Vol. 274, Issue 4, H1152-H1162, April 1998
Opposing effects of
1-adrenergic receptor
subtypes on Ca2+ and pH
homeostasis in rat cardiac myocytes
Giovanni
Gambassi,
Harold A.
Spurgeon,
Bruce D.
Ziman,
Edward G.
Lakatta, and
Maurizio C.
Capogrossi
Laboratory of Cardiovascular Science, Gerontology Research
Center, National Institute on Aging, National Institutes of Health,
Baltimore, Maryland 21224
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ABSTRACT |
We examined the
effect of
1-adrenergic receptor
(AR) subtypes on contraction, cytosolic
Ca2+ concentration
([Ca2+]i),
and cytosolic pH (pHi) of rat
ventricular myocytes loaded with the
Ca2+ indicator indo 1 or the pH
indicator carboxy-seminaphthorhodafluor-1. Nonselective
1-AR stimulation was effected
with phenylephrine plus nadolol.
1-AR subtype stimulation was
achieved with
1-AR and
chloroethylclonidine (CEC) or with
1-AR and WB-4101. Cells were in
bicarbonate buffer with 0.5 mM Ca2+ and were electrically
stimulated at 0.5 Hz. Results show that 1) nonselective
1-AR stimulation increased
twitch and
[Ca2+]i
transient amplitudes, myofilament response to
Ca2+, and
pHi;
2)
1-AR plus CEC increased twitch
and
[Ca2+]i
transient amplitudes and also enhanced myofilament response to
Ca2+ via cytosolic alkalinization;
3)
1-AR plus WB-4101 decreased twitch and
[Ca2+]i
transient amplitudes and also pHi;
and 4) cytosolic acidification due
to
1-AR plus WB-4101 was
abolished by protein kinase C inhibition (staurosporine pretreatment)
or downregulation (prolonged exposure to phorbol esters). In summary,
the net effects of
1-adrenergic stimulation on contraction,
[Ca2+]i,
and pHi are due to opposing
WB-4101- and CEC-sensitive
1-AR subtype signaling pathways.
-adrenoceptor subtypes; chloroethylclonidine; WB-4101; indo 1; cardiac inotropy; cytosolic pH
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INTRODUCTION |
PHARMACOLOGICALLY DISTINCT
1-adrenergic receptor (AR)
subtypes have been described (6, 13, 18, 24, 28), and molecular cloning
and expression of the cDNA for three
1-AR subtypes has been reported
in rat myocytes and in the human heart (13, 28). Most studies that have
examined the functional role of
1-AR subtypes have relied upon
nonselective
1-AR stimulation
in the presence of receptor subtype antagonists such as
chloroethylclonidine (CEC; see Ref. 19) and
2-(2,6-dimethoxyphenoxyethly)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; see Ref. 19). Several studies have
described the effect of phenylephrine plus CEC and phenylephrine plus
WB-4101 on cardiac cell electrophysiological properties (1, 2, 7, 15,
20, 30) and growth (25). In the continued presence of phenylephrine,
WB-4101 decreased (1, 2, 20) and CEC enhanced (1)
phenylephrine-mediated arrhythmias. In a different study, CEC increased
spontaneously beating rate of canine Purkinje fibers, whereas the
opposite occurred upon WB-4101 addition (7).
Despite these reports, the functional role of
1-AR subtypes sensitive either
to CEC or to WB-4101 is still incompletely characterized in the heart.
Specifically, the effects of these receptor subtypes on contraction,
cell Ca2+ homeostasis, cytosolic
pH (pHi), and myofilament
responsiveness to Ca2+ of the
myocardium are still unknown. Furthermore, the interaction of these
receptor subtypes during nonselective
1-AR stimulation, as it
normally occurs in vivo, still remains to be determined. These
questions were addressed in the present study utilizing indo 1- or
SNARF-1-loaded adult rat ventricular myocytes.
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METHODS |
Myocyte isolation procedure.
Ventricular myocytes were enzymatically dissociated with minor
modifications of a technique previously described (27). Briefly, 2- to
4-mo-old male Wistar rats were anesthetized with an intraperitoneal
injection of pentobarbital sodium. The heart was quickly excised and
retrogradely perfused with 25 ml of a nominally
Ca2+-free bicarbonate buffer at 36 ± 1°C, continuously gassed with 95%
O2 and 5%
CO2 to keep the pH at ~7.4. The
perfusate was then switched to a similar solution to which collagenase
(1 mg/ml), protease (0.04 mg/ml), and bovine serum albumin (1 mg/ml)
had been added. After ~20 min of perfusion, the left ventricle was isolated, and cardiac myocytes were mechanically disaggregated and
resuspended in a bicarbonate buffer with 1.0 mM bathing
Ca2+ concentration
([Ca2+]).
Simultaneous measurement of length and indo 1 fluorescence. Cell length and indo 1 fluorescence were
measured simultaneously, as previously described (27). Briefly, single
myocytes bathed in bicarbonate-buffered medium were loaded with the
ester derivative [acetoxymethyl (AM) form] of the
fluorescent Ca2+ probe indo 1. After loading, cells were transferred to a Lucite chamber with a glass
coverslip on the stage of an inverted microscope and were continuously
superfused with buffer composed of (in mM) 116.4 NaCl, 5.4 KCl, 1.6 MgSO4, 26.2 NaHCO3, 1.2 NaH2PO4,
5.6 D-glucose, and 0.5 CaCl2. The buffer was gassed with
95% O2-5% CO2 (pH 7.36). Two platinum
electrodes placed in the bathing fluid and connected to a stimulator
(SD9; Grass Instrument, Quincy, MA) were used to field stimulate the
myocyte to twitch with pulses of 2-4 ms in duration at a rate of
0.5 Hz. Indo 1 fluorescence was excited by epi-illumination with 10-ms
flashes of 350 ± 5 nm light. Paired photomultipliers collected indo
1 fluorescence emission by simultaneously measuring spectral windows of
391 ± 434 and 457 ± 507 nm selected by bandpass interference
filters. The ratio of indo 1 emission at the two wavelengths was
calculated using a pair of fast integrator sample and hold circuits
under the control of a VAX 11/730 computer, and it was taken as an
index of cytosolic Ca2+
concentration
([Ca2+]i).
When isolated cardiac myocytes are loaded with indo 1-AM, there is
variable compartmentalization of the indicator into the mitochondria
(27) that prevents the use of a standard calibration curve. Thus the
present results in indo 1-AM-loaded myocytes are expressed as
fluorescence ratio rather than as absolute
[Ca2+] values. Cell
length was monitored simultaneously with indo 1 fluorescence ratio
using red light (650-750 nm) to form a bright-field image of the
cell, which was projected onto a photodiode array. Myofilament
responsiveness to Ca2+ was
assessed by the cell
length-[Ca2+]i
relation in the diastolic interval during 50 ms before the delivery of
the electrical stimulus (26). Myofilament
Ca2+ binding and
[Ca2+]i
achieve a quasi-equilibrium during the relaxation phase of a twitch,
and the phase-plane diagram of the cell
length-[Ca2+]i
relation, at the time of cell relengthening during a twitch, does not
change under a variety of conditions that alter the amplitude and time
course of the contraction and of the
[Ca2+]i
transient without affecting myofilament
Ca2+ sensitivity. Instead, the
cell
length-[Ca2+]i
relation is shifted in opposite directions by interventions that either
increase or decrease myofilament responsiveness to Ca2+ (26).
Both loading of the Ca2+ probe and
experiments were performed at 25°C to minimize loss of the
Ca2+ indicator from the cells
(27). Additionally, some experiments were performed with cells that had
not been loaded with indo 1, and only cell length was measured.
pHi measurements.
After enzymatic isolation, myocytes were bathed in
/CO2
buffer, unless indicated otherwise. Alternatively, cells were
superfused with a
/CO2-free
buffer of the following composition (in mM): 137.0 NaCl, 5.0 KCl, 1.2 MgSO4, 1.2 NaH2PO4,
10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 16 D-glucose, and 0.5 CaCl2 (pH 7.35). Experiments were performed at 25°C. Cells were loaded with the AM form of the
fluorescent H+-sensitive indicator
SNARF-1. pHi and cell length were
monitored on the stage of a modified inverted microscope, as previously described (3). After excitation at 530 ± 5 nm, the ratio of SNARF-1
emission at 590 ± 5 nm to that at 640 ± 5 nm was used as a
measure of pHi according to an in
vivo calibration.
Experimental protocols. Nonselective
1-AR stimulation was effected
with 10 µM phenylephrine, and
-AR was blocked with 1 µM nadolol.
Preliminary results had shown that, under our experimental conditions,
nadolol alone (1 µM) has no effect on the contractile properties of
isolated myocardial cells, whereas it provides an effective
-AR
blockade (not shown). Thus 1 µM nadolol was present in all buffers
with and without phenylephrine. In addition, experiments on the
concentration dependence of the positive inotropic action of
phenylephrine showed that, at 10 µM, phenylephrine caused a maximum
increase in twitch amplitude (not shown), and this concentration was
used for all experiments reported in this study. The effect of
1-AR subtype stimulation was
examined during
1-AR
stimulation with phenylephrine and nadolol in conjunction with either
the
1-AR subtype antagonist
WB-4101, with the
1-AR subtype
inactivator CEC, or with both WB-4101 and CEC.
Materials. Collagenase B was purchased
from Boehringer Mannheim (Indianapolis, IN). Protease type XIV was
purchased from Sigma (St. Louis, MO). Bovine serum albumin, fraction V,
fatty acid poor, was purchased from Calbiochem (La Jolla, CA). Indo
1-AM and SNARF-1 were purchased from Molecular Probes (Eugene, OR). Phenylephrine and prazosin were purchased from Sigma. CEC and WB-4101
were purchased from Research Biochemicals (Natick, MA). Nadolol was
obtained from Squibb (Princeton, NJ).
Statistical analysis. The results are
expressed as means ± SE. Paired and unpaired Student's
t-test were used for statistical analysis; P < 0.05 was taken to
indicate statistical significance. Concentration-response curves were
analyzed by means of analysis of variance for multiple comparisons and
by means of one-way repeated measures analysis of variance followed by
Bonferroni test.
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RESULTS |
Effect of phenylephrine plus WB-4101 or CEC on
contraction. As in previous studies (4, 10, 29), in the
present experimental conditions, superfusion of single ventricular
myocytes with phenylephrine increased contraction amplitude, and the
effect was maximal and achieved steady state in ~15 min (not shown).
The concentration dependence of the effect of WB-4101 and CEC on the
positive inotropic action of nonselective
1-adrenergic stimulation is
depicted in Fig. 1. On average,
nonselective
1-adrenergic
stimulation caused a twofold increase in twitch amplitude. After
addition of WB-4101 in the continued presence of phenylephrine, there
was a concentration-dependent decrease in twitch amplitude (Fig.
1A). The peak response was achieved at 2 µM WB-4101, and, at this concentration, twitch
amplitude was ~50% lower than in control. In contrast, CEC added in
the continued presence of phenylephrine caused a further increase in
twitch amplitude (Fig. 1B). This
effect saturated at 2 µM, and, at this concentration, twitch
amplitude was enhanced ~30% compared with phenylephrine alone.
Neither substance alone had any appreciable effect on cell function,
and, for all subsequent experiments reported in the present study,
either 2 µM WB-4101 or 2 µM CEC were used.

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Fig. 1.
Effect of phenylephrine alone and phenylephrine plus different
1-adrenergic receptor (AR)
antagonists on contraction of myocytes not loaded with indo 1. A: WB-4101 ( ) had a
concentration-dependent effect to oppose the positive inotropic action
of 10 µM phenylephrine ( ). Statistically significant effect was
achieved for concentrations of WB-4101 above 0.5 µM; WB-4101
decreased twitch amplitude to control at 1 µM and below control at 2 µM. B: chloroethylclonidine (CEC;
) had a concentration-dependent effect to further increase the
positive inotropic action of 10 µM phenylephrine ( ), and its
effect saturated at 2 µM. Concentrations of 2 µM and above reached
statistical significance. Both WB-4101 and CEC were added at the
concentrations indicated after achieving a maximal and steady response
to phenylephrine. Data are plotted as %control (C; ; twitch
amplitude in control was 4.1 ± 0.8% of diastolic cell length,
n = 9). Each point
represents average data obtained from at least 4 cells, and each cell
was exposed to only one concentration of phenylephrine and WB-4101 or
phenylephrine and CEC.
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Subsequent experiments were aimed at determining whether, under our
experimental conditions, CEC and WB-4101 were selective antagonists of
1-AR receptor subtypes.
Myocytes were incubated with 10 µM CEC for 30 min at 37°C. Such
an exposure to CEC is expected to fully and irreversibly inactivate
CEC-sensitive
1-AR subtypes
(19). Subsequently, CEC was extensively washed, and myocytes were
studied with the protocol depicted in Fig. 1. These cells responded to
phenylephrine with an increase in twitch amplitude, and the subsequent
addition of 2 µM CEC had no effect on contraction (Fig.
2). However, the response to WB-4101 was
preserved, and it reversed the effect of phenylephrine (Fig. 2). This
result indicates that, under our experimental conditions, 2 µM CEC
had no functional effect on the WB-4101-sensitive receptor subtype. Crossover binding of WB-4101 to the CEC-sensitive
1-AR receptor subtype also
appears unlikely because this should enhance rather than decrease
contraction amplitude.

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Fig. 2.
Representative example of the effect of phenylephrine and of the
subsequent addition of CEC or WB-4101. Before exposure to
phenylephrine, the cell was incubated for 30 min at 37°C with 10 µM CEC and extensively washed thereafter. Under these conditions,
phenylephrine had a positive inotropic action; the addition of CEC (2 µM) did not exert any effect, whereas WB-4101 (2 µM) decreased
twitch amplitude. Four myocytes were studied with this experimental
protocol.
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The results in Figs. 1 and 2 indicate that phenylephrine plus CEC and
phenylephrine plus WB-4101 have opposite effects on myocardial
contraction. The possible mechanisms for these responses were further
examined in indo 1- and SNARF-1-loaded myocytes.
Effect of phenylephrine plus WB-4101 or CEC on
contraction and indo 1 fluorescence. Figure
3 shows the effect of phenylephrine alone
and of phenylephrine plus WB-4101 on the simultaneously recorded
contraction and indo 1 fluorescence transient and on myofilament
Ca2+ response of a representative
myocyte. Nonselective
1-AR
stimulation with phenylephrine was associated with an increase in
twitch amplitude and
[Ca2+]i
transient amplitudes (Fig. 3A, compare
tracings a and
b). In addition, the inotropic
effect of phenylephrine was associated with a shift of the cell
length-[Ca2+]i
relation leftward of control (Fig.
3B) and with a reduction in
diastolic cell length, which occurred without a change in diastolic indo 1 fluorescence ratio (Fig. 3A,
tracings a and
b), another effect that suggests an
enhanced myofilament response to
Ca2+. These results are in
agreement with prior studies that have shown that the positive
inotropic action of
1-AR
stimulation is due both to an increase in the amplitude of the
[Ca2+]i
transient (4, 8, 10) and to an increased myofilament response to
Ca2+ (8, 10, 22, 29). Figure
3A (tracing
c) shows that WB-4101 in the continued presence of
phenylephrine decreased both contraction and
[Ca2+]i
transient amplitudes to values lower than control. In contrast, WB-4101
did not reverse the effect of phenylephrine on the myofilament response
to Ca2+ (Fig.
3B) and on diastolic cell length.
Figure 4 shows a representative example of
the effect of phenylephrine and phenylephrine plus CEC on contraction
and indo 1 fluorescence and shows the myofilament Ca2+ response of a single
myocyte. Similar to the result in Fig. 3, the positive
inotropic effect of phenylephrine alone was associated with an increase
in
[Ca2+]i
transient amplitude, with diastolic cell shortening without a rise in
diastolic indo 1 fluorescence (Fig.
4A, compare traces a and b) and with a
leftward shift of the cell
length-[Ca2+]i
relation (Fig. 4B). CEC superfusion
in the continuing presence of phenylephrine caused a further increase
in twitch amplitude and a further decrease in diastolic length without
changing either systolic or diastolic indo 1 fluorescence (Fig.
4A, compare traces b and c); in
addition, it also caused a further shift of the cell length-[Ca2+]i
relation (Fig. 4B).

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Fig. 3.
Representative example of the effect of phenylephrine and of the
addition of WB-4101 (WB) in the continued presence of phenylephrine on
an indo 1-loaded myocyte. A:
continuous cell length tracing (top)
and tracings of simultaneously recorded indo 1 fluorescence and cell
length (bottom). The latter are on
an expanded time scale and were obtained at the times indicated by the
letters below the upper continuous length record:
a, control;
b, effect of phenylephrine was
maximal; and c, after addition of
WB-4101. Phenylephrine increased both twitch and
[Ca2+]i
transient amplitudes. Additionally, phenylephrine decreased diastolic
cell length without raising diastolic indo 1 fluorescence (compare
traces a and
b). Addition of WB-4101 decreased
systolic
[Ca2+]i
and twitch amplitudes to values lower than in control (compare
traces a and
c). Reduction in diastolic length
without an increased indo 1 fluorescence ratio persisted during WB-4101
exposure (trace c). Four myocytes
were studied with this experimental protocol.
B: phase-plane diagram of the
length-[Ca2+]i
relation for the same cell shown in A.
Note the effect of phenylephrine to shift the relaxation phase of this
relation above and leftward of control, which is indicative of an
enhanced myofilament response to
Ca2+. Addition of WB-4101 did not
reverse the effect of phenylephrine alone. , 1-AR
stimulation.
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Fig. 4.
Representative example of the effect of phenylephrine and of the
addition of CEC in the continued presence of phenylephrine on
contraction and indo 1 fluorescence.
A: display of the results is similar
to that in Fig. 3. Phenylephrine alone increased twitch and
[Ca2+]i
transient amplitudes, decreased diastolic length, and did not raise
diastolic indo 1 fluorescence. CEC addition caused a further increase
in twitch amplitude and a decrease in diastolic cell length, which were
not associated with significant changes in systolic
[Ca2+]i
and diastolic indo 1 fluorescence (compare traces
b and c). Five
myocytes were studied with this experimental protocol.
B: phase-plane diagram of the
length-[Ca2+]i
relation. Relaxation phase of this relationship was shifted leftward
and above control by nonselective
1-AR stimulation ( ). This
shift was enhanced after addition of CEC.
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In other experiments, in contrast to those depicted in Figs. 3 and 4 in
which phenylephrine was given before subtype antagonists, myocytes were
first exposed either to WB-4101 or CEC alone, and phenylephrine was
added subsequently. Under these conditions, there was no effect of
either WB-4101 or CEC alone on contraction or indo 1 fluorescence.
However, phenylephrine plus WB-4101 decreased twitch and
[Ca2+]i
transient amplitudes below control without affecting the myofilament Ca2+ response
(n = 11, not shown). Phenylephrine in
the continued presence of CEC had a positive inotropic action
associated with an increase in
[Ca2+]i
transient amplitude, and this was also accompanied by an enhanced myofilament response to Ca2+
(n = 4, not shown).
The data presented so far show that nonselective
1-AR stimulation increases
contraction and
[Ca2+]i
transient amplitudes and are in agreement with prior reports that have
demonstrated an enhanced myofilament response to
Ca2+ (8, 10, 22, 29). These
effects are further enhanced by the addition of CEC in the continued
presence of phenylephrine. In contrast, phenylephrine plus WB-4101 has
a negative inotropic action that is due to a decreased
[Ca2+]i
transient amplitude.
Effect of phenylephrine plus WB-4101 and CEC on
contraction and indo 1 fluorescence. Because multiple
1-AR subtypes have been
identified in the rat heart (13, 28), it is necessary to establish
whether the effects of nonselective
1-AR stimulation with
phenylephrine on contraction and
[Ca2+]i
could be prevented by the simultaneous presence of both WB-4101 and
CEC. If WB-4101 and CEC, at the concentrations used in this study, were
truly selective blockers of only two
1-AR subtypes and if other
1-AR subtypes do not modulate
contraction, then the combined use of WB-4101 and CEC would be expected
to prevent all inotropic effects of nonselective
1-AR stimulation. This issue
was examined with the experimental protocol depicted in Fig.
5. The simultaneous addition of WB-4101 and
CEC had no effect on contraction and
[Ca2+]i,
and, in the continued presence of both substances, there was no effect
of phenylephrine on twitch amplitude, diastolic length, and indo 1 fluorescence. This result is representative of the average effect in
eight myocytes (see Table 1) and suggests
that only WB-4101- and CEC-sensitive
1-AR subtypes modulate
myocardial contraction and
[Ca2+]i.

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Fig. 5.
Representative example of the effect of combined WB-4101 and CEC
exposure and of subsequent phenylephrine addition. Display is similar
to that of Fig. 3. There was no effect of WB-4101 and CEC, and no
response occurred upon the addition of phenylephrine. Eight myocytes
were studied with this experimental protocol.
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Taken together, the data presented so far show that phenylephrine plus
WB-4101 and phenylephrine plus CEC have different effects on myocardial
cell contraction on systolic
[Ca2+]i
and on the myofilament response to
Ca2+. Because
pHi is a major determinant of
myofilament sensitivity to Ca2+
and because the nonselective
1-AR stimulation increases
pHi (10, 29), additional
experiments examined the effect of phenylephrine plus CEC or WB-4101 on
pHi and contraction.
Effect of phenylephrine plus CEC or WB-4101 on contraction and
pHi.
Neither CEC nor WB-4101 alone had an effect on
pHi. The addition of phenylephrine
in the continuing presence of CEC increased twitch amplitude and caused
cytosolic alkalinization (Fig.
6A). The
increase in twitch amplitude was correlated to that in
pHi (Fig.
6B). In contrast, phenylephrine plus
WB-4101 decreased pHi and
contraction below control values (Fig.
6C). The degree of cytosolic
acidification exhibited cell-to-cell variability and was related to the
magnitude of the decrease in twitch amplitude (Fig.
6D). Previous studies had shown that
nonselective
1-AR stimulation
increases pHi in myocardial cells
via activation of sarcolemmal
Na+/H+
exchange (10, 29). Therefore, the results of the present study suggest
that the CEC-sensitive receptor may be responsible for the effect of
nonselective
1-AR stimulation
to enhance pHi. In contrast, that
a different
1-AR subtype could
decrease pHi was unexpected.

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Fig. 6.
Effects of phenylephrine plus either CEC or WB-4101 on intracellular pH
(pHi) and contraction of
isolated myocardial cells. A:
phenylephrine plus CEC enhanced both
pHi and contraction amplitude
(n = 7). There was no effect of CEC
alone. Average increase in pHi was
0.06 ± 0.01 pH units (P < 0.005;
pHi in control 7.33 ± 0.06).
Average increase in twitch amplitude was 269.3 ± 59.9% of control
(P < 0.01; twitch amplitude in
control was 5.9 ± 1.9% of diastolic cell length).
B: cytosolic alkalinization was
correlated to the increase in twitch amplitude in response to
phenylephrine plus CEC (r = 0.72, P < 0.05).
C: phenylephrine plus WB-4101
decreased both pHi and contraction
amplitude (n = 9). There was no effect
of WB-4101 alone. Average decrease in
pHi was 0.04 ± 0.01 pH
units (P < 0.05;
pHi in control 7.36 ± 0.02),
and, in the same myocytes, twitch amplitude was 75.5 ± 6.3% of
control (P < 0.05; twitch amplitude
in control was 6.5 ± 1.2% of diastolic cell length).
D: cytosolic acidification was
correlated with the decrease in contraction amplitude in response to
WB-4101 and phenylephrine (r = 0.79, P < 0.05). TA,
difference in twitch amplitude, expressed as % of twitch amplitude
under control conditions.
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At least three different mechanisms appear to be responsible for
maintaining pHi in myocardial
cells:
Na+/H+
exchange, Na+-independent
Cl
/
exchange, and Na+-dependent
Cl
/
exchange (17). Because two of these control mechanisms are dependent on
the presence of
in the buffer, we
examined whether the effect of phenylephrine plus WB-4101 to decrease
pHi was maintained in a
/CO2-free solution. The results show that, in these experimental conditions, the
effects of phenylephrine plus WB-4101 to decrease
pHi and contraction persisted
(Fig.
7A). In
addition,
Na+/H+
exchanger inhibition with 10 µM ethylisopropylamiloride (EIPA) decreased pHi and prevented
cytosolic acidification due to phenylephrine plus WB-4101 stimulation
(not shown). Because protein kinase C (PKC) modulates
Na+/H+
exchange, its potential role as the signal transduction mechanism for
the effect of phenylephrine plus WB-4101 was addressed. PKC inhibition
with staurosporine (Fig. 7B; see Ref.
10) or PKC downregulation with prolonged exposure to 4
-phorbol
12-myristate 13-acetate (PMA; Fig. 7C;
see Ref. 10) abolished the phenylephrine plus WB-4101-mediated decrease
in both pHi and contraction
amplitude.

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Fig. 7.
Effects of WB-4101 plus phenylephrine in a
/CO2-free
buffer. A: representative example of
the effect of WB-4101 plus phenylephrine to decrease
pHi and contraction in
/CO2-free
buffer. Similar result was obtained in 6 myocytes; average
pHi was 0.03 ± 0.01 pH units (P < 0.05;
pHi in control 7.39 ± 0.05),
and twitch amplitude was 78.5 ± 7.5% of control
(P < 0.05; twitch amplitude in
control was 6.2 ± 1.5% of diastolic cell length). Both
responses were comparable to those in
/CO2-buffered
solution. B: representative example of
a myocyte preincubated for 3 h with 5 nM staurosporine. This cell
did not respond to phenylephrine plus WB-4101 with a decrease
in pHi or contraction
amplitude, and a similar result was obtained with 3 other myocytes.
C: representative example of prolonged
(12-24 h) incubation with 0.2 µM PMA. This myocyte did not
respond to phenylephrine plus WB-4101 with a decrease in
pHi and contraction amplitude.
Four myocytes were studied with this experimental protocol.
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To further characterize this effect, we examined whether phenylephrine
plus WB-4101 also modulated pHi
recovery from an acid load induced by
NH4Cl prepulse. In contrast to
control conditions (Fig.
8A) in
which pHi fully recovers,
superfusion with phenylephrine plus WB-4101 delayed
pHi recovery after an initial
NH4Cl pulse (Fig.
8B and Table
2). In addition, the peak decrease in
pHi achieved upon a second
NH4Cl washout was also enhanced
and delayed by phenylephrine plus WB-4101 (Fig.
8B and Table 2). These effects were
abolished by preincubation with staurosporine (Fig.
8D and Table 2) and were qualitatively
similar to those obtained with Na+/H+
exchange inhibition by EIPA (Fig. 8C
and Table 2). Taken together, these results suggest that the effect of
the WB-4101-insensitive
1-AR
subtype to decrease pHi in
myocardial cells is due to PKC-mediated inhibition of
Na+/H+
exchange.

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Fig. 8.
Representative tracings of the effect of phenylephrine plus WB-4101 on
pHi recovery from an acid load
induced by exposure and washout of a solution containing 20 mM
NH4Cl. All experiments were
implemented in
/CO2-free
buffer. Arrows indicate exposure to either phenylephrine plus WB-4101
(B and
D) or EIPA
(C); thereafter, 10 min were allowed
before the next NH4Cl pulse.
A: under control conditions, a second
exposure to NH4Cl yielded effects
on pHi similar to the initial
exposure (n = 6).
B: left-hand tracing shows the effect
of NH4Cl exposure and washout in
control. In the same myocyte, phenylephrine plus WB-4101 enhanced and
delayed the peak decrease in pHi
upon NH4Cl washout. In addition,
there was a sustained decrease in
pHi at a time when full recovery
had already occurred in control (n = 5).
C:
Na+/H+
exchange inhibition with 10 µM EIPA abolished
pHi recovery and enhanced
cytosolic acidification after
NH4Cl washout
(n = 4); these effects were
qualitatively similar to those of phenylephrine plus WB-4101.
D: effects of phenylephrine plus
WB-4101 on pHi were abolished by
preincubation with 5 nM staurosporine for 3 h
(n = 5).
|
|
Under physiological conditions,
1-AR agonists such as
norepinephrine and epinephrine will bind
1-AR receptor subtypes, and the
net resultant effect on pHi and
contraction will be due to the interaction between CEC and
WB-4101-sensitive
1-AR
subtypes. Thus we examined the effect on
pHi and contraction of CEC
addition during nonselective
1-AR stimulation with
phenylephrine. Under these conditions, exposure to CEC further enhanced
the increase in twitch amplitude and cytosolic alkalinization due to
phenylephrine (Fig. 9). Note also that the
decrease in diastolic length due to phenylephrine was further augmented
by CEC. This result suggests that
1-AR subtypes have opposing
effects on myocardial pHi and contraction and that the CEC-sensitive
1-AR subtype antagonizes the
effects of the WB-4101-sensitive
1-AR subtype on
pHi.

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|
Fig. 9.
Representative example of the effects of CEC added in the continued
presence of phenylephrine on contraction and
pHi. Phenylephrine enhanced
contraction amplitude and pHi.
Upon addition of 2 µM CEC, both
pHi and contraction amplitude
increased further. Four myocytes were studied with this experimental
protocol. Display of results is similar to that in Fig. 3.
|
|
 |
DISCUSSION |
Although recent evidence indicates that
1-AR subtypes are present on
adult rat myocytes, the pharmacological properties of these receptors
have not been completely elucidated, and no specific agonists are
available. As in prior studies, the effects of
1-AR subtypes were dissected in
the present studies using nonselective
1-AR stimulation with
phenylephrine in conjunction with
1-AR subtype blockers WB-4101
and CEC, at concentrations that provided maximal and opposite
physiological effects. Because confusion still exists between the
pharmacological properties and the molecular classification of
1-AR subtypes, in the
present study,
1-AR subtypes have been referred to as WB-4101- and CEC-sensitive, and
attribution of the physiological effects to a specific receptor subtype
has been avoided.
The results of the present study provide the first evidence of the
functional role of WB-4101- and CEC-sensitive
1-AR subtypes on myocardial
contraction, cell Ca2+
homeostasis, myofilament responsiveness to
Ca2+, and
pHi. The specific following
conclusions can be drawn from the results:
1) the positive inotropic action of
nonselective
1-AR stimulation
is associated with both an increased
[Ca2+]i
transient amplitude, pHi, and an
enhanced myofilament response to
Ca2+;
2) a similar response is obtained
with phenylephrine plus CEC; 3)
phenylephrine plus WB-4101 causes a negative inotropic action that is
associated with a decrease in
[Ca2+]i
transient amplitude and pHi;
4) the decrease in
pHi caused by phenylephrine plus
WB-4101 appears to be due to inhibition of
Na+/H+
exchange and is abolished by interventions that either inactivate or
downregulate PKC; 5) when
1-AR subtypes are stimulated
simultaneously, they effect opposing actions on cell
Ca2+ homeostasis and
pHi, and the CEC-sensitive
receptor attenuates the positive inotropic action of the
WB-4101-sensitive receptor subtype; and
6) because all effects of
nonselective
1-AR stimulation with phenylephrine are abolished by simultaneous addition of WB-4101 plus CEC, it is likely that only two
1-AR subtypes modulate
myocardial inotropy in rat ventricular myocytes.
That
1-AR subtypes have
opposing effects on myocardial inotropy,
[Ca2+]i,
pHi, and on
Na+/H+
exchange is a novel discovery and may provide an explanation to prior
studies which showed that nonselective
1-AR stimulation can either
enhance, have no effect on, or decrease myocardial contraction (4, 9,
23, 31). These different responses, at least in part, may be related to
preferential activation of one of the two receptor subtypes under
varying experimental conditions or among species under a given
experimental condition. Prior studies have shown that nonselective
-adrenergic stimulation causes cytosolic alkalinization via
PKC-mediated activation of
Na+/H+
exchange (10, 29). Here we report that the WB-4101-sensitive
1-AR subtype (i.e.,
phenylephrine plus CEC) mediates such an increase in
pHi. In addition, the effect of
the CEC-sensitive subtype (i.e., phenylephrine plus WB-4101) to
decrease pHi seems also to be
mediated by PKC modulation of the
Na+/H+
exchange as it is abolished either by staurosporine, prolonged exposure
to PMA, or by EIPA. Indeed, in mammalian myocardium, there is evidence
that the WB-4101-sensitive subtype is mainly coupled to
phosphoinositide hydrolysis and PKC activation (14). This is also
confirmed by previous results obtained in similar experimental
conditions. In high bathing
[Ca2+], a condition
known to cause a downregulation of PKC, stimulation of the
WB-4101-sensitive
1-AR
subtypes had no appreciable effect on twitch amplitude,
cytosolic Ca2+, and
pHi (11). Interestingly, in the
same experimental conditions, the effect of CEC-sensitive
subtypes to reduce pHi was
abolished, whereas the residual negative inotropic effect correlated
with a reduced cytosolic
Ca2+ (11). Thus the present
results suggest the involvement of PKC in the effect of both
CEC- and WB-4101-sensitive
1-AR
receptor subtypes.
It is uncertain how PKC may decrease
pHi via
Na+/H+
exchange. However, recent studies have identified different
Na+/H+
exchanger isoforms (32), and one member of the
Na+/H+
exchanger gene family, NHE-3, has been reported to decrease
pHi upon acute stimulation with
PMA (16). Thus it is tempting to suggest that different
Na+/H+
exchanger isoforms may be expressed within the rat myocardium and that
CEC- and WB-4101-sensitive
1-AR
subtypes may be coupled via PKC to
Na+/H+
exchangers that possess opposite modulatory effects on
pHi. Alternatively,
1-AR subtypes may be coupled to
different PKC isoforms that have different effects on
Na+/H+
exchange.
Although phenylephrine enhances myofilament responsiveness to
Ca2+, and this effect can be
prevented by preexposure in WB-4101, why this effect was not reversed
by WB-4101 remains to be explained. It has been shown that cytosolic
alkalinization (10, 29) and possibly a change in the phosphorylation
state of myosin light chain 2 (5) are mechanisms for the
1-AR effect on myofilament responsiveness to Ca2+. However,
the reversibility of the enhanced
myofilament-Ca2+ sensitivity due
to
1-AR stimulation has not yet
been examined. Thus, once myofilament responsiveness to
Ca2+ has been increased, it may
not be promptly reversed, even if some of the mechanisms for this
response are no longer operative.
From our functional studies, phenylephrine plus WB-4101 or CEC appeared
to act on specific receptor subtypes without evidence of crossover
binding. However, the selectivity of CEC and WB-4101 has been
questioned (14, 21). CEC has been reported to bind irreversibly to sites identified as
1B,
1C, and
1D, and
WB-4101 has been reported to have a relatively high affinity for both
1A and
1C subtypes (12). Nonetheless, both antagonists have been
used extensively, and results similar to ours have been found by
several authors employing rat myocardial preparations. In addition, as
illustrated in Fig. 2, the adoption of the protocol of incubation and
cell pretreatment with CEC as originally devised by Minneman et al.
(19) elicited a pattern of responses virtually identical to our
conventional preparations.
In summary, the present study indicates that WB-4101- and CEC-sensitive
1-AR subtypes have opposite
modulatory actions on myocardial contraction, cell
Ca2+ homeostasis, and
pHi. Furthermore, our results
complement those of other studies that have shown opposite effects of
phenylephrine plus CEC and phenylephrine plus WB-4101 on the
electrophysiological properties of different cardiac preparations (1,
2, 7, 20).
 |
ACKNOWLEDGEMENTS |
We thank Sharon Wright for excellent secretarial assistance.
 |
FOOTNOTES |
Present address of G. Gambassi: Istituto di Medicina Interna e
Geriatria, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.
Address for reprint requests: M. C. Capogrossi, Laboratorio di
Patologia Vascolare, Istituto Dermopatico dell'Immacolata, Istituto di
Ricovero e Cura a Carattere Scientifico, Via dei Monti di Creta 104, 00167 Rome, Italy.
Received 17 October 1996; accepted in final form 10 December 1997.
 |
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