrenergic agonists, TPA, endothelin, and
AngII (8, 9, 11, 12, 13, 42) .
Since activation of the Na
/H
exchanger is frequently a mechanism underlying these responses,
it was unexpected to find in the present study that AngII had no effect
on this antiporter. The physiological significance of signaling
pathways that favor one transporter compared to another is not defined
at present, although the selective action of AngII reported here
underscores the importance of such a diversity. In part the answer may
lie in the notion that activation of transporters subserve a metabolic
role within the cell beyond that of pH regulation alone.
In summary,
these studies provide new insight into the regulation of
pH. AngII stimulates intracellular alkalinization
through an unusual pathway that is independent of phosphoinositide
signaling and the Na
/H
exchanger and
is likely to involve products of fatty acids such as ARA. Since it has
been established that several of the PKC isoforms found in heart are
activated by fatty acids, focused experiments with PKC isoforms are
warranted to further define the action of ARA and AngII in this
context.